Isolation and Characterization of Collagenase from Bacillus Subtilis

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ScienceDirect Aquatic Procedia 7 (2016) 76 – 84

2nd International Symposium on Aquatic Products Processing and Health, ISAPPROSH 2015

Isolation and Characterization of Collagenase from Bacillus subtilis (Ehrenberg, 1835); ATCC 6633 for Degrading Fish Skin Collagen Waste from Cirata Reservoir, Indonesia Emma Rochimaa*, Nadia Sekara , Ibnu Dwi Buwonob, Eddy Afriantoa, Rusky Intan Pratamaa a Laboratorium of Fisheries Processing Technology, b Laboratorium of Aquatic Biotechnology, Faculty of Fisheries and Marine Science, Padjadjaran University, Jl. Raya Bandung-Sumedang, Jatinangor 45363, West Java, Indonesia

Abstract The objective of this research was to isolate and characterize collagenase from Bacillus subtilis ATCC 6633 collection of Microbiology Laboratory, Department Pharmacy Biology, Faculty Pharmacy Padjadjaran University. The substrate collagen derived of Tilapia fish skin waste from Cirata Reservoar which has’nt exploited fully yet. The experimental design used and the data analysed descriptively. Collagen as substrate from Tilapia skin waste had extracted by Yuniarti (2010) method in Luria Broth media. The production time of collagenase used Rahmayanti (2014) methods which incubated the isolate for 24 hours and the OD of absorbances from 0.2 to 0.8 evaluated. The effect of temperature on collagenase activity evaluated by temperature from 20 to 70oC. The effect of pH collagenase activity evaluated pH from 5 to 10. The conclusion of the research that B. subtilis ATCC 6633 has colagenolitik activity showed by the clear zone in the Luria media. The optimum production time of collagenase was 24 h of incubation. Collagenase activity reached the optimum temperature was 50 ° C (1,298 Unit mL–1), while the pH optimum collagenase obtained in the range of 7-9 (from 1.298 Unit mL–1 to 1,321 U mL–1. © Published by Elsevier B.V.B.V. This is an open access article under the CC BY-NC-ND license ©2016 2016The TheAuthors. Authors. Published by Elsevier (http://creativecommons.org/licenses/by-nc-nd/4.0/). Peer-review under responsibility of the Science and Editorial Board of ISAPPROSH 2015. Peer-review under responsibility of the science and editorial board of ISAPPROSH 2015 Keywords: Bacillus subtilis (Ehrenberg, 1835); Cirata; collagenase; reservoir.

* Corresponding author. Tel.: +62 817 924 4109. E-mail address: [email protected]

2214-241X © 2016 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). Peer-review under responsibility of the science and editorial board of ISAPPROSH 2015 doi:10.1016/j.aqpro.2016.07.010

