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Sep 30, 2017 - 1Department of Biochemistry, Government College Women University, Faisalabad-38000, Pakistan, ...... In outlines of biochemistry. 287-. 298.

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ISSN: 2230-9926

International Journal of Development Research Vol. 07, Issue, 09, pp.15335-15343, September, 2017

ORGINAL RESEARCH ARTICLE

ORIGINAL RESEARCH ARTICLE

OPEN ACCESS

STUDY OF EFFECT OF PHYSICAL MUTAGENSIS ON PRODUCTION OF GLUCOSE OXIDASE IN

ASPERGILLUS NIGER *1Tanzila

Sahar, 2Muhammad Anjum Zia, 3Anum Sahar, 1Naila Rafiq, 1Sobia Aleem, 1Sadia Aslam, 1Kausar Parveen, 4Nadia Noor and *4Nusrat Shafiq

1Department

of Biochemistry, Government College Women University, Faisalabad-38000, Pakistan, of BioChemistry, University of Agriculture, Faisalabad-38000, Pakistan 3Department of Chemistry, University of Agriculture, Faisalabad-38000, Pakistan 4Department of chemistry, Government College Women University, Madina Town, Faisalabad-38000, Pakistan 2Department

ARTICLE INFO

ABSTRACT

Article History:

Glucose Oxidase has get marked attention due to its wide range of applications in different industries and it is naturally produced in fungi, bacteria and insects where its catalytic product, hydrogen peroxide acts as an anti-microbial agent in food, diagnostic and pharmaceutical preparations. To meet its increasing applications of glucose oxidase, especially in food industries; large-scale production of this enzyme is possible by mutagenesis of various microbes including Aspergillus niger. This work describes that UV rays as physical mutagen were used to induce mutagenesis in Aspergillus nigerfor enhanced production of glucose oxidase. TS-UV-200 mutant derived isolate of Aspergillus nigerscreened out as best positive mutants with the 225.62% marked increase in glucose oxidase activity corresponding to its parent wild strain. Production of glucose oxidase from wild type and mutant strain of Aspergillusnigerwas carried outby preoptimized conditions using corn steep liquor as substrate. In current study, it was found that UV rays as a physical mutagen could be efficiently used for the hyper-production of glucose oxidase from Aspergillusniger. It was also revealed that UV mutant strains of Aspergillus niger has a great potential for enhanced production of glucose oxidase and could be valuable for further advance research investigations and commercial scale production of glucose oxidase especially in food and pharmaceutical preparations.

th

Received 28 June, 2017 Received in revised form 04th July, 2017 Accepted 07th August, 2017 Published online 30th September, 2017

Key words: Aspergillus niger (A. niger), Glucose oxidase (GOX), Mutagenesis, UV-Rays.Corn steep Liquor (CSL).

*Corresponding author Copyright ©2017, Tanzila Sahar et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Citation: Tanzila Sahar, Muhammad Anjum Zia, Anum Sahar, 1Naila Rafiq, Sobia Aleem, Sadia Aslam, Kausar Parveen, Nadia Noor and Nusrat Shafiq. 2017. “Study of effect of physical mutagensis on production of glucose oxidase in aspergillus niger”, International Journal of Development Research, 7, (09), 15335-15343.

INTRODUCTION Diabetes mellitus is one of the most common disease that is present in nearly all over the world and continues to increase in numbers and significance at an alarming rate. It is considered as one of the main threats to human health in the 21st century, in both developed and developing nations. The study of glucose level in diabetic patients needs the key enzyme glucose oxidase where its optimized production by mutant derived strain could affect the economic burden and accuracy of the test. Glucose oxidase (GOx) catalyzes the oxidation of D-glucose to D-gluconolactone and hydrogen peroxide.

