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ISOLATION, CHARACTERIZATION CHARACTERIZATION AND OPTIMIZATION OF AMYLASE PRODUCING PRODUCING BACTERIA FROM MUNICIPAL WASTE BY

CHANDRIMA SINHA DIVISION OF MICROBIOLOGY DEPARTMENT OF PHARMACEUTICAL TECHNOLOGY

THESIS SUBMITTED IN THE PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF PHARMACY IN THE FACULTY OF ENGINEERING AND TECHNOLOGY JADAVPUR UNIVERSITY KOLKATA - 700032 2010

JADAVPUR UNIVERSITY CERTIFICATE May, 2010 This is to certify that Chandrima Sinha , Jadavpur University, has carried out the research work on the subject entitled “ISOLATION, CHARACTERIZATION AND OPTIMIZATION OF AMYLASE PRODUCING BACTERIA FROM MUNICIPAL WASTE” under my direct supervision and guidance, in the Division of Microbiology, Department of Pharmaceutical Technology of this university. I am satisfied that she has carried out this work independently with proper care and attention.

……………………………… Dr. Amalesh Samanta Forwarded:

…………..…………..……………………………

Prof (Dr.) T. Jha Head of the Department Department of Pharmaceutical Technology Jadavpur University Kolkata – 700032

Reader, Division of Microbiology Department of Pharmaceutical Technology Jadavpur University Kolkata - 700032

………………………………………………..

Prof Niladri Chakraborty Dean, Faculty of Engineering & Technology Jadavpur University Kolkata - 700032

DEDICATED TO BABAA… AND MAA…

ACKNOWLEDGEMENT

I deem it a privilege to work under the guidance of Dr. Amalesh Samanta, Department of Pharmaceutical Technology, Jadavpur University. I am greatly indebted to his valuable guidance throughout the work that has enabled me to complete the project work. I am also grateful to Prof. (Dr.) T. Jha, Head of the Department of Pharmaceutical Technology, Jadavpur University, for providing me the facilities to carry-out this work. I would also like to express my gratitude to all other respected teachers for their help and support. I am indeed glad to convey cordial thanks to Ananya Banerjee, Durbadal Ojha, Arpita Sarkar, Anurup Mandal, Soma Ghosh and Pinaki Pal of our Laboratory for their help whenever needed. I am also grateful to Mr. Bira Kishore Parida, Senior Lab. Attendant, who has helped me immensely during my project work. I also thank my friends and all others who extended their co-operation to carry out this project successfully. And most importantly I would like to thanks my parents, sister, and all my family members for their patience, love and care without which I could not have completed this thesis work.

…………………………

Date: Place:

Jadavpur University

Chandrima Sinha Roll No. 000811402024 Pharmaceutical Technology Jadavpur University

INDEX Contents

Page No.

1.

Introduction

1 - 47

2.

Isolation and Characterization of Amylase Producing Bacteria

48 - 69

3.

Cultural and Nutritional Requirements For Optimum Amylase Production by The Two Selected Bacterial Strains

70 - 84

4.

Determination of Amylase Activity

85 - 94

And Stability 5.

Conclusions

95 - 97

6.

References

98 - 111

Preface: Municipal waste management becomes a hazardous problem nowadays and the wastes are responsible for environmental pollution as it’s consist of irritant industrial and domestic garbage. So the municipal wastes are one of the prime targets of the environmentalists for their crusade against pollution. In this regard, enzymes, the catalyst of biological systems are remarkable molecular device to determine the pattern of chemical transformation without affecting the environment. The most striking characteristics of the enzymes are their catalytic power and specificity. For its low cost and diversity microbial enzymes are enormously used in the field of pharmaceuticals, fuel, agriculture, food, Mining, chemical, bioengineering and other biotech industries in our country and abroad. In the context microbial amylase, break down starches into sugars has gained profound importance in the pharmaceutical and fine-chemical industries if enzymes with suitable properties could be prepared. With the advent of new frontiers in biotechnology, the spectrum of amylase application has widened in many other fields, such as clinical, medicinal and analytical chemistries, as well as their widespread application in starch saccharification and in the textile, food, brewing and distilling industries. The fungal strains are able to produce amylase extracellularly or intracellularly or both by different fermentation process. A few bacteria also found to produce extracellular amylase with a short period of cultivation. As municipal waste highly consists of different substances suitable for different microbes to grow, we use it for the isolation of amylase producing bacteria. Two bacterial strains were identified; their enzyme producing capabilities were compared. The physic- chemical and culture conditions of the two strains were optimized for maximum amylase production and the characterization of the crude amylase was also done. The production of sugars from starch by the action of amylase was determined to produce a justified investigation from the applied point of view. The thesis is presented in six chapters. The introductory part is confines to a review of amylase including its source, synthesis, regulation and application. The subsequent chapters deal with the isolation and identification of amylase producing bacteria, optimizations of the culture condition for maximum amylase production, determination of activity and stability of the enzyme, conclusions and lastly the references which helped me to carry out the work. The present investigation has widened the scope for research and development of amylase from bacterial origin. The bacterial enzymes has favorable properties like thermostability and salt tolerance that make it promising for the application in analytical fields and pollution control mechanisms. I hope that my work has made some contribution towards this.

Chandrima Sinha

Introduction

1

1.1.

GENERAL INTRODUCTION:

Biotechnology are considered as a useful alternative to conventional process technology in the industrial and analytical fields because of biological systems can accomplish complex chemical conversions under mild environmental conditions with high specificity and efficiency unlike the chemical catalysis. More over biological systems help in ingredient substitution, less undesirable products, increased plant capacity, increased product yields and at the same time they are less energy intensive and less polluting. The variety of chemical transformations catalyzed by biocatalysts i.e. enzymes, which are now a prime target of exploitation by the emerging biotechnology based industries. Enzymes are mainly proteins, that catalyze (i.e., increase the rates of) chemical reactions.[1][2] In enzymatic reactions, the molecules at the beginning of the process are called substrates, and the enzyme converts them into different molecules, called the products. Almost all processes in a biological cell need enzymes to occur at significant rates. Since enzymes are selective for their substrates and speed up only a few reactions from among many possibilities, the set of enzymes made in a cell determines which metabolic pathways occur in that cell. Like all catalysts, enzymes work by lowering the activation energy (Ea‡) for a reaction, thus dramatically increasing the rate of the reaction. Most enzyme reaction rates are millions of times faster than those of comparable un-catalyzed reactions. As with all catalysts, enzymes are not consumed by the reactions they catalyze, nor do they alter the equilibrium of these reactions. However, enzymes do differ from most other catalysts by being much more specific. Enzymes are known to catalyze about 4,000 biochemical reactions.[3] A few RNA molecules called ribozymes also catalyze reactions, with an important example being some parts of the ribosome.[4][5] Synthetic molecules called artificial enzymes also display enzyme-like catalysis[6]. Enzyme activity

[7]

can be affected by other molecules. Inhibitors are molecules that

decrease enzyme activity; activators are molecules that increase activity. Many drugs and poisons are enzyme inhibitors. Activity is also affected by temperature, chemical 2

environment (e.g., pH), and the concentration of substrate. Some enzymes are used commercially, for example, in the synthesis of antibiotics. In addition, some household products use enzymes to speed up biochemical reactions (e.g., enzymes in biological washing powders break down protein or fat stains on clothes; enzymes in meat tenderizers break down proteins, making the meat easier to chew). The activity of an enzyme depends, at the minimum, on a specific protein chain. In many cases, the enzyme consists of the protein and a combination of one or more parts called cofactors. This enzyme complex is usually simply referred to simply as the enzyme. Apoenzyme: The polypeptide or protein part of the enzyme is called the apoenzyme and may be inactive in its original synthesized structure. The inactive form of the apoenzyme is known as a proenzyme or zymogen. The proenzyme may contain several extra amino acids in the protein which are removed, and allows the final specific tertiary structure to be formed before it is activated as an apoenzyme. Cofactors: A cofactor is a non-protein substance which may be organic, and called a coenzyme. The coenzyme is often derived from a vitamin with specific examples discussed later. Another type of cofactor is an inorganic metal ion called a metal ion activator. The inorganic metal ions may be bonded through coordinate covalent bonds. The major reason for the nutritional requirement for minerals is to supply such metal ions as Zn+2, Mg+2, Mn+2, Fe+2, Cu+2, K+1, and Na+1 for use in enzymes as cofactors. Final Enzyme: The type of association between the cofactor and the apoenzymes varies. In some cases, the bonds are rather loose and both come together only during the course of a reaction. In other cases, they are firmly bound together by covalent bonds. The activating role of a cofactor is to either: activate the protein by changing its geometric shape, or by actually participating in the overall reaction. The overall enzyme contains a specific geometric shape called the active site where the reaction takes place. The molecule acted upon is called the substrate. 3

1.2.

Industrial applications of enzymes;

Enzymes are used in the chemical industry and other industrial applications when extremely specific catalysts are required. However, enzymes in general are limited in the number of reactions they have evolved to catalyze and also by their lack of stability in organic solvents and at high temperatures. Consequently, protein engineering is an active area of research and involves attempts to create new enzymes with novel properties, either through rational design or in vitro evolution

[8]9]

. These efforts have begun to be

successful, and a few enzymes have now been designed "from scratch" to catalyze reactions that do not occur in nature.[10] Table1.1. Industrial applications of enzymes:

Application

Enzymes used

Uses

Production of sugars from starch, such as in making high-fructose corn syrup.[11] In

baking,

catalyze

Amylases from fungi and breakdown of starch in the Food processing

plants.

flour

to

sugar.

Yeast

of

sugar

fermentation Amylases catalyze the release

produces

of simple sugars from starch

dioxide

the that

carbon raises

the

dough.

Biscuit manufacturers use Proteases

them to lower the protein level of flour.

4

Trypsin

Baby foods

To predigest baby foods.

Enzymes from barley are released

during

mashing Brewing industry

stage

of

the beer

production.

They degrade starch and proteins to produce simple sugar, amino acids and peptides that are used by yeast for fermentation.

Widely used in the brewing Industrially

produced process to substitute for the

barley enzymes

natural enzymes found in barley.

Germinating barley used for malt.

Amylase,

glucanases, Split polysaccharides and

proteases

proteins in the malt.

Betaglucanases

and Improve the wort and beer

arabinoxylanases

Amyloglucosidase pullulanases

filtration characteristics.

and

Low-calorie adjustment

beer

and of

fermentability.

Brewing industry Remove Proteases

cloudiness

produced during storage of beers.

5

Acetolactatedecarboxylase (ALDC)

Fruit juices

Increases

fermentation

efficiency

by

reducing

diacetyl formation.[12]

Cellulases, pectinases

Clarify fruit juices

Rennin, derived from the stomachs

of

young Manufacture

ruminant

animals

of

cheese,

(like used to hydrolyze protein.

calves and lambs). Dairy industry Microbially enzyme

produced Now finding increasing use in the dairy industry.

Is implemented during the

Lipases

production

of

cheese

enhance

to

Roquefort the

ripening of the blue-mould

Roquefort cheese

cheese.

Lactases

Break

down

lactose

to

glucose and galactose.

Meat tenderizers Papain

To soften meat for cooking.

6

Amylases, amyloglucosideases and glucoamylases

Converts

starch

glucose and various syrups.

Converts Starch industry Glucose

into

glucose

into

fructose in production of high fructose syrups from

Fructose Glucose isomerase

starchy

materials.

syrups

have

These

enhanced

sweetening properties and lower calorific values than sucrose for the same level of sweetness.

Degrade starch to lower viscosity, aiding sizing and coating paper. Xylanases

Paper industry

reduce bleach required for decolorising; Amylases,

Xylanases, smooth

Cellulases and ligninases

cellulases

fibers,

water

enhance

drainage,

promote

ink

and

removal;

lipases reduce pitch and A paper mill in South Carolina.

lignin-degrading

enzymes

remove lignin to soften paper.

Biofuel industry

Cellulases

Used

to

break

down

7

Cellulose in 3D

cellulose into sugars that can

be

fermented

(see

cellulosic ethanol).

Ligninases

Primarily

Use of lignin waste

proteases,

produced in an extracellular form from bacteria

Used

for

presoak

conditions and direct liquid applications helping with removal of protein stains from clothes.

Detergents Amylases Biological detergent

for

machine

dish washing to remove resistant starch residues.

Used Lipases

to

assist

in

the

removal of fatty and oily stains.

Cellulases

Used in biological fabric conditioners.

To remove proteins on Contact lens cleaners

Proteases

contact lens to prevent infections.

8

To generate oxygen from Rubber industry

Catalase

peroxide to convert latex into foam rubber.

Dissolve gelatin off scrap Photographic industry

Protease (ficin)

film, allowing recovery of its silver content.

Used to manipulate DNA in genetic

engineering,

important in pharmacology, agriculture and medicine. Molecular biology Part of the DNA double helix.

Restriction enzymes, DNA Essential ligase and polymerases

digestion

for

restriction and

the

polymerase chain reaction. Molecular biology is also important

in

forensic

science.

The demand for industrial enzymes, particularly microbial origin, is ever increasing owing to their applications in a wide variety of processes. Besides that, the microbial enzymes can be produced in sufficient quantity to meet all demands of the market. Seasonal fluctuations of raw materials do not count and there are possibilities of genetic and environmental manipulations of microorganisms to give increased yield of desired enzymes in a way that is not possible with plant and animal cell enzymes. Moreover the diversity of enzymes available from microorganisms is great. Lastly, microbial enzymes

9

present a wild spectrum of characteristics that make them utilizable for quite specific applications. The first step in the production of microbial enzyme involves the selection of microorganism, which is suitable to produce the desired enzyme. next the physical and chemical environment of the organism must be optimized for the enzyme synthesis as large as possible. In this context, extracellular enzyme is preferred as because it is cost effective and present in a relatively pure form in the culture liquor. Microbial enzymes are now widely used in a large number of fields such as pharmaceutical, food, diary, detergent, textile and cosmetic industries. The current total consumption of all the enzymes in India including penicillin acylase, alpha amylase, amylo- glycosidase, papain, pancreatin, alkaline protease, lipase, fungal diastase ete, in monetary terms is over 450 million (Rs). In the above scenario, amylase, a starch degrading enzyme have gained importance in various industrial process like pharmaceutical, food, brewing, paper , textile

and

chemicals. It is extensively used in pharmaceutical industries in digestive tonics, for hydrolysis of starch to produce different sugars like glucose and maltose which have several applications. Starchy substances constitute the major part of the human diet for most of the people in the world [13], as well as many other animals. They are synthesized naturally in a variety of plants. Some plant examples with high starch content are corn, potato, rice, sorghum, wheat, and cassava. It is no surprise that all of these are part of what we consume to derive carbohydrates. Similar to cellulose, starch molecules are glucose polymers linked together by the alpha-1, 4 and alpha-1,6 glucosidic bonds, as opposed to the beta-1,4 glucosidic bonds for cellulose. In order to make use of the carbon and energy stored in starch, the human digestive system, with the help of the enzyme amylases, must first break down the polymer to smaller assimilable sugars, which is eventually converted to the individual basic glucose units.