Emma Rochima et al. / Aquatic Procedia 7 (2016) 76 – 84

1. Introduction Enzymatic hydrolisis are commonly used to increase nutritional and functional properties from food protein (Zu et al., 2006). Fish protein hydrolisate has been known to have antioxidative, antihypertension, antimicrobial and immunomodulatory properties (Fujita and Yoshikawa, 1999; Shahidi et al., 1995). Protein hydrolisate antioxidative properties specifically has been a major topic which attract attention from pharmacy, food and health field (Alasalvar et al., 2002; Hagen and Sandes, 2004). Protein hydrolisate with fish as its raw material which showed antioxidant activity were Alaska Pollack skin gelatin (Buusan et al., 2008); yellowfin sole (Jun et al., 2004), Allaska Pollack, tuna backbone, yellow stripe trevally (Je et al., 2005), round scad (Thuansiakul et al., 2007), gelatin hydrolisate (Khantaphant and Benjakul, 2008). Collagen manufactures main source up until now are derrived from cow’s and pork’s skin and bones. However, since the spreading of mad cow disease, the consumers of cow’s collagen have grow worry, and other than that the consumption of pork’s collagen have been banned in some areas due to religion reason. Therefore fish waste such as bones, scales and skin which contain many collagen are now becoming safer alternative choice. Collagen in industry is produced thermochemically with strong alkali and high temperature. The product from this process has not fully satisfying due to varying collagen quality produced. Further than that, thermochemical process requires energy in great amount to produce and maintain high temperature and also it produces waste and by product in form of high concentration alkali which could have the potential to become toxic to environment. As an alternative, collagen hydrolisis is able to accomplish enzymatically using collagenase enzym from microbial sources. Protein hydrolisate produced by this enzymatic process is expected to be more controlable, more efficient, specific and enviromental friendly. Several bacterial isolates which produce collagenase are Clostridium perfringens and Clostridium histolica, Bacillus subtilis FS-2 (Nagano, 1999), Bacillus subtilis CN (Tran and Nagano, 2002), Bacillus subtilis AS1.398, (Rui et al., 2009), Bacillus pumilus Co-J (Wu et al., 2010) , Bacillus cereus (Liu et al., 2010) and Streptomyces sp. Strain 3B (Petrova, 2006). The bacteria which have the potential as collagenase’s source are Bacillus subtilis which isolated from Rambatan river, Indramayu, Indonesia. This isolate was the collection of Laboratorium of Aquatic Biotechnology Faculty of Fisheries and Marine Science Padjadjaran University collection. Considering the importance of all of those things describes, collagen hydrolisate production from fish waste source enzymatically as an effort to handle enviromental problem and to increase economicalvalue is important to perform. Technological fish waste (skin, bone and scale) enzymatically from local isolates are expected to increase its selling value. Through this research, isolation and characterization of collagenase from Bacillus subtilis ATCC 6633 (especilly to evaluate the effect of temperature and pH on collagenase activity) has been conducted. 2. Materials and methods The main research location for samples producing and testing were at Fishery Product Processing Laboratory, Aquatic Biotechnology Laboratorium and Biochemical Laboratory Padjadjaran University. The main materials used in this research were: collagen from Tilapia fish skin, B. subtilis ATCC 6633 and various chemical reagents: 100 mL acetc acid 1.5 %, 20 g Luria Agar, 20 g Luria Broth, 10 mL buffer fosfat, 10 mL leusin 5 mM, 15 mL TCA 0.5 %, 15 mL ninhidrin 0.1 %; 30 ml iso-propanol 50 %, 100 mg Comassie Briliant Blue (CBB) G-250, 50 mL ethanol 50 %, 100 mL asam fosfat 85 %, 100 mg BSA, aquadest, spirtus, alcohol, aluminium foil, plastic wrap, filter paper and cotton. The main equipments used: spectrophotometer Genesis 10 UV, cold centrifuge Sigma, incubator VWR Scientific, homogenizer, vorex Digisystem. The experimental methods used and the data analysed descriptively. Collagen as substrate from Tilapia skin waste had extracted by Yuniarti (2010) method in Luria Broth media. The production time of collagenase used Rahmayanti (2014) methods which incubated the isolate for 24 h and the OD of absorbances from 0.2 to 0.8 evaluated. The effect of temperature on collagenase activity evaluated by temperature from 20 oC to 70 oC.

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3. Result and discussions 3.1. Extraction of collagen from fish skin Tilapia Extraction of collagen was conducted using Yuniarti (2010) methods. The weight of the raw material Tilapia was 51.95 g (Fig.1). The skin of Tilapia as raw materials must be freshness so that the collagen produced have a good quality (Peranginangin et al., 2014). The skin of fish was a part of the body as byproduct of the Tilapia fillet production and have a yield of 8.7 % of the total weight of the fish (Peranginangin et al., 2006).