GOx is produced naturally in some fungi and insects where its catalytic product is hydrogen peroxide which acts as an antibacterial and anti-fungal agent. Large-scale production of GOx is enabled with mutagenesis of microorganism that may cause over-production of GOx to meet its increasing applications in near future. Diabetes mellitus is a general metabolic problem which ultimately leads to death. Diabetes mellitus affects 1– 2% of the population worldwide (Nessar Ahmed.2005). WHO predicts that developing countries will bear the brunt of this epidemic in the 21st century. Currently, more than 70% of people with diabetes live in low- and middle income countries. An estimated 285 million people, corresponding to 6.4% of the world's adult population, will live with diabetes in 2010. The

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number is expected to grow to 438 million by 2030, corresponding to 7.8% of the adult population (World diabetes foundation, 2012). According to World Health Organization (WHO), prevalence of Type 2 diabetes in Pakistan for the year 2000 was 5.2 million and for 2030 it would be around 13.8 million (Rashid M. Ansari. 2009). The enzymatic method using glucose oxidase enzyme was most advantageous for the diagnosis of glucose in diabetes patients (Giampietro et al., 1982). The total cost for the diabetes care has been investigated for the Pakistan in year of 2007 is 515.8 per visit in outpatient clinics (Liaqat et al., 2007). The decrease in cost of diagnosis could help the patients in terms of the financial burden. Now a day, research has been focused on the increased production of enzymes by using strains improvement techniques like mutagenesis (Khattab and Bazaraa, 2005; Haq et al., 2001). Several attempts have been made to improve glucose oxidase production through Aspergillusnigerby strain selection using mutagenesis classical screening techniques (Fiedureket al., 1986; Markwellet al., 1989; Witteveenet al., 1990). The enhancement of commercially important microbial strains has been exercise from long time. Great advantage of screening method is its simplicity; it does not require any profound understanding of molecular biology and physiology of the micro-organisms being manipulated. Meanwhile, strain improvement of the enzyme producer offers the greatest opportunity for cost reduction without significant capital outlay (Stanburyet al., 1995). In the present research an effort has been made to improve the enzyme glucose oxidase production from mutagenization of Aspergillusniger, resistant to a range of metabolic inhibitors, as mutants isolateshaving capability of high yields of glucose oxidase production that could be used for glucose oxidase production at industrial scale. The best mutants were compared with the wild strain for the production of glucose oxidase using fermentation biotechnology methods.

MATERIAL AND METHODS Present research work was carried out in Enzyme Biotechnology Laboratory (EBL), Department of Biochemistry, University of Agriculture, Faisalabad, Pakistan. Microorganism selection and culture maintenance: Aspergillus niger was used as parental strain and pure culture of Aspergillus niger (isolated from soil) was obtained from First Fungal Culture Bank, University of the Punjab, Lahore, Pakistan. The stock culture of wild strain of Aspergillus niger was preserved on potato dextrose agar media (PDA) (Table.1) and stored in refrigerator at 4oC, sub-cultured after 15-20 days. Inoculum Preparation: While Vogel’s medium (Table 1) was used as inoculum medium for Aspergillus niger strains. Aspergillus nigerspores from PDA culture were aseptically transferred in laminar air flow toVogel’s medium (Zia et al., 2010; Sahar et al. 2015). For homogeneous microbial colonies growth, small marble beads(3 - 5 in number) were added in inoculum flask and autoclaved for sterilization for 15 min at 15 psi pressure and 121oC temperature. Inoculum medium was incubated for 36 hours at 30ºC on an orbital shaker (150 rpm) that was then used for further mutagenesis experiments or to inoculate the glucose oxidase fermentation medium (Rasul et al., 2011; Sahar et al. 2015). Strain Improvement Technique/ Mutagenesis: Physical mutagenesis was used as strain improvement technique for the