10

Because of the existence of two types of linkages, the alpha-1, 4 and the alpha-1,6, different structures are possible for starch molecules. An unbranched, single chain polymer of 500 to 2000 glucose subunits with only the alpha-1, 4 glucosidic bonds is called amylose. On the other hand, the presence of alpha-1, 6 glucosidic linkages results in a branched glucose polymer called amylopectin. The degree of branching in amylopectin is approximately one per twenty-five glucose units in the unbranched segments. Another closely related compound functioning as the glucose storage in animal cells is called glycogen, which has one branching per 12 glucose units. The degree of branching and the side chain length vary from source to source, but in general the more the chains are branched, the more the starch is soluble. Starch is generally insoluble in water at room temperature. Because of this, starch in nature is stored in cells as small granules which can be seen under a microscope. Starch granules are quite resistant to penetration by both water and hydrolytic enzymes due to the formation of hydrogen bonds within the same molecule and with other neighboring molecules. However, these inter- and intra-hydrogen bonds can become weak as the temperature of the suspension is raised. When an aqueous suspension of starch is heated, the hydrogen bonds weaken, water is absorbed, and the starch granules swell. This process is commonly called gelatinization because the solution formed has a gelatinous, highly viscous consistency. The same process has long been employed to thicken broth in food preparation. Depending on the relative location of the bond under attack as counted from the end of the chain, the products of this digestive process are dextrin, maltotriose, maltose, and glucose, etc. Dextrins are shorter, broken starch segments that form as the result of the random hydrolysis of internal glucosidic bonds. A molecule of maltotriose is formed if the third bond from the end of a starch molecule is cleaved; a molecule of maltose is formed if the point of attack is the second bond; a molecule of glucose results if the bond being cleaved is the terminal one; and so on. As can be seen from the exercises in Experiment No. 3, the initial step in random depolymerization is the splitting of large chains into various smaller sized segments. The breakdown of large particles drastically reduces the viscosity of gelatinized starch solution, resulting in a process called 11

liquefaction because of the thinning of the solution. The final stages of depolymerization are mainly the formation of mono-, di-, and tri-saccharides. This process is called saccharification, due to the formation of saccharides. Since a wide variety of organisms, including humans, can digest starch, alpha-amylase is obviously widely synthesized in nature, as opposed to cellulase. For example, human saliva and pancreatic secretion contain a large amount of alpha-amylase for starch digestion. The specificity of the bond attacked by alpha-amylases depends on the sources of the enzymes. Currently, two major classes of alpha-amylases are commercially produced through microbial fermentation. Based on the points of attack in the glucose polymer chain, they can be classified into two categories, liquefying and saccharifying. Because the bacterial alpha-amylase to be used in this experiment randomly attacks only the alpha-1,4 bonds, it belongs to the liquefying category. The hydrolysis reaction catalyzed by this class of enzymes is usually carried out only to the extent that, for example, the starch is rendered soluble enough to allow easy removal from starch-sized fabrics in the textile industry. The paper industry also uses liquefying amylases on the starch used in paper coating where breakage into the smallest glucose subunits is actually undesirable. (One cannot bind cellulose fibers together with sugar!) On the other hand, the fungal alpha-amylase belongs to the saccharifying category and attacks the second linkage from the non reducing terminals (i.e. C4 end) of the straight segment, resulting in the splitting off of two glucose units at a time. Of course, the product is a disaccharide called maltose. The bond breakage is thus more extensive in saccharifying enzymes than in liquefying enzymes. The starch chains are literally chopped into small bits and pieces. Finally, the amyloglucosidase (also called glucoamylase) component of an amylase preparation selectively attacks the last bond on the non reducing terminals. The type to be used in this experiment can act on both the alpha-1, 4 and the alpha-1, 6 glucosidic linkages at a relative rate of 1:20, resulting in the splitting off of simple glucose units into the solution. Fungal amylase and amyloglucosidase may be used together to convert starch to simple sugars. The practical

12

applications of this type of enzyme mixture include the production of corn syrup and the conversion of cereal mashes to sugars in brewing. Thus, it is important to specify the source of enzymes when the actions and kinetics of the enzymes are compared. Four types of alpha-amylases from different sources will be employed in this experiment: three of microbial origin and one of human origin. The effects of temperature, pH, substrate concentration, and inhibitor concentration on the kinetics of amylase catalyzed reactions will be studied. Finally, the action of the amylase preparations isolated from microbial sources will be compared to that from human saliva. A number of microorganisms like fungi

[14-18]

, yeast

[19-21]

and bacteria

[22-25]

have been

reported to degrade starch and thrive on them by producing sugars (ref). In this present treatise, an account of extracellular amylase, including its assay and production have been considered from the newly isolated bacterial strain.

1.3.

Present knowledge about amylase [26]:

Amylase is a digestive enzyme classified as a saccharidase (an enzyme that cleaves polysaccharides). It is mainly a constituent of pancreatic juice and saliva, needed for the breakdown of long-chain carbohydrates (such as starch) into smaller units. Amylase is a digestive enzyme made primarily by the pancreas and salivary glands. Enzymes are substances made and used by the body to trigger specific chemical reactions. The primary function of the enzyme amylase is to break down starches in food so that they can be used by the body. Amylase is also synthesized in the fruit of plants during ripening, causing them to become sweeter. Amylases are enzymes that catalyze the hydrolysis of alpha-1, 4-glycosidic linkages of polysaccharides to yield dextrins, oligosaccharides, maltose and D-glucose. Amylases are derived from animal, fungal and plant sources. Pancreatin and pancrelipase contain amylase derived from the pancreas of animals, usually porcine pancreas. Amylase is also 13

derived from barley malt and the fungus Aspergillus oryzae. There are a few different amylases. These enzymes are classified according to the manner in which the glysosidic bond is attacked. 1. α- Amylase (EC 3.2.1.1 ) (Alternate names: 1, 4-α-D-glucan glucanohydrolase; glycogenase) The αamylases are calcium metalloenzymes, completely unable to function in the absence of calcium. By acting at random locations along the starch chain, α-amylase breaks down long-chain carbohydrates, ultimately yielding maltotriose and maltose from amylose, or maltose, glucose and "limit dextrin" from amylopectin. Because it can act anywhere on the substrate, α-amylase tends to be faster-acting than β-amylase. In animals, it is a major digestive enzyme and its optimum pH is 6.7-7.0. In human physiology, both the salivary and pancreatic amylases are α-Amylases. They are discussed in much more detail at alpha-Amylase. Also found in plants (adequately), fungi (ascomycetes and basidiomycetes) and bacteria (Bacillus). 2. β- Amylase (EC 3.2.1.2 ) (Alternate names: 1, 4-α-D-glucan maltohydrolase; glycogenase; saccharogen amylase) Another form of amylase, β-amylase is also synthesized by bacteria, fungi, and plants. Working from the non-reducing end, β-amylase catalyzes the hydrolysis of the second α-1, 4 glycosidic bond, cleaving off two glucose units (maltose) at a time. During the ripening of fruit, β-amylase breaks starch into maltose, resulting in the sweet flavor of ripe fruit. Both α-amylase and β-amylase are present in seeds; β-amylase is present in an inactive form prior to germination, whereas α-amylase and proteases appear once germination has begun. Cereal grain amylase is the key to the production of malt. Many microbes also produce amylase to degrade extracellular starches. Animal tissues do not contain β-

14

amylase, although it may be present in microorganisms contained within the digestive tract. 3. γ- Amylase (EC 3.2.1.3 ) (Alternative names: Glucan 1, 4-α-glucosidase; amyloglucosidase; Exo-1, 4-α-glucosidase;

glucoamylase;

lysosomal

α-glucosidase;

1,

4-α-D-glucan

glucohydrolase) In addition to cleaving the last α (1-4) glycosidic linkages at the nonreducing end of amylose and amylopectin, yielding glucose, γ-amylase will cleave α (1-6) glycosidic linkage. Unlike the other forms of amylase, γ-amylase is most efficient in acidic environments and has an optimum pH 3. Amylase is also used in industry. It is used in brewing and fermentation industries for the conversion of starch to fermentable sugars, in the textile industry for designing textiles, in the laundry industry in a mixture with protease and lipase to launder clothes, in the paper industry for sizing, and in the food industry for preparation of sweet syrups, to increase diastase content of flour, for modification of food for infants, and for the removal of starch in jelly production. The pancreas has the highest amylase concentration and largest total amount of amylase of any organ in the body. Unfortunately, other tissues, particularly the parotid salivary glands, also contain large quantities. Pancreatic or salivary amylase is not absorbed by intact gut mucosa, and normal serum amylase activity levels are low. Any amylase that is present presumably comes from enzyme leakage directly into the blood from the ascinar cells or via lymphatics draining the glands. With ductular obstruction and/or inflammation of either the pancreas or parotid glands, enzyme leakage directly into the blood or via the lymphatics increases. In rare situations gut permeability changes, whereby a large quantity of amylase leaks into the peritoneal fluid and is eventually delivered via thoracic duct into the circulation causing hyperamylasemia. When amylase levels are too high in the urine and someone has abdominal pain then they are automatically diagnosed with pancreatitis.

15

The major role of the calcium-containing metalloenzyme amylase in the oral cavity is the hydrolysis of the alpha-1, 4 linkages of starch. Amylase can play an important role in the colonization and metabolism of oral bacteria leading to dental plaque formation. Amylase bound to the bacterial surface retains its enzymatic activity. The bacterial-bound amylase can hydrolyze starch to glucose, which is then metabolized to lactic acid by the bacteria, thereby leading to dental plaque formation and progression.

1.4.

Review of literature:

The enzyme amylase is of great significance in biotechnology due to its application interestingly, the first enzyme produced industrially was amylase from fungal source in 1984, which was used as a pharmaceutical aid for the treatment of digestive disorders. Although they can be derived from several sources such as plants, animals and micro organisms, the enzyme from microbial sources generally meet the industrial demands. With the advent of new frontiers in biotechnology, the spectrum of amylase application has widen in many other fields, such as in clinical, medicinal and analytical chemistry, starch saccharification, textile, food, brewing and distilling industries. Given below is the literature available on microorganisms reviewed, scince any attempt on other sources is beyond the scope of the present study. It is reported that several species of bacteria, yeast and fungi produce amylase.

Table 1.2. Sources of microbial amylase: Organisms

References

Bacteria: Aeromonas caviae

De-Almeida et al. (1997)

16

Alicyclobacillus acidocaldarius

Schwermann et al. (1994)

Alteromonas haploplanetis

Feller et al. (1994)

Archeobacterium pyrococcus

Melasniemi (1988)

Bacillus acidocaldarius

Boyer et al. (1979) Kanna (1986)

Bacillus amyloliquefaciens

Milner et al. (1997) Syu & Chen (1997) Hillier et al. (1997) Lin et al. (1997)

Bacillus brevis

Konishi et al. (1990) Tonkova et al. (1994) Ariga et al. (1997) Stefanova et al. (1998)

Bacillus caldovelox

Fogarty et al. (1991) Kelly et al. (1991)

Bacillus circulans

Napier (1977) Ivanova and Debreva (1994)

Bacillus coagulans

Medda and Chandra (1980) Babu and Satyanarayana (1995)

Bacillus globisporus

Bandhopadhyay et al. (1994)

17

Bacillus licheniformis

Medda and Chandra (1980) Morgan and Priest (1981) Krishan and Chandra (1983) Priest and Thirunavukkarasu 91985) Ramesh and Lonsane (1989) Decordt Medda et al. (1992) Padmanabhan et al. (1992) Varlan et al. (1996) Dobreva et al. (1998)

Bacillus megaterium

Stark et al. (1982) David et al. (1987) Brumm et al. (1991)

Bacillus staerothermophilus

Srivastava and Baruah (1986) Omidiji et al. (1997)

Bacillus subtilis

Orlando et al. (1983) Gayal et al. (1990) Nishimura et al. (1994) EI Helow and EI Gazaerly (1996) Butz et al. (1996) Krishna and Chandrasekharan (1996)

18

Gayal et al. (1997) Bacillus flavothermus

Kelly et al. (1997)

Chloroflexus aurantiacus

Ratankhanokchai et al. (1992)

Clostridium thermohydrosulfuricum

Hyun and Zeikus (1985) Melasniemi (1988)

Clostridium acetobutylicum

Annous and Blaschek (1990) Paquet et al. (1991) Soni et al. (1992)

Clostridium butricum

Tanaka et al. (1987)

Clostridium thermosulfurogenes

Hyun and Zeikus (1985) Bahl et al. (1991) Swamy ans Seenayya (1996)

Eubacterium spp.

Delahaye et al. (1991)

Flavobacterium spp.

Sugita et al. (1996)

Halobacterium halobium

Patel et al. (1996)

Halobacterium salinarium

Ramesh and Lonsane (1989)

Lactobacillus amylophilus

Vishnu et al. (1998)

Lactobacillus fermentum

Sanni et al. (2002)

Lactobacillus plantarum

Giraud et al. (1991) Sanni et al. (2002)

19

Lysobacter brunescens

Tigerstrom and Stelmaschuk et al. (1987)

Micrococcus luteus

Illori et al. (1997)

Micrococcus vanans

Adeleye (1990)

Myxococcus coralloides

Farezvidal et al. (1990) Farezvidal et al. (1995)

Pseudomonas stutzeri

Robyt and Ackerman (1971) Sakano et al. (1982) Sakano et al. (1983) Kimura et al. (1989)

Pseudomonas saccharophila

Zhou et al. (1989)

Pyrococcus furiosus

Brown and Kelly (1993)

Pyrococcus woesei

Koch et al. (1991)

Streptomyces albus

Hyslop and Sleeper (1964) Andrews and Ward (1987)

Streptomyces albaduncus

Chadha et al. (1995)

Streptomyces rimosus

Vukelic et al. (1992)

Streptomyces praecox

Wako et al. (1978)

Streptomyces hygroscopicus

Hidaka and Adachi (1980)

Streptomyces limosus

Pairbairn et al. (1985)

Streptomyces thermoviplaceus

Goldberg and Edwards (19900

20

Streptomyces bovis

Satoh et al. (1997)

Thermus spp.

Abramov et al. (1986)

Thermactinimyces thalophilus

Uguru et al. (1997)

Fungi: Aspergillus awamorei

Bhella and Altosaar (1985) Siedenberg et al. (1997)

Aspergillus flavus

Ali and Adbelmoneim (1989) Fakhoury and Woloshuk (1999)

Aspergillus ficum

Hayashida and Teramoto (1986)

Aspergillus foetidus

Michelena and Castillo (1984)

Aspergillus fumigates

Reade and Gregory (1975) Goto et al. (1998)

Aspergillus kawachi

Mikami et al. (1987) Ohnishi et al. (1990)

Aspergillus oryzae

Kundu and Das (1970) Thorbek and Eplov (19740 Shibuya et al. (1992) Torrado et al. (1994) Murado et al. (1997)

21

Agger et al. (1998) Francis et al. (2002) Aspergillus niger

Venkataramu et al. (1975) Iqbal and Zafar (1994) Hoshino et al. (1994)

Aspergillus flavipes

Frolova et al. (2002)

Aspergillus usanii

Suganuma et al. (1996)

Botryodiplodia theobromae

Ray (2004)

Daedalea flavida

Das et al. (1978)

Ganoderma lucidum

Das et al. (1978)

Gloeophyllum striata

Das et al. (1978)

Gloeophyllum saepiarum

Das et al. (1978)

Filobasidium capsuligenum

Narayanan and Shanmugasundaram (1967)

Fuusarium oxysporum

Kurchenko et al. (2001)

Humicola insolens

Fergus (1969)

Humicola lanuginose

Fergus (1969)

Humicola stellata

Fergus (1969)

Lentinus subnudes

Das et al. (1978)

Lentinus palisoti

Das et al. (1978)

Lipomyces spp.