Figure 1. Raw material of collagen from skin Tilapia

Fish skin contains 69.6 % water content; 26.9 % protein content; 2.5 % ash content and 0.7 % fat content (Rush 2004). Fish skin composed of two main layers of the epidermis and the dermis. Dermis is a connective tissue that is thick and contains a number of collagen fibers (Lagler et al. 1977 in Azizah 2013). Nearly 80 % of the dermis layer composed of collagen fiber network built by woven binder (Setiawati 2009). Extraction is the process of separating a substance based on the difference in solubility of the two are not mutually soluble liquids of different, usually water and other organic solvents. The principle of enzyme extraction method considered several things, including: the source of the enzyme, the type, nature and form of extracts or preparations desired. Animal enzymes are generally located in specific organs such as the digestive organs and tissues. Enzyme extraction of organs is done by separating the crude enzymes of fat then destroyed by controlling the temperature so that no protein denaturation of enzymes (Suhartono 1989). Skin collagen substrate to determine whether the bacteria can produce the enzyme collagenase. Their enzyme activity is indicated by the appearance of clear zone around the colony of bacteria (Agustin 2013). This study used a Luria Agar medium containing casein, yeast extract, sodium chloride, and order appropriate for the type of Bacillus growth. Production of collagen substrate by mixing 90 % of Luria Agar with 10 % solution of Tilapia skin collagen then incubated at 37° C for 24 h. The presence of a clear zone around the colonies showed that Bacillus subtilis ATCC 6633 can produce collagenase activity, for a clear zone indicates that the enzymes secreted by the bacteria can break down collagen into collagen molecules smaller so that the bacteria can take up the nutrients in the form of small molecules. Extraction is the process of separating a substance based on the difference in solubility of the two are not mutually soluble liquids of different, usually water and other organic solvents. The principle of enzyme extraction method considered several things, including: the source of the enzyme, the type, nature and form of extracts or preparations desired. Animal enzymes are generally located in specific organs such as the digestive organs and tissues. Enzyme extraction of organs is done by separating the crude enzymes of fat then destroyed by controlling the temperature so that no protein denaturation of enzymes (Suhartono, 1989). Several bacterial isolates which produce collagenase are Clostridium perfringens and Clostridium histolica, Bacillus subtilis FS-2 (Nagano, 1999), Bacillus subtilis CN (Tran and Nagano, 2002), Bacillus subtilis AS1.398, (Rui et al., 2009), Bacillus pumilus Co-J (Wu et al., 2010) , Bacillus cereus (Liu et al., 2010) and Streptomyces sp. Strain 3B (Petrova, 2006).

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3.2. Collagenolitic activity A clear zone appears after the isolates were incubated for 24 h (Fig.2). The existence of a clear zone indicates that these isolates produced collagenases due to the bacteria break down the collagen substrate into smaller molecules. According Susanti (2003), the wide of a clear zone around the growth of the bacteria can not represent the amount of protease enzyme produced by a microbe. This is because the clear zone produced will increase with increasing the time of incubation. Therefore, a clear zone only indicate the presence of enzyme activity qualitatively.

Clear zone around the colony

Figure 2. The clear zone of Bacillus subtilis ATCC 6633 collagenase

A clear zone appears after the isolates were incubated for 24 h. The existence of a clear zone indicates that these isolates produce collagenas. This is because the bacteria break down the collagen substrate into smaller molecules. According Susanti (2003), the width of clear zone around the growth of the bacteria can not represent the amount of protease enzyme produced by a microbe. This is because the width of the clear zone will increase with the increasing of incubation time. Therefore, a clear zone only indicate the presence of enzyme activity qualitatively. Collagenase have three domains involved in the hydrolysis of collagen molecules. The first called the catalytic domain that contain zinc so that metalloprotease group; The second, collagen binding domain (CBD) and the third was Polycystic Kidney Diseases domain-like (PKD). The role of CBD to recognize the structure of the collagen triple helix, whereas PKD for preparing collagen by developing a triple helix structure without a stick on the collagen molecule. The catalytic domain of collagenase molecule that attached to collagen molecules and would cut the collagen at specific point (Brandsetter and Eckhard, 2011 in Rahmayanti, 2014). The mechanism of degrading collagen by collagenases described by Figure 3.

Figure 3. The stages of collagenolytic (Chung et al., 2004)

Based on Fig. 3 showed that catalityc domain had the active site which is bounded into the collagen substrate. Then, it will promote the rigid triple helix substrate of collagen so that the binding of Gly775-Ile776 dan Gly775-Leu776

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amino acid residu on α2(I) would be hidrolized and degraded. The chain of amino acid degraded into two parts (¼ and ¾ fragments) which is N terminal located on fragments ¼, meanwhile two chains of α1(I) degraded not caused by collagenolitic activity, but due to conformation changes and collagenase stability partialy on a part of collagen I which is complete (Chung et al., 2004).