hyper production of glucose oxidase (GOX) enzyme from Aspergillus niger.UV rays was used as physical mutagen, to induce mutagenesis in Aspergillus niger for the improved GOX production. UV rays known in literature as the potent inducers of mutations in microbial strains. Spore suspension (1x107 spores ml-1) of 36 hours fresh inoculum culture of wild Aspergillus niger was exposed to ultraviolet rays for selected exposure times that was 0, 40, 80, 120, 160, 200, 240 and 280 min (Gromada and Fiedurek, 1997,Khattab and Bazaraa, 2005) at 37oC. Exposure was carried out at distance of 20cm from the center of UV germicidal lamp (UV Lamp: Type A-409, P.W. Allen and Co., 253-Liverpool, RD., London-N.1). Afterwards, all the fractions of spore’s suspension of Aspergillus nigercollected after selected time intervals given above, wrapped with aluminum foil and kept in dark in laminar air flow. (Zia et al., 2010). After mutagenization for each exposure time, diluted volume (100 times dilution) of 100 μL of surviving spores were spread on the petri plates containing PDA supplemented with 1% triton X-100 (colony restrictorthat limited the microbial growth by permitting the highly resistant colonies to grow.) with the help of sterilized spreader in laminar air flow and these plates were kept at 30oC in incubator till colonies growth appeared (5-9 days). Once microbial colonies appeared, colonies were counted for each exposure time. All experiments conducted in sterilized environment. Then efficiency of mutagen was assessed by drawing three-log killcurve. Kill/ survival curve was drawn for the selection of best mutation time for the hyperproduction of glucose oxidase enzyme (Petruccioliet al., 1999; Zia et al., 2010). Screening and Selection Methods for the Isolation of Positive Mutants Selection by Colony Restrictor using 3 Log Kill Curve; Optimal exposure time of UV rayswas screened by drawing three-log-kill curve (Zia et al., 2010). At this phase almost ninety percent decrease in viability was determined. Few colonies were screened and selected from optimal exposure time and were isolated on PDA plates for further study of their enzyme activities.Microbial colonies present on optimal exposure time were then again subject to grow on triton X-100 (1%) for further screening in order to investigate the potential of selected colonies.Triton X-100 is a synthetic surfactant that is capable to reduce microbial growth. It causes a delay in logarithmic growth phase of microbes. In plate screening method, above screened colonies were grown on PDA media containing 1% triton-X-100 and these plates were kept at 30 C in incubator for growth. After 3-5 days of incubation, the size of clearing zones was determined. The colonies having more fast growing potential and bigger zone were selected and further sub-cultured. Only few colonies were obtained showing large clearance zones than wild type (Petruccioliet al., 1999). For precise screening, mutant microbial colonies confirmed on colony restrictor were then subjected to selective marker, 2-Deoxy-D-glucos (1mg/mL) that is an analogue of glucose (Gromada and Fiedurek, 1997, Zia et al., 2012b).Afterwards, mutant fungal colonies that showed potential growth on 2-Deoxy-D-glucos comprising PDA medium were selected for further screening and verification by enzyme diffusion zone test for optical and spectrophotometrically quantifiable investigation. It is a simple and reliable test for the estimation of enzyme production. Above selected potential mutant fungal colonies were then

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examined on 2% agar comprising petri plates using peroxidase (225 U/mL) and O-dianisidine (0.1 g/L) for glucose oxidase (GOX) production.Clear brown color zone was formed around the fungal colonies due to the presence and production of GOX that was clearly perceived and measured (El-Enshasy, 1998). The colonies producing greatestdiameter ofzone with higher enzyme activity corresponding to its parental strain were selected for additional scrutinization for GOX production. Results of enzyme diffusion zone test reconfirmed by well test (Fiedureket al., 1986; Gromada and Fiedurek, 1997). In well test, 2% agar plates were prepared and wells (approximately 0.3 mm) are formed by using sterilized glass pipettes. In these wells, fungal spores were kept and added 0.1 mL reaction mixture (peroxidase + phosphate buffer + Odianisidine). After few minutes, clear brown zone formed around the fungal colonies,measure the zone diameter. Colonies having larger zone size have more ability to produce enzyme. The above screened colonies producing larger zonewere measured and scratched, liquefied in (0.1 M) phosphate buffer (pH 6), filtered and then enzyme activity was quantitatively measured spectrophotometically at 460 nm (Zia et al., 2010).

quantity of enzyme needed to oxidize one unit of glucose in micromole per minute at 30oC. In glucose oxidase enzyme assay, enzyme was analyzed by using1% orthodianisidinesoln, peroxidase enzyme and 18% D-glucose solnas a substrate. All the data given in this study is the average of measurements.

RESULTS AND DISCUSSION Strain improvement becomes a one of the most essential steps in the development of microbial production processes on industrial scale. Among the other available techniques, the established method of mutagenesis and selection is still a symbol of good probability for success and successively used for the production of industrially important mutant strains (Haq et al., 2002). The area of microbial enzyme applications are constantly increasing in various industries and it considers as a vital phase in enzyme biotechnology field. Various fields of genetic engineering have worked together to achieve exceedingly dynamic enzyme producers. However, customary procedures i.e mutagenesis are still successfully used for the improved productionof different enzymes (Semashkoet al., 2000) because of its simplicity and cost effectiveness.

Table 1. Composition of Potato dextrose Agar (PDA) and Vogel’s medium for A. nigergrowth and Composition of Fermentation Media for Glucose oxidase Production Sr. No.