Spencer-Martins and Vanuden (1979)

22

Lipomyces spp.

Spencer-Martins (1982) Spencer-Martins (1984) Kelly et al. (1985)

Mucor spp.

Mohapatra et al. (1998)

Mucor pusillus

Loginova et al. (1970)

Myceliophthora thermophila

Sadhukhan et al. (1993)

Penicillum amagaskiense

Doyle et al. (1988)

Penicillium brunneum

Hasha and Ohta (1994)

Penicillium expansum

Doyle et al. (1988)

Piromyces communis

Yanke et al. (1993)

Polyporus anthelminiticus

Das et al. (1978)

Polyporus grammocephalous

Das et al. (1978)

Polyporus luzonensis

Das et al. (1978)

Polyporus sanguineus

Das et al. (1978)

Polyporus tricholoma

Das et al. (1978)

Polyporus zonalis

Das et al. (1978)

Rhizopus spp.

de Souza et al. (1996)

Rhizopus oryzae

Ray (2004)

Neocallimastix patrician

Yanke et al. (1993)

Orpinomyces joyonii

Yanke et al. (1993)

23

Schizophyllum commune

Das et al. (1978)

Sclerotium rolfsii

Nwufo and Fajola (19880

Sporotrichum thermophile

Adams (1997)

Schwanniomyces alluvius

Simoes-Mendes (1984)

Saccharomyces cerevisiae

Soni and Sandhu (1990) Shibuya et al. (1992) Olasupo et al. (1996)

Saccharomyces kluyveri

Moller et al. (2004)

Saccharomycopsis fibuligera

Gogoi et al. (1987) Gogoi et al. (1998)

Saccharomycopsis capsularis

Soni et al. (1996)

Schwanniomyces occidentalis

Park et al. (1992) Kocher and Katyal (2004)

Schwanniomyces castellii

Clementi et al. (1980) Oteng- Gyang et al. (1981) Wilson and Ingledew (1982) Sills et al. (1984)

Trichoderma harzianum

De Azevedo et al. (2000)

Trichosporon beigelii

Soni and Sandhu (1990)

Trichosporon pullulans

De Mot and Verachtert (1986)

24

Trametes persooni

Das et al.(1978)

Trametes lactinea

Das et al.(1978)

Torulopsis candida

Soni and Sandhu (1990)

Brettanomyces naardensis

Gautam et al. (1991)

Botryodiplodia theobromae

Nwufo and Fajola (1988)

Candida antartica

De Mot and Verachtert (1987)

Candida tsukubanensis

De Mot et al. (1985)

Candida utilis

Moreton (1978)

Cryptococcus spp.

Lefuji et al. (1996)

Debaromyces hansenii

Soni and Sandhu (1990)

Hansenula anomala

Soni and Sandhu (1990)

Oosporidium margaritiferum

Soni and Sandhu (1990)

Phaffia rhodozyma

Gautam et al. (1991)

1.5.

Chemical structure and distribution of starch:

Starch or amylum is a carbohydrate consisting of a large number of glucose units joined together by glycosidic bonds. This polysaccharide is produced by all green plants as an energy store. It is the most important carbohydrate in the human diet and is contained in such staple foods as potatoes, wheat, maize (corn), rice, and cassava. Pure starch is a white, tasteless and odorless powder that is insoluble in cold water or alcohol. It consists of two types of molecules: the linear and helical amylose and the branched amylopectin. Depending on the plant, starch generally contains 20 to 25% 25

amylose and 75 to 80% amylopectin.[27] Glycogen, the glucose store of animals, is a more branched version of amylopectin. Starch is processed to produce many of the sugars in processed foods. When dissolved in warm water, it can be used as a thickening, stiffening or gluing agent, giving wheatpaste. The word "starch" is derived from Middle English sterchen, meaning to stiffen. "Amylum" is Latin for starch, from the Greek "amulon" which means "not ground at a mill". The root amyl is used in biochemistry for several compounds related to starch. Energy store of plants In photosynthesis, plants use light energy to produce glucose from carbon dioxide. The glucose is stored mainly in the form of starch granules, in plastids such as chloroplasts and especially amyloplasts. Toward the end of the growing season, starch accumulates in twigs of trees near the buds. Fruit, seeds, rhizomes, and tubers store starch to prepare for the next growing season. Glucose is soluble in water, hydrophilic, binds much water and then takes up much space; glucose in the form of starch, on the other hand, is not soluble and can be stored much more compactly. Since starch is a reserve sugar for the plant, glucose molecules are bound in starch by the easily hydrolyzed alpha bonds. The same type of bond can also be seen in the animal reserve polysaccharide glycogen. This is in contrast to many structural polysaccharides such as chitin, cellulose and peptidoglycan, which are bound by beta-bonds and are much more resistant to hydrolysis. Biosynthesis Plants produce starch by first converting glucose 1-phosphate to ADP-glucose using the enzyme glucose-1-phosphate adenylyltransferase. This step requires energy in the form of ATP. The enzyme starch synthase then adds the ADP-glucose via a 1, 4-alpha glycosidic bond to a growing chain of glucose residues, liberating ADP and creating amylose. Starch 26

branching enzyme introduces 1, 6-alpha glycosidic bonds between these chains, creating the branched amylopectin. The starch debranching enzyme isoamylase removes some of these branches. Several isoforms of these enzymes exist, leading to a highly complex synthesis process.[28]. While amylose was traditionally thought to be completely unbranched, it is now known that some of its molecules contain a few branch points.[29] Glycogen and amylopectin have the same structure, but the former has about one branch point per ten 1, 4-alpha bonds, compared to about one branch point per thirty 1, 4-alpha bonds in amylopectin.[30] Another difference is that glycogen is synthesized from UDPglucose while starch is synthesized from ADP-glucose.

Properties Structure Starch molecules arrange themselves in the plant in semi-crystalline granules. Each plant species has a unique starch granular size: rice starch is relatively small (about 2µm) while potato starches have larger granules (up to 100µm). Although in absolute mass only about one quarter of the starch granules in plants consist of amylose, there are about 150 times more amylose molecules than amylopectin molecules. Amylose is a much smaller molecule than amylopectin.

Fig.1.1. Representative Partial Structure of Amylase [31] 27

Fig.1.2. Representative Partial Structure of Amylopectin [31] Starch becomes soluble in water when heated. The granules swell and burst, the semicrystalline structure is lost and the smaller amylose molecules start leaching out of the granule, forming a network that holds water and increasing the mixture's viscosity. This process is called starch gelatinization. During cooking the starch becomes a paste and increases further in viscosity. During cooling or prolonged storage of the paste, the semicrystalline structure partially recovers and the starch paste thickens, expelling water. This is mainly caused by the retrogradation of the amylose. This process is responsible for the hardening of bread or staling, and for the water layer on top of a starch gel (syneresis). Some cultivated plant varieties have pure amylopectin starch without amylose, known as waxy starches. The most used is waxy maize, others are glutinous rice, waxy potato starch. Waxy starches have less retrogradation, resulting in a more stable paste. High amylose starch, amylomaize, is cultivated for the use of its gel strength. Hydrolysis The enzymes that break down or hydrolyze starch into the constituent sugars are known as amylases. 28

Alpha-amylases are found in plants and in animals. Human saliva is rich in amylase, and the pancreas also secretes the enzyme. Individuals from populations with a high-starch diet tend to have more amylase genes than those with low-starch diets; chimpanzees have very few amylase genes. It is possible that turning to a high-starch diet was a significant event in human evolution [32]. Beta-amylase cuts starch into maltose units. This process is important in the digestion of starch and is also used in brewing, where the amylase from the skin of the seed grains is responsible for converting starch to maltose (Malting, Mashing). Dextrinization If starch is subjected to dry heat, it breaks down to form pyrodextrins, in a process known as dextrinization. Pyrodextrins are brown in color. This process is partially responsible for the browning of toasted bread. Chemical tests

Iodine test Iodine solution is used to test for starch; a dark blue color indicates the presence of starch. The details of this reaction are not yet fully known, but it is thought that the iodine (I3− and I5− ions) fit inside the coils of amylose, the charge transfers between the iodine and the starch, and the energy level spacing in the resulting complex correspond to the absorption spectrum in the visible light region. The strength of the resulting blue color depends on the amount of amylose present. Waxy starches with little or no amylose present will color red.

29

Fig.1.3. Starch, 800x magnified, under polarized light. Starch indicator solution consisting of water, starch and iodine is often used in redox titrations: in the presence of an oxidizing agent the solution turns blue, in the presence of reducing agent the blue color disappears because tri-iodide (I3−) ions break up into three iodide ions, disassembling the starch-iodine complex. A 0.3% w/w solution is the standard concentration for a starch indicator. It is made by adding 3 grams of soluble starch to 1 liter of heated water; the solution is cooled before use (starch-iodine complex becomes unstable at temperatures above 35°C). Microscopy of starch granules - Each species of plant has a unique shape of starch granules in granular size, shape and crystallization pattern. Under the microscope, starch grains stained with iodine illuminated from behind with polarized light show a distinctive Maltese cross effect (also known as extinction cross and birefringence). Starch as food Starch is the most important carbohydrate in the human diet and is contained in many staple foods. The major sources of starch intake worldwide are rice, wheat, maize (corn), potatoes and cassava. Widely used prepared foods containing starch are bread, pancakes, cereals, noodles, pasta, porridge and tortilla [33]. Depending on the local climate other starch sources are used for food, such as arrowroot, arracacha, buckwheat, barley, oat, millet, rye, banana, breadfruit, canna, colacasia, katakuri, kudzu, malanga, oca, polynesian arrowroot, sago, sorghum, sweet potato, taro, 30

water chestnut and yams. Chestnuts and edible beans, such as favas, lentils, mung bean and peas are also rich in starch. Digestive enzymes have problems digesting crystalline structures. Raw starch will digest poorly in the duodenum and small intestine, while bacterial degradation will take place mainly in the colon. Resistant starch is starch that escapes digestion in the small intestine of healthy individuals. In order to increase the digestibility, starch is cooked. Hence, before humans started using fire, eating grains was not a very useful way to get energy. Starch industry The starch industry extracts and refines starches from seeds, roots and tubers, by wet grinding, washing, sieving and drying. Today, the main commercial refined starches are cornstarch, tapioca and wheat and potato starch. To a lesser extent, sources include rice, sweet potato, sago and mung bean. Historically, Florida arrowroot was also commercialized. Still starch is extracted from more than 50 types of plants. Untreated starch requires heat to thicken or gelatinize. When a starch is pre-cooked, it can then be used to thicken instantly in cold water. This is referred to as a pregelatinized starch. Starch sugars Starch can be hydrolyzed into simpler carbohydrates by acids, various enzymes, or a combination of the two. The resulting fragments are known as dextrins. The extent of conversion is typically quantified by dextrose equivalent (DE), which is roughly the fraction of the glycosidic bonds in starch that have been broken. These starch sugars are by far the most common starch based food ingredient and are used as sweetener in many drinks and foods. They include: •

Maltodextrin, a lightly hydrolyzed (DE 10–20) starch product used as a blandtasting filler and thickener.

31



Various glucose syrup / corn syrups (DE 30–70), viscous solutions used as sweeteners and thickeners in many kinds of processed foods.



Dextrose (DE 100), commercial glucose, prepared by the complete hydrolysis of starch.



High fructose syrup, made by treating dextrose solutions with the enzyme glucose isomerase, until a substantial fraction of the glucose has been converted to fructose. In the United States, high fructose corn syrup is the principal sweetener used in sweetened beverages because fructose has better handling characteristics, such as microbiological stability, and more consistent sweetness/flavor. High fructose corn syrup has the same sweetness as sugar.



Sugar alcohols, such as maltitol, erythritol, sorbitol, mannitol and hydrogenated starch hydrolysate, are sweeteners made by reducing sugars.

Modified starches A modified food starch is a starch that has been chemically modified to allow the starch to function properly under conditions frequently encountered during processing or storage, such as high heat, high shear, low pH, freeze /thaw and cooling. The modified starches are E coded according to the International Numbering System for Food Additives (INS): [34] •

1401 Acid-treated starch



1402 Alkaline-treated starch



1403 Bleached starch



1404 Oxidized starch



1405 Starches, enzyme-treated



1410 Monostarch phosphate



1412 Distarch phosphate



1413 Phosphated distarch phosphate



1414 Acetylated distarch phosphate



1420 Starch acetate

32



1422 Acetylated distarch adipate



1440 Hydroxypropyl starch



1442 Hydroxypropyl distarch phosphate



1443 Hydroxypropyl distarch glycerol



1450 Starch sodium octenyl succinate



1451 Acetylated oxidized starch

INS 1401, 1402, 1403 and 1405 are in the EU food ingredients without an E-number. Typical modified starches for technical applications are cationic starches, hydroxyethyl starch and carboxymethylated starches. 1.6.

Exoenzymes and their cellular localization:

In biological systems enzymes act as reaction catalyst which are present in organized sequences, catalyzes the stepwise reaction in metabolic pathway by which nutrient molecules are degraded, chemical energy is conserved and transformed. Very small cellular processes of the microorganisms are able to synthesize and activate enzymes within cell (intracellular) or secrete it into surrounding (extracellular) medium. The intracellular group of enzymes may either be purely located inside the cell (cytoplasmic) or attached to the cytoplasmic membranes. Whereas, extracellular or exoenzymes are completely dissociated from cell and found free in the surrounding medium. Therefore, various enzymes, on the basis of their location, maybe classified as 1. Intracellular i) Cytoplasmic ii) Membrane bound 2. Extracellular It is very difficult for intracellular enzyme to determine whether the enzyme lies free inside the cytoplasm or bound to the cytoplasmic membrane. But now a days refined techniques have been developed to decide about the localization. Extracellular enzymes

33

localization is full of controversy and mingled with confusion. Earlier biochemist thought that the extracellular enzymes set free into the medium after autolysis of the death cells. In support of these view, Gorbach and Pirch (1936) [35] reported the release of enzyme by B. liquefaciens was proportionate to the number of death cells. Van Heyningen (1940) [36] also suggested that extracellular enzymes comes out of the cells presumably due to cell lysis. Rogers (1971)

[37]

proposed that a cell population might evolve to liberate an

enzyme into medium by controlled autolysis of small proportion of cell membrane. Wood and Tristram (1970)

[38]

mentioned that enzymes may be membrane bound in young cell

and released as exoenzymes when the culture enter into the stationary phase or solubilized by relatively mild procedures including washing of cells with water or concentrated salt. In this regard it must be appreciated that a number of enzymes, including amylase are synthesized during growth, and the rate of synthesis declines as the culture enters stationary phase

[39]

.even functionally similar types of enzymes may be

found cell bound in one species of micro organism and extreacellular in other. In some cases the same strain of organism produce enzymes which are intracellular and extracellular forms. Amylase has been reported as extracellular enzyme by Bacillus subtilis [40] whereas the same has been reported as intracellular in other strains of Bacillus subtilis

[41]

. Both Extracellular and intracellular amylases have been purified from a

thermophilic Bacillus stearothermophilus

[42]

. In our study we measured the activity of

the extracellular amylase dissociated in the surrounding media. Priest (1977)

[43]

described the clear location of extracellular enzymes in the genus Bacillus. He

also suggested that the periplasm of the gram negative bacteria is recognized as an enzyme- containing compartment bounded inside by the cytoplasmic membrane and outside by the outer membrane of the cell wall. The absence of an outer membrane in the gram positive cell wall precludes the possibility of a comparable location. The exact

location of these enzymes and their relationship with true extracellular enzymes remain obscure in gram positive bacteria, but it should be noted that at least some exoenzymes do have a periplasmic counterpart which may be released by protoplasting. Exoenzymes are not synthesized simultaneously as cytoplasmic and extracellular molecules. There are three lines of evidence that suggest that at no time do exoenzymes occur within the cytoplasm. First, the intracellular levels of a-amylase and protease in Bacillus subtilis are 34

virtually negligible and any activity that can be detected probably represents trapped exoenzyme [44]. Second, there exists in the cytoplasm of B. amyloliquefaciens an inhibitor of extracellular ribonuclease (RNase)

[45]

. Since the formation of the enzyme inhibitor

complex is essentially irreversible it seems unlikely that the native enzyme could exist within the cell. Finally, there is a close coupling between the synthesis of exoenzymes and their secretion, suggesting that there is no sizable "pool" of internal enzyme [46].