3.3. Characterization of collagenase The main factors that affect the activity of the enzyme is the enzyme concentration, substrate, product, and activator inhibitor compounds, pH and type of solvent contained in the environment, ionic strength and temperature (Suhartono, 1989). Characterization of collagenase conducted to determine the catalytic properties of the enzyme so that it can also optimum condition of enzyme activity. Collagenase from various sources, having catalytic properties are different. The difference is caused by factors such as species, age, type of food, water quality, ambient temperature. Differences in the catalytic properties of enzymes are also found in the same species caused by factors interspesies. These factors include age, size sex, spawning phase, spawning history, the composition of the food, a history of stress and others (Haard, 2000). Differences in the catalytic properties of collagenase from various sources, including microorganisms are presented in Table 1. Table 1. Catalytic properties of collagenase from various sources No

Sources

pH opt

Temp opt o C

Inhibitor

Metal ion Inhibitor

1

2

3

4

5

6

7

8

Invicera maccarel (Scomber japanicus) (Park et al., 2002). Invicera filefish (Novoden modestrus) (Kim et al., 2002) . Hepatopancreas shrims (Pandalus eous) (Aoki et al., 2003) . Piloric caeca tunna (Thunnus thynnus) (Byun et al., 2002) Rainbow trout Oncorhynchus mykiss tail (RTT) (Saito et al., 2000) Streptomyces strain 3B (Petrova et al., 2005)

55

PMSF, TLCK, Soybean trypsin inhibitor

Hg2+, Zn2+

7.0 to 8.0

55

TLCK

Zn2+, Cd2+, Cu2+ Ni2+

K+, Li+, Ba2+, Ca2+, Mg2+

7.5 to 8.5

40 to 45

PMSF , antipain

-

-

7.5

55

PMSF, TLCK, Soybean trypsin inhibitor

Hg2+, Zn2+

-

20

1,10-phenanthroline, cysteine±zinc

37

1,10-phenanthroline, EDTA

Bacillus subtillus FS-2 (Nagano and Kim, 1999)

9

50

EDTA, Soybean tripsin inhibitor, iodoecetamidaiodoacetic acid

Clostridium perfringens (Matshishita et al., 1994)

7.2

42

1,10-phenanthroline

Continued on the next page

Type colagenase

Collagen type I

14.8

serin

-

27.0

serin

Collagen type I

Collagen type I

Collagen type I

Cu2+, Zn2+ Hg2+, Fe2+

Molecule Weight kDa

Activator

7.5

7.5

Substrate spesifici ty

22.0 23.0

to

serin

15.0

serin

29; 27; 26

metallo

Mg2+, Ca2+, Ba2+

gelatin dan Collagen type I

116. 97

metallo

Ca2+ , Mg2+ , dan Zn2+

gelatin

125

metallo

Ca2+ , Mg2+ dan Zn2+

120

metallo

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Emma Rochima et al. / Aquatic Procedia 7 (2016) 76 – 84 Table 1. Continued

pH opt

Source

9

10

Photorhabdus luminescens (Marokhazi et al., 2004) Fish meat Pasific rockfish (Sebastes sp.) (Brocho and Haard, 1995)

Temp opt ºC

Inhibitor

7.0

1,10-phenanthroline and EDTA

7.5 to 8.5

1,10-phenanthroline and EDTA

60 to 70

Substrate spesifici ty

Metal ion Inhibitor

Activator

Mn2+ dan Ca2+

Zn2+, Co2+

Ca

Type colagenase

kDa

Lingcod skin type I collagen Gelatin

2+

Molecule Weight

74

metallo

47.95

metallo

3.3.1. Effect of temperature on enzyme activity Temperature characterization was conducted to determine the effect of different levels of temperature on the activity of the enzyme collagenase. By characterizing the temperature can be determined according to the optimum temperature of the enzymes work. Determining the optimum temperature collagenase carried out by reacting the collagenase with collagen skin Tilapia at different levels of temperature is (20, 30, 40, 50, 60 and 70) oC, then measured the activity based methods ninhydrin according Moore and Stein (1954) in Yuniarti (2010) were modified. According to Winarno (1986), collagenase is an enzyme capable of hydrolyzing collagen which has not been denatured at neutral pH so the pH is used to characterize this temperature is 7. The results of the characterization of temperature on the enzyme activity is presented in Figure 4.