However at least two exoenzymes, penicillinase and α-amylase, can be detected as both membrane-bound and truly extracellular molecules. At neutral pH approximately half of the penicillinase synthesized by B. licheniformis is secreted into the environment in a very stable hydrophilic form. Of the remaining cell bound enzyme, 50% is tightly associated with the cytoplasmic membrane and 50% is associated with periplasmic vesicles [47]. Evidence is now accumulating to suggest that α-amylase also exists as a membraneassociated and as an extracellular enzyme [48]. Nagata et al. [49] have described cell bound α-amylase in a variety of strains including B. subtilis Marburg, B. natto, B. amyloliquefaciens, and B. subtilis var. amylosacchariticus. Both the extracellular and cell-bound α-amylases of B. subtilis are derived from the same structural gene, and point mutations in amyE (the structural gene for a-amylase) prevent the synthesis of both forms of the enzyme. The cell-bound α-amylase can be separated into three fractions by chromatography on Sephadex G-75. These correspond to native extracellular enzyme, believed to represent molecules en route to the exterior, and two higher-molecular-weight species which consist of enzyme associated with membrane fragments.

1.7. Process of Enzyme secretion: Prokaryotic exoenzymes do not occur, at least in their native configuration, in the cytoplasm, and they are secreted as they are synthesized. Furthermore, in many bacteria a considerable but variable fraction of the ribosomal population has been reported to be

35

associated with the cytoplasmic membrane. In exponential-phase B. licheniformis cells 96% of the total ribosomal material is membrane bound, and no polysomes are present in the

cytosol

[50]

. In B. megaterium

[51]

and B. amyloliquefaciens

material is associated with the membrane. Coleman

[53]

[52]

35 to 50% of the ribosomal

studied the distribution of a-

amylase-forming ability between the soluble and membrane-bound ribosomal fractions of B. amyloliquefaciens. Both types of ribosome incorporated labeled amino acids equally efficiently. However, the synthesis of a-amylase was fivefold greater in the membranebound fraction than in the soluble fraction, suggesting that the membrane-bound polysomes were preferentially synthesizing exoenzymes. One hypothetical model [54] for the synthesis of exoenzymes for prokaryotic cell proposed that exoenzyme mRNA migrates from a nuclear transcription site towards the membrane. In late stationary-phase cells a pool of migrating mRNA accumulates. The messenger may be associated with a migratory protein, possibly the 30S ribosomal subunit. As the signal sequence emerges from the 50S ribosomal subunit, it is recognized by membrane proteins. Recognition results in a loose association of these proteins, which is subsequently stabilized by interaction of sites on the large ribosomal subunit with sites on the membrane proteins. After vectorial discharge of the nascent chain through this membrane "tunnel" the enzyme assumes its native configuration. Endoproteolytic removal of the signal sequence results in the formation of stable hydrophilic extracellular enzyme. Experimental results confirm this prediction. A membrane associated from of α-amylase has recently been purified from the cytoplasmic membrane of B. amyloliquefaciens [55]. It is larger than the extracellular enzyme and differs in both electrophoretic mobility and sedimentation velocity. This may be an extracellular molecule bearing an intact signal sequence; the examination of more exoenzyme precursors and, in particular, the aminoterminal sequences may prove these speculations. Another studies show that the membrane bound penicilinase of B. licheniformis is larger than the hydrophilic extracellular enzyme by a sequence of 25 amino acid residues, terminating with a phospholipid group [56].

36

1.8. Regulation of Amylase Synthesis:

1.8.1. Growth and amylase synthesis:

A number of bacteria, fungi and yeast have been reported to produce extracellular amylase. The extracellular enzyme synthesis by the genus bacillus generally not occurs during lag phase, which is correlated with the increase of biomass. The kinetics of aamylase production by B. subtilis Marburg growing in complex medium adhere to the former pattern, and maximal synthesis occurs after growth has ceased [57,58]. However, B. subtilis W23 grown in minimal medium containing starch as the main carbon source synthesizes a-amylase at a low and linear rate throughout the growth cycle

[59]

. Of the

several possible explanations for this anomaly, the most plausible relates to the different media. The minimal salts-starch medium supported a lower growth rate and reduced final cell density compared with nutrient broth. This would have the effect of “flattening" the growth curve and consequently minimizing any variation in the rate of α-amylase synthesis. B. licheniformis

[60]

, B. macerans

[61]

, and the highly amylolytic bacilli, B.

amyloliquefaciens, B. subtilis SAC, and B. subtilis NAT

[62, 63]

synthesize amylase

maximally during the latter stages of the growth cycle. Thus amylase synthesis among these bacilli differs primarily in magnitude rather than the pattern of secretion throughout the growth cycle. The differences in the physiological state of bacteria growing in batch and continuous culture can be, at least partially, resolved by the use of multistage continuous systems [64]. The multistage chemo stat consists of a growth vessel into which medium is entering and from which culture is fed into a second vessel. The transferred culture is, to a certain extent, “shifted down," and a part of the growth curve after the exponential phase is adopted. The growth rate of the culture in the second or subsequent stages may be modified by the addition of fresh nutrient. Fencl et al.

[65]

arranged two-

stage chemo stat culture of B. subtilis for the production of a-amylase. Optimal results were obtained when the growth rate in the first stage was high and in the second stage relatively low. This system produced better enzyme levels in the second stage than in a single-stage fermentor, but the activity was still 40% lower than that of equivalent batch

37

cultures. Nevertheless, optimization of multistage continuous fermentors and the use of heterogeneous continuous fermentors may provide a valuable economic alternative to the batch culture production of exoenzymes.

1.8.2. Genetic analysis of amylase The α-amylase (amy) cistron of B. subtilis first analyzed in detail by Yuki and Ueda [66], although the α-amylase structural gene been recognized as a transformable element some 6 years previously

[67]

. Transformational analysis of the cistron was facilitated the

isolation of two linked markers, phe-96 and aro-116. By using these markers Yuki and Ueda placed 16 Amy- mutants in a single locus, amyE, on the B. subtilis 168 chromosomes. amyE is the structural gene for α-amylase. Similarly, Yamaguchi et al.[68] isolated 28 Amy- mutants in which α-amylase undetectable in 11 and reduced in the remaining 17. Every amy marker was found to be cotransformable with aro-116 at a frequency of to 40%. Three-point crosses were used to place aro-116 to the right o famyE, and a tentative genetic map was proposed. The enzymological properties of four thermo sensitive α-amylases from strains possessing point mutation in amyE have been studied in detail and compared with the parental α-amylase

[69]

.More recently, the amyE

region has been placed on the genetic map of B. subtilis as revised by LepesantKejzlarova et al.[70]. aro-116 has been shown to lie in the arol cistron which has been located on the early replicating portion of the chromosome between lin and narB Threepoint transformation crosses have been used to confirm the location of amyE and provide a map order, lin amyE aroI narB [71]. The yield of saccharifying α-amylase from B. subtilis Marburg, B. subtilis NAT, and B. subtilis SAC of 10, 50, and 150 U per mg of cells,respectively, reflects the relative rates of synthesis of this enzyme in these strains[72]. The amylases from B. subtilis and B. subtilis NAT can be differentiated according to their electrophoretic mobilities. A character that determines the high level of α-amylase synthesis B. subtilis NAT can be transferred to B. subtilis by transformation. About 10% of the transformants with the Amyh phenotype produce the recipient-type enzyme, Marburg (M) amylase, and the 38

remaining 90% produce the donor type, NAT (N) α-amylase

[73]

. Linkage map of B.

subtilis based on the revised location of markers was reported by Lepesant Kejzlarovi et al.

[70]

. Loci affecting the synthesis of extracellular enzymes are placed on the outside of

the circle; reference markers on the inside. Genetic analysis has shown that (i) the structural genes amyEm and amyEn, for types M and N α-amylases, respectively, are allelic to each other and (ii) a gene, closely linked to the structural gene, regulates the rate of α-amylase synthesis. This gene has been designated amyR where amyR1 of B. subtilis and amyR2 of B. subtilis NAT direct a low and high rate of α-amylase synthesis, respectively. Further genetic studies have shown that (i) amyR3 is closely linked to amyEs and that both genes are linked to aroI116 and (ii) the gene order around the αamylase cistron in B. subtilis SAC, B. subtilis NAT, and B. subtilis Marburg is very similar

[72]

. The genetic determinant(s) conferring the "extra high" production of α-

amylase by B. subtilis SAC does not therefore lie in the amyR3 locus but, may be the result of a gene that affects the rate of synthesis of both α-amylase and protease. A second gene affecting the quantity of α-amylase produced by B. subtilis has been described by Sasaki et al.

[74]

. Of seven mutants of B. subtilis NA64 (amyR2) that were

resistant to the antiviral antibiotic tunicamycin (TM), two produced two- to fivefold increased levels of α-amylase. The mutation is specific for α-amylase and has no effect on RNase or protease synthesis. Transformation analysis revealed that the Tmr Amyh phenotype was the result of a single mutation in the tentatively named tmr gene. This gene did not cotransform with amyR. TM selectively inhibits cell wall synthesis in B. subtilis, and thus, the hyperproduction of α-amylase may be the result of an alteration in the structure of the cell surface. The reason for the selective effect on α-amylase remains unknown.

1.9.

Mechanism of Regulation:

Bacteria can utilize a large variety of nutrient for their growth. Therefore, they have to synthesize the necessary enzymes for utilization of the various nutrients. The microbial

39

enzyme synthesis and secretion is mainly under the control of substrate induction, catabolic repression and feedback regulation.

a)

Substrate induction :-

Early studies on the factors affecting enzyme synthesis in microorganism lead to hypothesis that there are two classes of enzymes, differing in their occurrence under different metabolic conditions. i.The constitutive enzymes are produced at constant rates and in constant amounts regardless of the metabolic state. Constitutive enzymes are held to be a part of the permanent and basic enzymatic machinery of cell. ii.Inducible enzymes in normally present in a trance amount in microorganism. Its concentration can quickly increased in several folds or more when it’s suitable substrate or substrate analogue are present in the medium, particularly when the substrate is the only carbon source of the cell.

The majority of exoenzymes synthesized by the bacilli appear to be at least partially inducible

[43]

. Catabolic enzymes are normally induced by the enzyme substrate, but

exoenzymes are an exception in that they are presumably excreted because the substrate is unable to enter the cell. The substrate cannot, therefore, be directly involved in the induction process although it is conceivable that it could indirectly induce the enzyme via a cell wall-membrane binding site. It is now generally accepted however, that a low basal level of constitutive exoenzyme degradesits exogenous substrate and the resultant lowmolecular-weight products enter the cell and induce further exoenzyme synthesis. α-amylase synthesis is regulated in some bacilli by such a mechanism, and maltotetraose is the most effective inducer of this enzyme in both B. stearothermophilus licheniformis

[76]

[75]

and B.

. It has long been known that higher yields of α-amylase are obtained

from bacteria grown on medium containing complex starch materials than on artificial defined media. Not only do these carbon sources fail to exert catabolite repression, but it would seem that they also provide maximal induction of α-amylase. However, some bacilli appear to synthesize a-amylase constitutively. Unfortunately, the effect of malto oligosaccharides on the differential rate of α-amylase synthesis in these bacilli has not 40

been determined, and it is possible that a relatively high basal level of constitutive enzyme is being secreted that could be increased by induction. The Hyperthermophilic Sulfolobus solfataricus produces α-amylase which hydrolyzed starch, dextrin, and acyclodextrin with similar efficiencies

[77]

. Addition of exogenous glucose represses

production of a-amylase, demonstrating that a classical glucose effect is operative in this organism. α-amylase is constitutively produced at low levels but can be induced further by starch addition. Aspartate was identified as the most repressing sole carbon source for α-amylase production, while glutamate was the most depressing. Incubation of embryo less barley (Hordeum vulgare) half seeds for 24 hours with 0.1 M glutamate or aspartate resulted in the release of 17 to 48% as much α-amylase as did incubation with 260 mum gibberellins

[78]

. Two patterns were noted in the active compounds which induce the α-

amylase production (a) all of the active compounds contain an amino group and are biosynthetically derived from citric acid cycle intermediates; and (b) biosynthetic precursors of the amino acids arginine, proline, threonine, and lysine were active whereas these amino acids were not. Total extracellular amylases were studied from the promising production strain NRRL Y12,974[79]. Growth rates and yields were equivalent in cultures grown on glucose, maltose, soluble starch, or cornstarch. Total amylase levels were low and varied only three-fold, from 0.01 IU ml−1 in glucose-grown cultures to 0.03 IU ml−1 in soluble-starchgrown cultures. In its original host, the thermophilic Streptomyces strain sp. TO1, the amy TO1 gene was expressed during growth but only in the presence of starch in the growth medium. When cloned in Streptomyces lividans, on a low copy number replicative plasmid, amy TO1 expression was detectable in fructose-, mannitol- and galactose-grown cultures but not in glucose- or glycerol-grown cultures. This basal expression could be further induced by maltotriose

[80]

. The mechanism of induction remains completely unknown, and in no

instance has a non metabolizable, gratuitous inducer been described. Such a molecule would be an invaluable aid in the elucidation of the induction process.

41

b)

Regulation by Feedback inhibition :-

Feedback inhibition is the phenomenon by which the final metabolite of the pathway inhibits the synthesis of an enzyme; usually the first enzyme products of exoenzyme action may repress the synthesis of the enzyme in a manner resembling the end product repression of endocellular anabolic enzymes [81]. The tryptophan operon is a popular example of end product repression of enzyme synthesis [82]. The tryptophan operon of E. coli comprises the structural genes for five the five enzymes, which are involved in the conversion of chorismate to tryptophan. The regulatory gene is trp R, which code an effector protein, the apo-repressor. In presence of tryptophan in the medium, it binds with the apo-repressor, causing a conformational change, which permits the repressor to bind its operator and prevent transcription. Lowering of the tryptophan concentration leads to dissociation of the repressor and frees the operator for the synthesis of mRNA Alpha-amylase production by Bacillus licheniformis M27 [83] in submerged fermentation was reduced from 480 to 30 units/ml when soluble starch concentration in medium was increased from 0.2 to 1.0%. In contrast, the enzyme production increased by 29 times even with 42 fold increase in the concentration of soluble starch and other starchy substrates in solid state fermentation system. The data establish regulation of the enzyme formation by enzyme end-product in submerged fermentation and ability of solid state fermentation to minimize it significantly.

c)

Catabolic repression :-

This involves the inhibition of the enzyme synthesis by the catabolic product of the readily utilized carbon sources [81]. It provides and attractive explanation of many aspects of exoenzyme synthesis. Catabolic repression is the cause of the well-known phenomenon of diauxie. The pair of substrates, e.g. glucose and sorbitol; glucose and lactose or glucose and acetate is not utilized simultaneously by E. coli. Glucose utilized first and repressed the enzyme formation for the catabolism of the other substrate. An understanding of the phenomenon was provided when Markman and Sutherland

[84]

showed that a rapid decrease in the

intracellular level of cyclic-3-5-adenosine monophosphate (cAMP) occurs in the presence 42

of glucose. The evidence in favor of the catabolic repression is best evaluated in βgalactosidase synthesis in E. coli, where only lactose induces enzyme synthesis. But synthesis of this enzyme is inhibited when glucose and lactose present simultaneously in the mediu,. The mechanism behind this type of inhibition was clearly described by Jacob and Manod (1961)

[85]

they mentioned that the intracellular cAMP along with CAP

(catabolic gene activator protein) enhances the binding of RNA polymerase in the promoter site to transcribe the mRNA of β-galactosidase. By an independent mechanism, the bacterium accumulates cAMP only when organism is starved for a source of carbon. In the presence of glucose or glycerol, the bacteria will lack sufficient cAMP to bind to CAP, so that DNA dependent RNA polymerase cannot initiate the transcription of lac operon. The rate of α-amylase synthesis in B. subtilis 168 is dependent on the carbon source in the order lactate> glutamate > maltose > glycerol > glucose, with glucose providing the fastest growth but lowest rate of α-amylase synthesis

[86]

. Starch, glycerol, and glucose

support doubling times for B. amyloliquefaciens of 2.2, 1.7, and 1.6 h, respectively, and the rate of α –amylase synthesis is inversely related

[87]

. Carbon-limited continuous

cultures of B. licheniformis with glucose, glutamate, or alanine as carbon source produce high levels of a-amylase; in contrast, nitrogen limited cultures contain excess glucose and a barely detectable level of enzyme activity [88]. Furthermore, under carbon-limited conditions, the amount of α-amylase produced by B. Iicheniformis in continuous culture is inversely proportional to the growth rate

[88]

. The

addition of glucose to a constitutive or fully-induced α-amylase producing B. subtilis culture growing on a non repressing carbon source cells synthesizing results in a severe transient repression; thereafter, the normal glucose-repressed rate of synthesis is adopted [89]

. Amyle protein is an amylase repressor protein in B. subtilis to regulate amylase

synthesis by inhibiting the initiation of the amyE transcription from amylase promoter [90].

43

1.10. Industrial Application of Microbial Amylase: Amylase is one of the most important hydrolytic enzymes for all starch based industries and the commercialization of amylases is the oldest with first use in 1984, as a pharmaceutical aid for the treatment of digestive disorders. Now a day’s amylases are applied in all the industrial processes such as food, detergents, textiles and paper industries, for the hydrolysis of starch. In this context, microbial amylases have completely replaced chemical hydrolysis in the starch processing industry. They can also be of potential use in pharmaceutical and fine chemical industries.

1.10.1. Application of enzyme based detergents Enzyme based detergents also known as green chemical find a wide range of application in laundry, dishwashing, textile and other such industries. The enzyme preparation like amylases, proteases, lipases and cellulases are considered as indispensable ingredients in these detergents. The amylase in the detergent mainly degrades the residues of starchy food like porridge, potatoes, gravies, custard, chocolate etc to dextrins [91]. The main advantage of the enzyme application in detergents is due to much milder conditions than with enzyme free detergents. The early automatic dishwashing detergents were very harsh, caused injury when ingested and were not compatible with delicate china and wooden dishware. This forced the detergent industries to search for milder and more efficient solutions. The demand for α- amylase for automatic dishwashing detergents are growing [92].

1.10.2. Textile industry Modern production processes for textiles introduce a considerable strain on the warp during weaving. The yarn must, therefore, be prevented from breaking. For this purpose a removable protective layer is applied to the threads. The materials that are used for this size layer are quite different. Starch is a very attractive size because it is cheap, easily available in most regions of the world and it can be removed quite easily. Good desizing of starch sized textiles is achieved by the application of α- amylases, which selectively remove the size and do not attack the fibers. It also randomly cleaves the starch into

44

dextrins that are water soluble and can be removed by washing. α- amylases is used in warp sizing of textile fiber for manufacturing fibers with great strength [93]. Amylase are now widely used to remove starch which is used as an adhesive or size on threads of certain fabrics to prevent damage during weaving. Currently in the textile industry, there is a wide spread demand for faded jeans. This involves subjecting such clothes to amylases, a process commonly referred to as biowashing or biobleaching, an alternative to the term enzyme fade. This allows elegant softness and unique shades to be given to the cloth which overcomes the traditional methods of bleaching by sodium hypochlorite or trembling with pumice stones and also offer better safety as well as economy [91].

1.10.3. Clarification of fruit juice for jelly manufacture Jellies made from apple juice are hazy in appearance because of the high starch content. Treating the juice with amylase for one hour at 80-95oF and filter gives a clear juice suitable for making sparking jelly.

1.10.4. Use in baby foods Fungal amylase finds extensive use in the preparation of dried baby foods and cereal product. The cereal is heated to a temperature of 150-160OF and subsequently fungal amylase is added. Digestion is then allowed to take place for about four minutes, the mass being agitated throughout the digestion period. The cereal is then dried in stream heated rolls at a temperature of 100OC, which both dries the product and inactivates the enzyme. The treatment helps to impart a malt syrup flavor to the product and also helps to produce a smoother sheet on the drying rolls.

1.10.5. Production of chocolate syrup Chocolate syrup made by treating cocoa slurries with amylase, produces a product with which does not tend to “layer” in storage, eliminate appreciable stiffening or set back and give rise to a product with an improved flavored and solubility in milk. 45

1.10.6. Production of high conversion syrup They are produced from liquefied starch by hydrolysis with α-amylase and glucoamylase. This syrup comprises about 40% glucose, 45% maltose and the remainder maltotriose. They are used extensively in the brewing, baking, confectionary and soft drink industries.

1.10.7. Alcohol industry Bacterial amylase find a wide spread application for the hot liquification of starchcontaining materials before fermentation. This saves the purchase and storage of malt, which moreover may contain undesirable micro organisms that could cause an excessive pH drop during fermentation. Alcohol yields are increased by 1.0-3.0% [94].

1.10.8. Paper industry The use of α-amylase for the production of low viscosity, high molecular weight starch for coating of paper is reported [95]. The use of amylase in the pulp and paper industry is in the modification of starches for coated paper. As for textiles, sizing of paper is performed to protect the paper against mechanical damage during processing. It also improves the quality of the finished paper. The size enhances the stiffness and strength in paper. Starch is also a good sizing agent for the finishing of paper.

1.10.9. Analysis in medical and clinical chemistry There are several processes in the medicinal and clinical areas that involve the application of amylase. A process for the detection of higher oligosaccharides, which involved the application of amylase was, developed

[96]

. This method was claimed to be

more efficient than the silver nitrate test. Biosensors with an electrolyte isolator semiconductor capacitor (EIS-CAP) transducer for the processing monitoring were also developed [97].

46

1.11. Aim of the present study: Though municipal waste is one of the most hazardous components of our society, it consists of different components which can be utilized by various kinds of microorganism some of which may have economically beneficial roles. As municipal wastes are rich in components which support the luxurious growth of different bacteria, we choose the waste as a source for isolation of amylase producing bacteria. After isolation the amylase production was carried out in liquid media by shake flask method. Thorough investigation on the physicochemical factors affecting the enzyme production enzyme activity and stability was also performed. The specific objectives include: 1. Isolation of amylase producing bacteria from municipal waste collected from Dhapa, Kolkata and select suitable bacterial strains that can produce appreciable amount of amylase. 2. Biochemical and non biochemical characterization of the selected bacteria and final identification by 16S rDNA based molecular technique. 3. Optimization of the media component for maximum amylase production and comparison of the growths of the bacteria with enzyme productivity. 4. Study on the effects of the physical factors on the enzyme production and growth. 5. Study on the bacterial growth and enzyme production with respect to time at optimum physic-chemical condition. 6. Investigation on activity and stability of crude enzyme from the isolated bacteria.

47

Isolation and Identification of Amylase Producing Bacteria

48

2.1. Introduction: Extraction of chemically active compounds from natural resources by enzymatic conversion is becoming popular in modern-day technology. In these aspect microbial enzymes is proving to be an important tool in various industrial process for bioconversion, since it can increase the efficiency of production without affecting the environment. Enzymes of microbial origin have several advantages than those of plants and animals. In microbial system the specific extracellular enzymes can easily overcome the cell barrier and get released into the medium from which the desired enzyme can be easily obtained. Moreover extracellular enzymes are easy to produce in huge amounts by micro organism and its controlling mechanism is very simple. Efficiency of production can also be increase several fold by genetic manipulation, by controlling the culture condition and by the use of cheaper substrates. Limitless quantities of enzymes maybe produced through microbial sources, whereas in other sources the availability of base material may be a limiting factor. Among industrially important enzyme amylase has wide spread of application in pharmaceutical, chemical, food, paper and textile industries. Extensive screening studies have been conducted by different workers to select the potent cultures for amylase production from different environment [98, 99]. In this regard the solid waste can be one of the best reservoirs of such organism as because it contains different components that fulfill the nutritional requirement for different micro organism. The amylase from bacterial source have some advantages over fungus, because maximum amount of enzyme is produced with in a very short time of cultivation, synthesis occurred as extracellular form and it is very easy to separate. Keeping this in view, I have tried to isolate some suitable amylase producing bacterial strain from municipal waste rich in different types of micro organisms.

49

2.2. Material and Method:

2.2.1. Collection of samples: Freshly collected municipal waste from Dhapa, Kolkata

2.2.2. Date of collection: 28.07.09 1 gm of freshly collected municipal waste was suspended in 10 ml of sterile distilled water in a sterile test tube. The suspension is diluted 10 times in another sterile test tube. They were cotton plugged and preserved at 4oC until use for isolation.

2.2.3. Media for selection of amylase producing bacteria: 1. For isolation of amylase producing bacteria form the municipal waste, media containing 1%starch and 2% agar was used. Only the amylase producing bacteria can utilize the starch as sole carbon source and able to grow on this media. 2. After isolation of amylase producing bacteria, they were streaked subsequently in different media for getting pure isolated colonies. The compositions of those media are,

Medium I – Peptone

- 2%

Yeast extract

-1%

NaCl

-1%

Starch

- 1%

Agar

- 2%

50

Medium II Starch

-1%

Yeast extract

-0.2%

Peptone

-0.5%

MgSO4

-0.1%

NaCl

-0.1%

CaCl2

-0.02%

Agar

-2%

Medium III Peptone

-0.6%

MgSO4

- 0.05%

KCl

-0.05%

Starch

-1%

Agar

-2%

2.2.4. Dilution Plating: The waste material was suspended at approximate ratio of 1gm to 10ml sterile waster in an Erlenmeyer flask and it was shaken thoroughly on a rotary shaker for 1hr and was allowed to stand for some time to settle down the heavy particles. For serial dilution of the solution one ml of this clear supernatant was added to 9ml of sterilized distilled water in a culture tube and shaken for 2min on a vortex rotary shaker. 1ml of this suspension was then diluter serially up to 10-9 and for every dilution five replicates were made. Each tube was thoroughly shaken in a vortex rotary shaker. Then 1ml of each dilution was poured in a sterile petridish aseptically and to it 20ml of sterile and melted starch agar media (at 45oC) was taken and was allowed to solidify and

51

incubated at 37oC for 72 hrs. Plates with up to 30 developed colonies were generally selected for screening of the desired isolates.

2.2.5. Selection of bacteria: Micro organisms that survived in the starch – agar media and produce a clear zone around the colonies when the plate was flooded with iodine solution, were considered and selected as amylase producing bacteria

[99]

. The clear zone is form due to

degradation of the starch at the surrounding area by the amylase produced by the micro organism. By identifying the morphological characteristics two bacterial strains which were giving largest clear zones were taken for further studies. The bacteria were streaked for several times on the above mentioned media for getting pure isolated colonies. Then the isolated colonies were inoculated in the above mention media without ager as liquid broth and incubated for 24 hrs. The culture fluids of each isolate were then centrifuged separately and supernatant were used for studying the amylase production. The medium, for which the production was the maximum, was used for further studies.

2.2.6. Identification of the bacteria: The isolated bacteria were identified according to standard procedure and various tests necessary for identification were followed from the Collins and Lyne [100].

2.2.6.1. i.

Morphological Characteristics

Shape, size and character: for determining the shape and gram character,

bacterial film were stained by Gram’s method- A standard bacterial smear was prepared and heat fixed on a glass slide. The primary stain, crystal violet was poured on the smear. After 1 minute it was washed by tap water and iodine solution is added as mordant. After 1 min the slide was washed with water and rapid decolorization with alcohol or acetone was done. After washing with tap water a counter stain, safranine was added to it .it was 52

washed after 45 seconds. After drying, examine the slide under microscope using cider wood oil to maintain the refractive index.

ii.

Motility: The motility test is not a biochemical test since we are not looking at

metabolic properties of the bacteria. Rather, this test can be used to check for the ability of bacteria to migrate away from a line of inoculation thanks to physical features like flagella. Craigie’s method is used to perform this test. The semi solid nutrient agar medium ( contains 0.2%-0.5% of agar) containing test tube is inoculated with the test organism into the central glass tube Incubate at the relevant temperature for 18-24 h. Motile organism will grow outside the inner tube while non motile organism remains in the inner tube.

2.2.6.2.

Cultural Characteristics

Colony morphology and pigment formation was studied from the growth on solid media

2.2.7. Biochemical Characteristics

2.2.7.1.

Catalase test :

One of the by-products of oxidation-reduction in the presence of O2 during aerobic respiration is hydrogen peroxide (H2O2). This compound is highly reactive and must be degraded in the cytoplasm of the cell producing it. It can be especially damaging to molecules of DNA. Most aerobes synthesize the enzyme catalase, which breaks down H2O2 into water and oxygen (see background information on aerobic spore-formers).

53

catalase

2 H2O2

2 H 2O

+

O2 ↑

The O2 gas is identified by the production of bubbles from a concentrated cell suspension. The test for catalase is simple and usually very reliable. It is a major method of distinguishing between Staphylococcus (catalase positive), Streptococcus (catalase negative), and Enterococcus (catalase negative), although some strains of Enterococcus faecalis may be positive.