1.4

Unit Activity (Unit per mL)

1.298

1.2 1 0.86

0.8 0.6

0.568

0.4

0.105 0.2

0.153

0 20

30

40 50 Temperature ° C

60

70

Figure 4. Effect of temperature on enzyme activity

Figure 4 showed the change in enzyme activity at 30 °C to 70 °C. The enzyme-catalyzed reaction rate increases with increasing temperature and the highest of collagenase from Bacillus subtilis ATCC 6633 activity was at 50 °C (1.298 unit per mL). Based on Rahmayanti (2014) that collagenase from Bacillus licheniformis F11.4 which has the optimum temperature was 40 oC to 50 oC. Nagano and To (1999) stated that collagenase of Bacillus subtilis FS-2 which was isolated from the traditional fish sauce had the optimum temperature of 50 °C to degrade gelatin, meanwhile, Baehaki et al. (2012) concerning at pure collagenase which produced by Bacillus licheniformis F11.4 has the optimum temperature of 50 °C so that classified as thermostable moderate bacteria.

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Collagenase activity of 30 ° C and 40 ° C tend to be very small and then increased activity at 50 °C drastically. The optimum activity occured in temperature of 50 °. Enzymes are protein compounds which is very sensitive to temperature changes. Kurnia (2010) states that the higher the temperature will change the structure of the enzyme, followed by the loss of the catalytic activity of the enzyme, while at low temperatures the rate of inactivation of the enzyme to be slow and very small. Generally, the enzyme has the maximum activity value at a certain temperature, called the optimum temperature. The enzyme activity will increase until the optimum temperature is reached. Enzymes are a class of proteins that has the physical and chemical properties similar to protein, and denaturation is a natural thing happened on the enzyme, especially when there is an increase in temperature. Denaturation at collagenase cause collagenase lose its shape so that the collagen can not be bound to the catalytic enzyme (Rahmayanti 2014). 3.3.2. Effect of pH on enzyme activity Characterization of the pH is performed to determine the effect of different levels of pH on the activity of the enzyme collagenase. By doing characterization of pH optimum pH can be determined in accordance with these enzymes work. The determination of optimum pH collagenase collagenase reacting it with collagen skin Tilapia at different levels, namely pH 6, pH 7, pH 8, pH 9, pH 10, and then measured the activity based methods ninhydrin. The temperature used for optimization of pH is previously obtained optimum temperature, ie 50 oC. The characterization results of pH on the enzyme activity is presented in Figure 5.

1.8 1.696

Unit Activity (Unit per mL)

1.6 1.4

1.321

1.298

1.2 1.011

1 0.8

0.6 0.4 0.2

0

0 5

6

7

8

9

10

pH Figure 5. Effect of pH on enzyme activity

Based on Fig.5 it can be concluded that the optimum pH for collagenase enzyme produced by Bacillus subtilis ATCC 6633 in the range of pH 7 to pH 9. In accordance with the results of research Rahmayanti (2014) on collagenase Bacillus licheniformis F11.4 having a pH optimum of pH 8 pH 9, research Baehaki et al. (2012) concerning pure collagenase produced by Bacillus licheniformis F11.4 has an optimum pH 7, and research Nagano and To (1999) regarding the collagenase produced by Bacillus subtilis FS-2 is isolated from the traditional fish sauce has an optimum pH 9 to degrade gelatin. In most enzymes are indeed the optimum pH range wherein optimum enzyme activity takes place and has a high stability (Kusuma, 2010). Unlike the temperature graph, the graph pH enzyme activity at various levels of higher value is above 1. This is due to the temperature used to characterize the pH is already an optimum temperature is 50 oC. However, at pH 6, no activity was shown by collagenase when reacted with collagen substrate so that the collagenase produced by Bacillus subtilis ATCC 6633, including the type of protease from neutral to alkaline. According Rahmayanti (2014), acidic conditions can cause changes in the amino acid composition of the charge on the side of the catalytic


collagenase so that collagen can not be attached to the side of the catalytic enzyme. Enzymes have many functional groups that can be ionized, so that this pH change can affect the shape of the enzyme and its ability to bind to the substrate. 4. Conclusion The conclusion of the research that Bacillus subtilis ATCC 6633 have the collagenase activity which signed of the clear zone in Luria Media. The optimum production time of collagenase was 24 h of incubation. Collagenase activity reached the optimum temperature was 50 ° C (1.298 unit per mL), while the pH optimum collagenase obtained in the range of pH 7 to pH 9 (from 1.298 unit per mL to 1.321 unit per mL) Acknowledgements This research funded by The Excellent Research Universiy Grant 2015 of Padjadjaran University from Directorate General of Higher Education Ministry of Research Technology and Higher Education Republic Indonesia References Alasalvar, C., Shahidi, F., Quantick, P., 2002. 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