Catalase production is generally associated with aerobic

organisms, since H2O2 is a toxic by-product of aerobic growth, but not always. Some anaerobes, particularly Bacteroides, a gut organism, produce catalase, especially if they are exposed to air. All of the Gram negative bacteria we use in this lab are catalase positive. 2.2.7.2.

Oxidase test :

Oxidase was examined by adding a few drops of 1% alpha-napthol in 95% ethanol and few drops of 1% aqueous dimethyl-p-phenylenediamine acetate to 3ml of culture. Appearance of purplish blue color indicates the positivity of the test.

2.2.7.3.

Sugar Fermentations - Glucose, Lactose, Mannitol , Maltose, Sucrose, Galactose:

Bacterial cells are able to generate energy from nutrients through respiration or through fermentation.

Respiration uses an external electron acceptor, like oxygen (aerobic

respiration) or some other exogenous source (anaerobic respiration) to generate high yields of ATP through complete oxidation of an organic compound. Fermentation, on 54

the other hand, only partially oxidizes the substrate and generates a relatively small amount of ATP. The terminal electron acceptor is usually produced as an intermediate in the pathway and so is internal instead of external. Different bacteria can ferment a wide variety of sugars and other compounds. The determination of a fermentation pattern for a series of different energy/carbon sources (usually sugars) by an unknown bacterial species is often a central part in the identification process.

For example, sugar fermentation patterns are used in the

identification of enteric bacteria. Acid products, which may be produced from the fermentation of a sugar, will cause a noticeable color change in the pH indicator (Andrade’s indicator) included in the medium.

Sugar fermentation does not produce alkaline products.

However, non-

fermentative hydrolysis of amino acids in the peptone, present in most fermentation media, may give an alkaline reaction, which will also cause a color change in the pH indicator. Gas production, H2 in particular, can be determined by placing a small, inverted Durham tube in the test medium. If gas is produced, it is trapped in the Durham tube and can be seen as a bubble. 2.2.7.4.

Hydrogen Sulfide Production:

Hydrogen sulfide (H2S) is produced by bacterial anaerobic degradation of the two sulfur-containing amino acids, cysteine and methionine. Hydrogen sulfide is released as a by-product when carbon and nitrogen atoms in the amino acids are consumed as nutrients by the cells.

Under anaerobic conditions the sulfhydryl (-SH) group on

cysteine is reduced by cysteine desulfurase.

The SIM agar medium (Hydrogen-Sulfide, Indole, Motility) is used to detect liberation of H2S gas by bacteria growing in a medium containing sodium thiosulfate and ferrous sulfate as an indicator. When H2S is produced, the ferrous ion reacts with it to give ferrous sulfide, an insoluble black precipitate.

55

Composition of SIM agar: Peptone-

30gm

Beef extract-

3gm

Ferrous ammonium sulfate- 0.2gm Sodium thiosulfate-

0.025gm

Agar-

3gm

Distilled water-

1000ml

FeS ↓

H 2S +

FeSO4

hydrogen

ferrous

ferrous

sulfuric

sulfide

sulfide

sulfate

acid

2.2.7.5.



+

H2SO4

Nitrate Reduction:

Under anaerobic conditions, some bacteria are able to use nitrate (NO3-) as an external terminal electron acceptor. This kind of metabolism is analogous to the use of oxygen as a terminal electron acceptor by aerobic organisms and is called anaerobic respiration. Nitrate is an oxidized compound and there are several steps possible in its reduction. The initial step is the reduction of nitrate (NO3-) to nitrite (NO2-).

Composition of nitrate broth: KNO3 -

1 gm 56

Peptone -

5gm

Beef extract-

3gm

Distilled water - 1000ml pH-

6.8-7.2

Composition of nitrate test reagent: Zinc Chloride-

20gm

Starch-

4gm

Potassium Iodide-

2gm

Distilled water-

1000ml

Nitrate reductase

NO3- + 2 H + + 2 e-

NO2- + H2O

Several possible products can be made from further reduction of nitrite.

Possible

reduced end products include the following: N2 (molecular nitrogen, a gas), NH3 (ammonia), N2O (nitrous oxide). Bacteria vary in their ability to perform these reactions, a useful characteristic for identification. A medium that will support growth must be used and the cells must be grown anaerobically (no shaking). Growth in the presence of oxygen will decrease or eliminate nitrate reduction.

57

Summary of Nitrate test results and interpretations

RESULT

INTERPRETATION

SYMBOL

Nitrate was reduced to nitrite

+1

Inky Blue after addition of Nitrate test reagent and diluted H2SO4 ( 1: 3 ; acid : water ) No color after

Nitrate was reduced to nitrite and then further

addition of zinc

reduced to another compound such as NH3

dust Blue color after

Nitrate is still present. The bacteria being tested

addition of zinc

did not reduce the nitrate

+2

_

dust

There are many possible end products of nitrate reduction such as nitrite, nitrogen gas (N2), nitrous oxides, ammonia, and hydroxylamine.

One could either assay for

disappearance of nitrate or the appearance of the end products. The standard test assays for the appearance of one of the products, nitrite. The test we use relies on the production of nitrous acid from the nitrite. This, in turn, reacts with the iodide in the reagent to produce iodine. The iodine then reacts with the starch in the reagent to produce a blue color. Since some of the possible products of NO3- reduction are gaseous, a Durham tube is sometimes inverted in the culture tube to trap gases.

This being the case, it is

important to pre-test the medium to ensure no detectable nitrite is present at the beginning, and, in the case of a negative test, to reduce any nitrate to nitrite to determine whether the nitrite was also reduced.

58

2.2.7.6.

Triple sugar Iron (TSI) test:

TSI medium containing tube when inoculated with the bacteria culture, the motile acid and gas producing bacteria will produce acid and gas by fermenting the sugars. As a result the whole media will turn yellow from red due to change in pH and the media will crack due to formation of gas. If no gas will be produced, the medium will just turn yellow but there will be no cracking. For non motile bacteria the upper surface of the medium only turn a little yellowish color, so no significant result will be found for non motile bacteria. The presence of a black color indicates that H2S was produced. Composition of TSI medium: Peptone-

20gm

Lactose-

10gm

Sucrose-

10gm

Dextrose-

10gm

NaCl-

5gm

Ferrous ammonium sulfate- 0.2 gm Sodium thio sulfatePhenol red-

0.2m 0.0025gm

Agar-

20gm

Distilled water-

1000ml

pH-

2.2.7.7.

7.3

Protein hydrolysis:

The bacteria were inoculated in skimmed milk and agar containing medium. If a clear zone is observed after incubation at 370C, then it is confirmed that the bacteria are able to hydrolyze protein. 59

2.2.7.8.

Citrate utilization test:

Citrate utilization test was carried out by inoculating the bacteria in Simmon's Citrate agar slant. Formation of blue color of the media indicates the positive result.

Composition of Simmon's Citrate agar:

Sodium citrate-

20.0gm

NaCl-

5.0gm

Magnesium sulfate-

0.2 gm

Di potassium hydrogen phosphate - 1.0 gm Ammonium di hydrogen phosphate - 1.0gm Bromothymol blue -

0.08gm

Agar-

15.0 gm

Distilled water-

1000ml

pH-

2.2.7.9.

7

Urease production test:

Slant containing urea agar was inoculated with the bacteria. Urea agar is prepared by autoclaving a media containing 1gm of peptone, 5gm of NaCl, 2gm of Di potassium hydrogen phosphate, and 20 gm of agar in 1000 ml of water, at pH-6.8. Then 1gm of glucose and 6.0ml of 0.2% phenol red solution were added to the media after bringing it at 50oC. Then steamed the media for 1hr and cooled it to 50oC. 20% urea solution was added to it after sterilization by filtration. Final media was used for the slant. If the media converted to red from yellow, the result was taken as positive.

60

2.2.7.10. Gelatin liquification:

Gelatin hydrolysis was examined with nutrient medium containing 12%gelatin. After inoculation of the tubes were incubated at 37oC for 2 days. Then the culture tubes were kept in a refrigerator for half an hour and the solidification of the gelatin was tested. Liquefied gelatin detectable over solid undecomposed gelatin represents a positive test.

2.2.7.11. Methyl red and Voges Proskauer (V.P) test:

Glucose phosphate broth at pH 7sterilized at 10lbs for 20 min. the tubes are then inoculated with the test organism. After incubation at 37oC for a week the culture broth was treated with a drop of 0.02% methyl red in 50% alcohol. For VP test, 0.5 ml of αnapthol solution in 95% alcohol and 0.5ml of 16% KOH were added to each tube containing 2ml of the liquid culture. Development of red color in broth of both cases indicated positivity of reaction

2.2.7.12. Indole production:

The test cultures were grown in sterile 1% tryptone broth for 5days and then few drops of Kovac”s reagent (5gm of β-dimethylamino benzaldehyde in a mixture of 75 mil amyl alcohol and 25 ml H2SO4) was added and shaken. A rose –pink color indicates the formation of indole.

2.2.8. Identification of a microbial culture using 16S rDNA based molecular technique 1.

DNA was isolated from the stab culture. Its quality was evaluated on 1.2% Agarose

Gel, a single band of high-molecular weight DNA has been observed.

2.

Fragment of 16S rDNA gene was amplified by PCR from the above isolated DNA. 61

A single discrete PCR amplicon band of 1500 bp was observed when resolved on Agarose Gel (Gel Image-1).

3.

The PCR amplicon was purified to remove contaminants.

4.

Forward and reverse DNA sequencing reaction of PCR amplicon was carried out

with 8F and 1492R primers using BDT v3.1 Cycle sequencing kit on ABI 3730xl Genetic Analyzer.

5.

Consensus sequence of 1428 bp of 16S rDNA gene was generated from forward and

reverse sequence data using aligner software.

6.

The 16S rDNA gene sequence was used to carry out BLAST with the nr-database of

NCBI gen bank database. Based on maximum identity score first ten sequences were selected and aligned using multiple alignment software program Clustal W. Distance matrix was generated using RDP database and the phylogenetic tree was constructed using MEGA 4.

2.3. Result:

2.3.1. Isolation of amylase producing bacteria: The production of amylase was confirmed by flooding the plate with gram iodine solution. Iodine will form blue coloration by reaction with starch. But the amylase producing bacteria degrade the starch by the action of amylase surrounding them and utilize it as solo carbon source. As a result they gave a transparent zone because due to utilization of starch iodine would not give any blue coloration. So a transparent zone in the blue plate appeared surrounding the amylase producing bacteria. Primary selections 62

of amylase producing bacteria were made on the basis of highest clear zone formation on iodine flooded starch agar plate.

The bacteria were then separately streaked on medium I, II and III for isolation of pure colonies. Both of the two bacteria grew luxuriously in these media. Maximum growth was seen in medium II and medium III. The two bacteria were named C1 and C2. C1 gave large colonies with greenish zone. C2 gave small yellowish colonies which turn white on storage These two bacteria were streaked in the middle of the plates containing medium III and incubate for 24 hrs at 370C. After incubation the plates were flooded with iodine solution. Both bacteria gave clear zones surrounding them. The amylase assay showed that the amylase production was the maximum for the medium III for both of the bacteria. So this media was taken for further studies.

63

C1 in medium I

C2 in medium I

C1 in medium II

C1 in medium III

C2 in medium II

C2 in medium III

Clear zone surrounding the C1 ( left) and C2 ( right) in a iodine flooded plate

64

2.3.2. Characterization of selected bacteria:

Table.2.1. Characterizations of C1 and C2

SI no.

Tests

C1

C2 “Gm -”

1

Gram straining

“Gm -”

2

Size

Rod

3

Colour (pigment)

4

Motility

Green pigmentation Non motile

Motile

5

Starch hydrolysis

+

+

6

Protein hydrolysis

+

_

7

Citrate utilization

+

+

8

Urea hydrolysis

+

_

9

Indole production

_

_

10

Methyl red test

_

_

11

VP test

_

+

12

Nitrate reduction

+

+

13

Gelatin liquification

+

_

14

Catalase

_

_

15

Oxidase

+

+

16

TSI media

_

+

17

SIM media

_

_

18

Sugar fermentation

Glucose

_

Acid + Gas

Sucrose

_

Acid + Gas

Galactose

_

Acid + Gas

Maltose

_

Acid + Gas

Mannitol

_

Acid + Gas

Lactose

_

Acid + Gas

Rod White

65

Gelatin Liquification

Voges Proskauer test

Protein hydrolysis

Citrate utilization

TSI agar test

Nitrate reduction

Oxidase test

Urease test

Motility test 66

Sugar Fermentation:

Maltose

Galactose

Sucrose

Lactose

Mannitol

Glucose 67

Table.2.2. Clear zone diameter (in mm) on iodine flooded starch agar plate

Strains no.

Type of microbes

Clear zone diameter (mm)

C1

Bacteria

6

C2

Bacteria

8

Table.2.3. Production of amylase by the two bacterial strains in different media after 24 hr incubation: Strains

Amylase activity (U/ml) Media I

Media II

Media III

C1

1.754

.73

1.90

C2

1.46

.584

2.193

2.3.3. Identification by 16S rDNA based molecular technique:

1. The culture, which was labeled as C1 was found to be Pseudomonas aeruginosa

(GenBank Accession Number: FJ985806.1) based on

nucleotide homology and phylogenetic analysis.

68

2.4. Discussion: In the present investigation, we are able to isolate two pure bacterial strains from municipal waste. The wastes, highly consists up starchy materials and we found bacteria isolated from such places may have better potential to produce enzyme under adverse condition. On the other hand, conversion of organic waste through microbial processes decreases the amount of waste disposed by land-filling

[101]

. In the present study, the

maximum amylase activity in medium III was lower than the values found for Bacillus subtilis

[102]

and B. licheniformis

[103]

, which under optimal culture conditions produced

535 and 252 U, respectively; however this value was higher than that reported for B. coagulans and B. circulans

[104]

, Aeromonas sp. and Pseudomonas sp.

[105]

. The

morphological, biochemical and cultural studies showed that all characters of both bacterial strains (C1 and C2) matches the character described in BERGEY’S manual systemic bacteriology (1986) Vol-2. The identification of the two strains also confirmed from Xcelris Labs Ltd. Ahmadabad 380054, India.

69

Cultural and nutritional requirements for optimum amylase production by the two selected bacterial strains

70

3.1. Introduction: For optimum enzyme production, it is necessary to standardize the nutritional, cultural and physiological conditions of the selected organism. The capacity to grow in a given habitat is determined by ability of the organism to utilize the nutrients from its surroundings. Nutrition as a whole serves at least three different functions: 1.

Providing the material required for synthesis of protoplasm.

2.

Supplying the energy necessary for cell growth and biosynthetic reactions.

3.

Serving as acceptor for the electrons released in the reaction that yields energy to the organism.

Fermentation medium basically contains sources of carbon, nitrogen, minerals, phosphates and some growth factors which constitute the working apparatus of the biological machines. There is a wide range of microbial diversity and each has particular type of nutritional habit. Therefore standardizations of physicochemical and nutritional conditions are essential that play a vital role in the growth and development of the organism as well as production of different kinds of metabolites. The literature survey indicates that amylase can be produced by liquid surface, submerged and solid state fermentations. The use of the submerged culture is advantageous because of the ease of sterilization and process control easier to engineer in these systems. Depending on the strain and the culture conditions, the enzyme can be constitutive or inducible, showing different production patterns [106]. Starch is used as sole substrate as well as carbon source for amylase production by most workers; however concentration of starch is an important factor for growth and amylase production [99]. Other Carbone sources like glucose, sucrose, maltose etc also effect the growth and amylase production to a large extent

[107]

. Different nitrogen, phosphates

sources were used by different workers. Trace amounts of mineral are also essential for amylase production. Among the physic chemical conditions, temperature and pH are of great importance for enzyme production [107].

71

3.2. Materials and Methods:

3.2.1. Micro organism: Isolated bacterial strains C1 and C2 have been used for this study. 3.2.2. Preparation of inoculums: An inoculum was prepared by growing the organism in the medium III (Peptone - 0.6%, MgSO4- 0.05%, KCl - 0.05%, Starch-1%). A loop full of fresh culture grown in 50ml of sterilized (10lb for 30 min) media in 250 ml Erlenmeyer flask for 20h on a rotary shaker (200 rpm) at 37oC. Then the broth was centrifuged (5000g, 10min) and pellet was washed twice in sterilized distilled water and used as inoculums. A1 % (v/v) inoculum was added in different culture medium. The composition and concentration of primary culture medium is same as the medium used for inoculums preparation. 3.2.3. Cultural condition: To study the effects of different pH and temperature on enzyme production and growth of the organisms, the organisms were grown in the basal media at different pH at 37oC and then at pH 7 at different temperature. The optimum temperature and pH for maximum enzyme production were obtained after working out a series of experiments for each bacterial stain. 3.2.4. Nutritional conditions for amylase production: Various carbon compounds were added to the culture media for studying their effects on amylase production. The carbon source giving the maximum amylase production was then added at different concentrations for getting the optimum concentration of sole carbon source, Similarly the effect of different inorganic and organic nitrogen sources and phosphate sources were studied by adding them in the basal medium at different concentrations. The effects of different metal ions on amylase production were observed by substituting the metal source MgSO4 of the basal medium for amylase production.

72

3.2.5. Assay of amylase: As described by Andrew D. Jamieson et al [108]. One ml of enzyme solution was added to 1 ml of substrate solution (10 gm/ L) and then incubated for three minutes at 37oC. Two ml of color reagent (100 mg of 3,5-dinitrosalicylic acid was dissolved in 20 ml of 2N NaOH and 50 ml of water; 30 gm of sodium-potassium tartrate (Rochelle salt) was added, and the solution was made up to 100 ml with water) was added to stop the enzyme reaction. Tubes were heated in a boiling water bath for five minutes to effect the color change, and then cooled with running tap water. Samples were diluted 1:10 with water. Absorbance was read in a spectrophotometer at 470mµ. The blank was prepared by substituting water for the amylase solution. A standard curve relating maltose concentration to the color reaction was obtained by reacting 2-ml samples of aqueous maltose solutions (concentrations of 0.1 to 1.0 mg/ml) with 2 ml of 3, 5 di nitro salicylic acid. The determination of amylase concentration an unknown sample was made by plotting the absorbance of the reaction product at 470mµ against a standard amylase preparation. Units of amylase activity were expressed as micromoles of maltose liberated per minute

3.3. Results: 3.3.1. Effect of temperature on amylase production:

Effect of temperature on growth and amylase production was studied in the basal medium (medium III) for amylase production at different temperature (25 – 45oC) for 24 h for both C1 and C2 (Fig. 3.1and 3.2). It was found that both of the organisms are able to grow between 25- 40 oC with significant production of amylase but no growth was found at 45oC. Maximum growth and amylase production was occurred for both organisms at 37oC.

73

Fig 3.1. Temperature optimization of C1 2

0.4

1.8

0.35

1.6

0.3

Growth (O.D) 0.25 at

1.4

0.15

0.8

1.2 1

0.2

0.6

0.1

0.4

0.05

0.2

0

0 25

30

34

37

41

45

Amylase activity (U/ml)

amylase activity (U/ml) growth (O.D) at 600mn

Temperature in ºC

0.3

Fig 3.2 Temperature optimization of C2

0.25

2.5

2

0.2

Growth (O.D) at 600nm 0.15

1.5

Amylase activity (U/ml)

1 0.1 0.5

0.05 0

0 25

30

34

37

41

amylase activity (U/ml) growth (O.D) at 600mn

45

Temperature in ºC

74

3.3.2. Effect of pH on amylase production:

The growth and amylase production in relation to initial medium pH was studied for both bacteria and represented in the Fig. 3.3 and 3.4. Significant growth and enzyme production obtained from pH 5.5 – 8.0 but maximum production for C1 at pH 7.0 and for C2

Fig 3.3. pH optimization of C1

0.4

2 1.8 1.6 1.4 1.2 1 0.8 0.6 0.4 0.2 0

0.35 0.3

Growth 0.25 (O.D) 0.2 at 0.15

0.1 0.05 0 5.5

6

6.5

7

7.5

8

Amylase activity (U/ml) amylase activity (U/ml) growth (O.D) at 600mn

pH

0.4

2

Fig 3.4. pH optimization of C2

1.8

0.35

1.6

0.3

1.4

Growth 0.25

1.2

(O.D) 0.2 at 600nm 0.15

1 0.8 0.6

0.1

0.4

0.05

0.2

0

0 5.5

6

6.5

7

7.5

8

Amylase activity (U/ml) amylase activity (U/ml) growth (O.D) at 600mn

pH

75

3.3.3. Effect of carbon source on amylase production: A number of carbohydrates (1%) had been tested for amylase production by C1 and C2 (Fig 3.5 and 3.6). Amylase production is the maximum for starch in both cases. But though in presence of glucose C2 showed highest growth and C1 also showed significant growth, there was no amylase production by both of the organisms when glucose was used as sole carbon source. For C1 maximum growth observed when sucrose was used as sole carbon source. Fig 3.5. Carbon source optimization of C1

0.5 0.45

1.8

0.4

1.6

0.35

1.4

Growth 0.3 (O.D) at 600nm 0.25

1.2 1

0.2

0.8

0.15

0.6

0.1

0.4

0.05

0.2

0

0

starch

0.6

glucose

mannitol

sucrose

Fig 3.6. Carbon source optimization of C2

0.5

Growth (O.D) at 600nm

2

Amylase activity (U/ml) amylase activity (U/ml) growth (O.D) at 600mn

2.5

2

0.4 1.5 0.3

Amylase activity (U/ml)

1 0.2 0.5

0.1 0

0

starch

glucose

mannitol

amylase activity (U/ml) growth (O.D) at 600mn

sucrose

76

Amylase production by the organisms were also studied at various concentrations (0.2 – 5%) of starch. For C1 growth was the maximum in 1% starch and amylase production was highest in 2% starch whereas for C2 growth was the maximum for 2% starch but amylase production was highest for 1% of starch (Fig 3.7 and 3.8).

Fig 3.7. Starch concentration optimization of C1 2.5

0.4 0.35

2

0.3

Growth (O.D) at

0.25

1.5

0.2 1

0.15 0.1

0.5

0.05 0

0 0.2

0.5

0.8

1

2

5

Amylase activity (U/ml) amylase activity (U/ml) growth (O.D) at 600mn

Starch concentration in %

Fig 3.8. Starch concentration optimization of C2 2.5

0.4 0.35

2

0.3 0.25

1.5

Growth 0.2 (O.D) 0.15 at 600nm

Amylase activity (U/ml)

1

0.1

0.5

amylase activity (U/ml)

0

growth (O.D) at 600mn

0.05 0 0.2

0.5

0.8

1

2

5

Starch concentration in %

77

3.3.4. Effect of nitrogen source on amylase production: The effect of nitrogen sources on amylase production and growth of the organisms were determined by using different organic and inorganic nitrogen sources (0.6%). Nitrogen compounds are very important for growth and enzyme production. Among different stimulatory nitrogen sources, bacto-tryptone gave the maximum growth for both. Though C1 the highest amylase for bacto-tryptone but for C2 production was the maximum for peptone (Fig 3.9 and 3.10). For NH4Cl there were no significant growth and no production of amylase for both organisms. Fig 3.9. Nitrogen source optimization of C1 2.5

0.8 0.7

2

0.6 0.5

1.5

Growth 0.4 (O.D) at 0.3 600nm

Amylase activity (U/ml)

1

0.2

0.5

0.1 0

0

peptone

0.45

beef extract

ammonium chloride

bacto tryptone

Fig 3.10. Nitrogen source optimization of C2

amylase activity (U/ml) growth (O.D) at 600mn

2.5

0.4 2

0.35

Growth 0.3 (O.D) 0.25 at 600nm 0.2

1.5 1

0.15 0.1

0.5

0.05 0

0

peptone

beef extract

ammonium chloride

bacto tryptone

Amylase activity (U/ml) amylase activity (U/ml) growth (O.D) at 600mn

78

Different peptone concentration (0.2- 2%) was also used to observe the effect on growth and amylase production (Fig 3.11 and 3.12). For C1 growth was increase with increase in peptone concentration but amylase production was the maximum at 0.6% peptone. For C2 growth was the maximum at 1% peptone but amylase production was the highest for 0.6% peptone. Fig 3.11. Peptone concentration optimization of C1 2

0.7

1.8

0.6

1.6 0.5

1.4

0.4

1.2 1

Growth 0.3

0.8

(O.D) 0.2 at 600nm 0.1

0.6

Amylase activity (U/ml)

0.4 0.2

0

0 0.2

0.4

0.6

0.8

1

2

amylase activity (U/ml) growth (O.D) at 600mn

Peptone concentration in %

Fig 3.12. Peptone concentration optimization of C2 0.35

2.5

0.3 2 0.25 1.5

0.2 0.15

1

Growth (O.D) 0.1 at 600nm

Amylase activity (U/ml)

0.5

0.05

0

0 0.2

0.4

0.6

0.8

Peptone concentration in %

1

2

amylase activity (U/ml) growth (O.D) at 600mn

79

3.3.5. Effect of phosphate source on amylase production: Different organic and inorganic phosphate source (0.05%) had been supplemented separately in the basal media to study the effect on growth and amylase production for C1 and C2 (Fig 3.13 and 3.14). Though for both organisms Adenosine mono phosphate gave highest growth but the amylase production was the maximum for K2HPO4.

Fig 3.13. Phoaphate source optimization of C1 0.7

1.8

0.6

1.6 1.4

0.5

1.2

Growth 0.4 (O.D) at 0.3

1 0.8

Amylase activity (U/ml)

0.6

0.2

0.4 0.1

0.2 0

0 Di potassium hydrogen phosphate

Potassium di Di ammonium hydrogen hydrogen phosphate phosphate

Adenosine mono phosphate

amylase activity (U/ml) growth (O.D) at 600mn

Fig 3.14. Phosphate source optimization of C2 2.5

0.7 0.6

2

0.5

Growth 0.4 (O.D) at 0.3 600nm

1.5 1

0.2

Amylase activity (U/ml)

0.5

0.1

0

0 Di potassium Potassium di Di ammonium hydrogen hydrogen hydrogen phosphate phosphate phosphate

Adenosine mono phosphate

amylase activity (U/ml) growth (O.D) at 600mn

80

3.3.6. Effect of metal ions on amylase production: Different metal ions (0.001%) were used to study the effect on the growth and amylase productions by C1 and C2 (Fig 3.15 and 3.16). It was found that in presence of calcium ions the growth and amylase production were the maximum for both of the organisms.

Fig 3.15. Metal ion optimization of C1 2

0.7

1.8

0.6

1.6 0.5

1.4

Growth 0.4 (O.D) 0.3 at 600nm

1.2 1 0.8 0.6

0.2

Amylase activity (U/ml)

0.4 0.1

0.2

0

0 Ferrus

0.6

Zinc

Manganese Molybdenum Calcium

Fig 3.16. Metal ion optimization of C2

0.5

amylase activity (U/ml) growth (O.D) at 600mn

2.5 2

0.4 1.5

Growth 0.3 (O.D) at 0.2 600nm

1

Amylase activity (U/ml)

0.5

0.1 0

0 Ferrus

Zinc

Manganese Molybdenum Calcium

amylase activity (U/ml) growth (O.D) at 600mn

81

3.3.7. Effect of incubation period on growth and amylase production: Growth kinetics of C1 and C2 were studied by using the medium optimized for highest amylase productions (Fig 3.17 and 3.18). The readings were taken for 26 h till the O.D become constant. Similarly the amylase production by them at different time interval was also recorded up to 48 h. the maximum amylase were produced after 24h for both organism and then the productions were declined. 1.4

Fig 3. 17. Time vrs. growth

1.2 1

Growth (O.D) 0.8 at 0.6 600 nm

Growth for C1

0.4

Growth for C2

0.2 0 0

5

10

15

20

25

30

Time in hours

3

Fig 3.18. Time vrs. Amylase Production 2.5

Amylase activity (U/ml)

2

1.5

Amylase for C1

1

Amylase for C2 0.5

0 0

10

20

30

40

50

60

Time in hours 82

3.4. Discussion: Optimization of growth condition is a prime step in using microorganisms in fermentation technology

[124]

. In the present study we observed as the optimum growth

temperature for the presently reported C1 and C2 strains and higher temperature (45oC) did not support any colonies (Fig 3.1 and 3.2). This could be due to the mesophilic nature of the species. As per earlier report

[125]

the high temperature may inactivate the

expression of gene responsible for the starch degrading enzyme. Most of the starch degrading bacterial strain revealed a pH range between 6.0 and 7.0 for normal growth and enzyme production

[126]

.The presently isolated strains also showed optimum growth in

between 6.5 – 7.5 (Fig 3.3 and 3.4). The composition and concentration of media greatly affect the growth and production of extracellular amylase production in bacteria Starch is ubiquitous and is an easily accessible source of energy

[129]

[127-128]

.

.In past studies, a

number of carbon and nitrogen sources have been examined for amylase production in several Bacillus species [127-129]. Similar to these past reports, the present study also observed increasing the starch concentration increase both growth kinetics and amylase production up to certain extent then decreased (Fig 3.5- 3.8). Similarly peptone is the best nitrogen source for amylase production for both organisms though bacto-tryptone supported the maximum growth (Fig 3.9- 3.12). Though Adenosine mono phosphate supported the maximum growth, the amylase production is the maximum for K2HPO4 (Fig 3.13 and 3.14). Maximum growth and amylase production for both C1 and C2 occurred in presence of calcium ion (Fig 3.15 and 3.16). The α- Amylase is known to be a calcium metalloenzyme having at least one calcium ion associated with its molecule. Enhanced bacterial growth and enzyme production may be the result of increased availability of calcium ions [130].

The standard medium lacking starch did not produce amylase

[131]

. Glucose did not

support amylase production presumably due to the catabolite repression by these sugars in the growing cells. Starch considerably induced amylase synthesis been reported to be an inducer for amylase synthesis

[134]

[132-133]

. Starch has

but there are few strains of 83

bacteria which produce amylase in media containing glucose or other mono saccharides [135]

. Glucose accelerated growth, but repressed amylase synthesis in the present study

which indicated that growth and amylase synthesis are controlled by separate regulatory mechanisms. . it was previously

[136]

observed that amylase activity in starch degrading

bacteria is non-growth related. Enzyme production from microorganism is directly correlated to the time period of incubation [137]. In our study it was found that both organisms yielded maximum amylase after 24 h of incubation and then the productions were decline may be due to the utilization of all necessary nutrients (Fig 3.17 and 3.18). The differences in nutritional requirements of various a-amylase producing organisms or microbial strains could be attributed to the difference in their genetics.

84

Determination of Amylase Activity and Stability

85

4.1. Introduction: The term "amylases" refers to a group of enzymes that break down starches and that are very widespread in Nature: they are found in animals and plants and are produced by many microorganisms. In order to make use of the plant-based starches present in food, large starch molecules first must be split into smaller units. It has been possible for a long time to produce amylases with a variety of fungal and bacterial cultures. As a rule, bacterial amylases are more stable in regard to temperature than are amylases derived from fungal cultures [109]. Amylase is used in brewing and fermentation industries for the conversion of starch to fermentable sugars, in the textile industry for designing textiles, in the laundry industry in a mixture with protease and lipase to launder clothes, in the paper industry for sizing, and in the food industry for preparation of sweet syrups, to increase diastase content of flour, for modification of food for infants, and for the removal of starch in jelly production. There are different types of enzyme assay 4.1.1. Continuous assays i.

Spectrophotometric

In spectrophotometric assays, you follow the course of the reaction by measuring a change in how much light the assay solution absorbs [110] ii.

Fluorometric

Fluorometric assays use a difference in the fluorescence of substrate from product to measure the enzyme reaction [111]. iii.

Calorimetric

Calorimetry is the measurement of the heat released or absorbed by chemical reactions [112]

.

86

iv.

Chemiluminescent

Chemiluminescence is the emission of light by a chemical reaction. Some enzyme reactions produce light and this can be measured to detect product formation. v.

Light Scattering

Static light scattering measures the product of weight-averaged molar mass and concentration of macromolecules in solution.

4.1.2. Discontinuous assays i.

Radiometric

Radiometric assays measure the incorporation of radioactivity into substrates or its release from substrates. ii.

Chromatographic

Chromatographic assays measure product formation by separating the reaction mixture into its components by chromatography. This is usually done by high-performance liquid chromatography (HPLC), but can also use the simpler technique of thin layer chromatography [113]. The colorimetric method for amylase assay has been developed by using starch as substrate [114]. An improved amylase assay has been developed by modifying the method of Bernfeld to increase accuracy and sensitivity [108]. The method described here differs from that of Bernfeld as follows: (1) The color reagent contained 1 mg/ml of 3, 5dinitrosalicylic acid instead of 10 mg/ml. (2) Incubation was performed at 25 C instead of 20 C as a matter of convenience. (3) Samples for spectrophotometric observations were diluted 1: 10 instead of 1:5 to reduce the total amount of light energy absorbed. (4)

87

Spectrophotometric readings were made at wavelength 470 mµ instead of 540 mµ because the absorbance peak of the reaction product occurs at 470 mµ. It is simple, reproducible and also satisfies to measure the amylase activity in relation to growth of the organism. The assay conditions of the amylase are also described in this chapter. Generally amylase obtained from bacterial sources is thermostable [115]. B stearothermophilus

[116]

retained

about 60% of its activity even after treatment at 80°C for 60 min. and the temperature optima for Rhodothermus marinus

[117]

were reported to be 85oC. The amylase produced

by B cadolyticus was reported to be thermostable up to 105oC o

species have been reported to give maximum activity at 45 C

[118]

[119]

Generally maximum amylase activity was found at neutral pH

Where some Bacillus

and 35oC [120].

[121]

. But the Bacillus sp.

often produces amylase that is active over a wide range of pH e.g. 2- 10

[115]

. The

amylases with high temperature and pH stability are proffered for industrial application. Enzymatic activity at high temperature suggests two possible models for balance of physical characteristics

[122]

. First, temperature drives structural recognition, modifying

the active center to create an environment for recognizing substrate. Second, the enzyme is maintain in a rigid structure at a low temperature but required increased temperature to provide the molecular flexibility for enzymatic activity. They also mentioned that the loss of α amylase activity of Pyrococcus furiosus is induced by lowering the temperature below 60 oC, cannot be attributed to cold denaturation. The loss of tertiary structure, indicative of cold denaturation, is usually a cooperation process associated with large enthalpy and entropy changes. From the municipal waste the micro organism isolated were able to survive in different adverse condition so they may produce industrially applicable enzymes like amylase.

4.2. Materials and Methods:

4.2.1. Micro organisms: Previously isolated two bacterial strains C1 and C2 were used throughout this study.

88

4.2.2. Preparation of inoculum: Several loop full of stock cultures of above strains were transferred to the medium III broth used as basal media for amylase production. They were incubated at 37oC for 24h on a rotary shaker. Then the broths were centrifuged (5000xg, for 10min) and the pellets were washed twice with sterile distilled water and used as inoculums. 4.2.3. Production of amylase: For enzyme production, the growths of the organisms were carried out in 250ml Erlenmeyer flasks containing 50 ml of the liquid medium (the medium for optimum amylase production as described in chapter 2). 1% (v/v) inoculum of each strain were added separately in the culture media and incubated at 37oC for 24h on a rotary shaker (200 rpm). The culture supernatants obtained by centrifugation (5000xg, for 10 min) were used as crude amylase for assay.

4.2.4. Different buffer solutions[123]:

1.

0.1 M Sodium acetate buffer (pH - 4): Take 874 ml of 0.1M acetic acid solution.

Dissolve 13.6 gm of sodium acetate ( tri hydrate) in 1L of water. Add 153ml of this solution to the acetic acid solution to obtain o.1M sodium acetate buffer of pH 4.

2.

0.1 M Sodium phosphate buffer (pH – 6): Add 15.6 gm sodium phosphate

monobasic dihydrate (NaH2PO4·2H2O, MW 155.99 gm/mol), in 500 mL dH2O. Add 53.65 gm sodium phosphate dibasic heptahydrate, in 1 L dH2O. Then add 263.1 ml of first solution and 36.9 ml of second solution and make up the volume up to 600ml to get 0.1M sodium phosphate buffer of pH 6.

3.

0.1 M Potassium phosphate buffer (pH – 7): Dissolve 34.0 gm of KH2PO4 in 250

ml of water and 45.6 gm of K2HPO4 in 200 ml of water. Add the 2nd solution to the 1st one to get 0.1M of potassium phosphate buffer of pH 7. 89

4.

0.1 M Tris HCl buffer (pH – 8): add 60.55gm of tris HCl in 19 ml of conc. HCl

and make up the volume with water up to 500ml to get 0.1M tris HCl buffer of pH 8.

5.

0.1 M Glycine NaOH buffer (pH – 10): add 75 gm of glycine and 10gm of NaOH

to 1L of water and pH is adjusted to 10 to obtain 0.1M glycine- NaOH buffer of pH 10.

4.2.5. Assay of amylase [108]:

4.2.5.1. Reagents employed: Enzyme - The crude enzyme solutions obtained as supernatant after centrifugation. Substrate - One percent soluble starch solution (10 gm/l) in 0.02M sodium phosphate buffer, pH 6.9 containing 0.006M NaCl. The starch solution was heated to boiling and filtered using Whatman no. 1 filter paper.

Color reagents.-One hundred milligrams of 3, 5- dinitrosalicylic acid was dissolved in 20 ml of 2N NaOH and 50 ml of water; 30 gm of sodium-potassium tartrate (Rochelle salt) was added, and the solution was made up to 100 ml with water.

4.2.5.2. Procedure: One ml of enzyme solution was added to 1 ml of substrate and then incubated for three minutes at 25 C. (2) Two ml of color reagent was added to stop the enzyme reaction. (3) Tubes were heated in a boiling water bath for five minutes to effect the color change, and then cooled with running tap water. (4) Samples were diluted 1:10 with water. (5) Absorbance was read in a spectrophotometer at 470 mµ. The blank was prepared by substituting media in use for the amylase solution instep 1.

90

4.3. Results:

4.3.1. Standard curve relating absorbance and maltose concentration: A standard curve relating maltose concentration to the color reaction was obtained by reacting 2-ml samples of aqueous maltose solutions (concentrations of 0.1 to 1.0 mg/ml) with 2 ml of 3, 5-dinitrosalicylic acid (Fig 4.1). The determination of amylase concentration in an unknown sample was made by plotting the absorbance of the reaction product at 470 mµ against a standard amylase preparation. Units of amylase activity were expressed as micromoles of maltose liberated per minute.

Fig 4.1. Absorbance vrs maltose concentration in mg/ ML 0.5 0.45 Optical 0.4 Density 0.35 at 470 0.3 0.25 nm 0.2 0.15 0.1 0.05 0

0.44 0.33 0.2 0.13 0.05 0

0.2

0.4

0.6

0.8

1

1.2

maltose concentration in mg / ML

4.3.2. Different incubation and storage temperature for determination of amylase activity and stability: The temperature optimum of the enzyme was evaluated by measuring the α-amylase activity at different temperatures (30-50oC). The effect of temperature on amylase stability was determined by measuring the residual activity after 24 h of pre-incubation in 0.02M sodium phosphate buffer, pH 6.9 at temperatures ranging from 30-50oC. Both of

91

the enzymes lost their stability at 50oC (Fig 4.2 and 4.3). They gave very little activity at 30oC. The enzyme from C1 and C2 gave maximum activity at 37oC but maximum stability at 35oC.

Fig 4.2. Effect of temperature on activity and stability of amylase from C1

Amylase activity(U/ml)

3

Activity (U/ml)

2.5 2

Stability after 24 hrs incubation(U/ml)

1.5 1 0.5 0 30

35

37

40

45

50

Temperature o C

Amylase activity(U/ml)

2.5

Fig 4.3. Effect of temperature on activity and stability of amylase from C2

2

Activity (U/ml) 1.5

Stability after 24 hrs incubation(U/ml)

1

0.5

0 30

35

37

40

45

50

Temperature in o C 92

4.3.3. Different incubation and storage pH for determination of amylase activity and stability: The pH optimum of the enzyme was determined by varying the pH of the assay reaction mixture using the following buffers (0.1 M): sodium acetate (pH 4), sodium phosphate (pH 6), potassium phosphate (pH 7), Tris-HCl (pH 8) and glycine-NaOH buffer (pH 10).

Amylase activity(U/ml)

3

Fig 4.4. Effect of pH on activity and stability of amylase from C1

2.5

Activity (U/ml)

2

Stability after 24 hrs incubation(U/ml)

1.5 1 0.5 0 4

6

7

8

10

pH

Amylase activity(U/ml)

Fig 4.5. Effect of pH on activity and stability of amylase from C2 3 2.5

Activity (U/ml)

2 1.5

Stability after 24 hrs incubation(U/ml)

1 0.5 0 4

6

7

8

10

pH

93

To determine the stability of a-amylase, the enzyme was pre-incubated in different buffers (pH 4-10) for 24 h. The residual enzyme activity was determined. Both enzymes had no activity at pH 4 and also had no stability (Fig 4.4 and 4.5). They lost their stabilities at pH 10. Enzyme from C1 possessed maximum activity at pH 7 where that from C2 at pH 8. Both of the enzymes were stable within the pH 6-8.

4.4. Discussion: Studies on crude a-amylase characterization revealed that optimum activity was at pH 7 and 37°C for C1 (Fig. 4.2 and 4.4). For C2 optimum activity was at pH 8 and 37°C (Fig. 4.3 and 4.5) The crude enzyme was stable for 24 h at pH range of 6-8 at 37°C for both (Fig. 4.2- 4.5). The enzymes were quite stable at 40°C, while at 50°C, the original activities were lost. Enzyme activity increased with temperature within the range of 4037°C. A reduction in enzyme activity was observed at temperatures above 40°C. Thermostability for 4 h at 100°C has been reported for α-amylase from B. licheniformis CUMC 30 [138]. Bacillus sp. ANT-6 amylase was stable after overnight (85.5%) and 24 h (55%) incubation at 100°C and pH 10.5

[139]

. A strain of Bacillus stearothermophilus

isolated from the samples of a potato processing industry had a highly thermostable aamylase. The temperature optimum for the activity of this enzyme was 70°C but pH optimum for activity was relatively low, in the range 5.5-6.0

[140]

. The α-amylases from

Bacillus genus are heat stable and this is a desirable property for industrial starch liquefaction. In our study the organisms were giving amylases which were able to tolerate temperature up to 40oC and pH 6-8. These characters are good enough to make them applicable for industrial purpose. They also can be used for the conversion of organic waste through microbial processes decreases the amount of waste disposed by land-filling [101].

94

Conclusions

95

Conclusions: Municipal garbage contains different house-hold, industrial and hospital waste. It is a rich source for different carbohydrate like starch, protein and other macro and micro nutrient which are essential for growth of different micro-organisms. We choose the municipal waste for isolation of amylase producing bacteria as bacteria can grow luxuriously in it. The municipal solid waste require disposal by incineration or land filling, from which many social and environmental problems have arisen recently. The conversion of organic waste through microbiological processes decreases the amount of waste disposed by land filling and fuel required for waste incineration; moreover, it produces compost that can be used to fertilize the soil. So our present investigation on amylase producing bacteria can be successfully used for bio conversation of starchy materials present in the municipal waste and thus may produce a beneficial role in waste management. On the other hand the micro organism isolated from such places may have better potential to produce enzyme under adverse condition. So the enzymes thus obtained can tolerate wide range of pH and temperature and can be used extensively in different industries like pharmaceutical, textile, food and chemical industries. In our investigation we have isolated two gram negative bacterial strains C1 and C2 which produce amylase within the pH 6-8 and temperature 30oC – 40oC. The enzyme production media is optimized for highest yield. The media contain 2% starch, 0.6% peptone, 0.01% CaCl2, 0.05% KCl, 0.05% MgSO4 and 0.05% K2HPO4. The amylase production was the maximum at 37oC after 24h of incubation and then the production gradually decreased due to the utilization of the media component for both C1 and C2. The crude enzyme stability studies revealed that both enzymes were stable for a pH range of 6-8 and a temperature range of 30o- 40oC. So the enzyme can be applicable for pharmaceutical and detergent industry as it has considerable temperature and pH stability. The bio chemical characterization of C1 and C2 also carried out. C1 possessed different enzymatic properties. It gives positive results for urea hydrolysis, skim milk hydrolysis, gelatin hydrolysis, citrate utilization, nitrate reduction, oxidase production and sugar fermentation with production of acid besides amylase production. It is indentified by 96

16SrDNA sequencing as Pseudomonas aeruginosa (GenBank Accession Number: FJ985806.1). These bacteria can be successfully used for bio conversation of municipal and other solid waste as it can solely convert different carbohydrate, protein and other nitrogenous and carbon containing components into environmentally beneficial compost. Thus the bacteria can be a useful tool for solid waste management and pollution control as it has multi enzymatic properties. The genetic identification of C2 has not been yet completed, but the production of amylase is more for it than for C1. C2 give positive results for citrate utilization, oxidase production, nitrogen reduction and fermentation of different sugars with production of acid and gas. The activity and stability of the enzyme obtained from C2 indicated its application in different biochemical, pharmaceutical and food industries.

These seems that the present findings may be considered as a start of new road which on further paving may lead to increase knowledge about enzyme, particularly with respect to their involvement in industries as well as in waste management and pollution control.

97

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