Components

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

Glucose Starch Agar Urea Potassium dihydrogen phosphate (KH2PO4) Potassium chloride (KCl) Magnesium sulfate heptahydrate(MgSO4.7H2O) Zinc sulphateheptahydrate(ZnSO4.7H2O) Yeast Extract Trisodium citrate Diammoniumsulphate [(NH4)2SO4] Ammonium nitrate (NH4NO3) Peptone CaCO3 Corn steep liquor (CSL) pH Temperature

PDA Quantity ( g/100 mL) 2 2 2 0.3 0.008 0.015 0.05 0.001 4 30°C

Enzyme production: Liquid state fermentation was used for glucose oxidase production from wild and mutant isolates of Aspergillus nigerusing pre-optimized medium conditions (Table1). Corn steep liquor (two percent) was employed as cheap substrate in fermentation medium. Fermentation media were aseptically inoculated by 5% inoculum (Table 1)and media flasks were kept on rotary shaker for 36 hours at 150 rpm in triplicate and incubation temperature was adjusted at 30ºC (Zia et al., 2010; Zia et al., 2012c). Resultant crude product kept in cell homogenizer for 15 min for homogenization, and then subsequent suspension was subjected to high speed centrifugation for the further removal of biomass at 10,000 rpm for 15 minutes (4oC), resultant suspension then clarified by Whatman filter paper (Gromada and Fiedurek, 1997; Rasulet al., 2011) then glucose oxidase activity was measured by enzyme assay. Glucose oxidase Assay: Enzyme activity of glucose oxidase was measured quantitatively in clarified suspension by spectrophotometric analysis (Worthington, 1988) at 460 nm for 3 to 5 minutes. Glucose oxidase activity was defined as

Vogel’s Medium Quantity ( g/100 mL) 2 0.5 0.02 0.2 0.5 0.4 0.2 0.1 5.5 30°C

Fermentation Media Quantity ( g/100 mL) 4 0.3 0.04 0.05 2 5 30°C

Aspergillus niger is successfully used for the production of various commerciallyimportant enzymes because of its effective production ability. Previously Aspergillus niger had been genotypically upgraded by using different phys-chemical mutagens for the improved production of various industrially important microbial products (Rasul et al., 2011; Zia et al., 2012b). This part of current work describes the improvement of microbial strain for the hyper prodution of glucose oxidase (Gox) by means of mutagenesis. Several efforts have been made in previous studies to get the overproduction of glucose oxidase in term mutagenesis (Semashkoet al., 2000; Khattab and Bazaraa, 2005; Khurshid, 2008; Rasulet al. 2011; Haqet al., 2014). The work of different disciplines of genetic engineering helps to attain the exceedingly dynamic glucose oxidase producers. On the other hand, the customary procedures for the induction of mutagenesis by using different mutagens and their mixtures are still effectively used for this purpose (Semashkoet al., 2000) because of its simplicity, easiness and cost effective nature of this method for screening and selection of highly potential mutant isolates. The advances in fermentation biotechnology are attained by using strain

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improvement techniques and it is a one the most important step in the progress of enzyme production process at industrial level (Rasul et al., 2011). Mutagenesis is a cost effective method for microbial strain improvement used to produce heritable deliberate variations in genome by using diverse mutagens that may control specific metabolic pathways. Mutation is a random event and is a major source of alteration in genome. Most of the mutations are lethal but some are found to be valuable particularly when happen in the nucleotide sequence of genome which regulate the production of vital biological products.Presentt investigation deals with the mutagenesis of Aspergillus niger for enhanced production of glucose oxidase by using ultraviolet rays (light) and N-nitrosoN N-methylurea. methylurea. Microbial enzyme production was also significantly enhanced by optimizing diverse cultivation culti conditions and microbial nutritional parameters. In the present investigation, Aspergillus niger is used as manipulating organism because of its metabolic versatility and their capability to yield huge amounts of specific and useful enzymes. Aspergillus nigeris is largely used microorganism for theproduction of glucose oxidase bothat laboratory and industrial level according to previously cited reports. Aspergillus niger is an effective producer of numerous industrially significant enzymes and it may genotypically ge upgraded by the exposure of different physical and chemical mutagens as previously described by Zia et al.(2012b). al Growth of Aspergillus niger was shown in Fig. 4.1.

Fig. 1. Growth of wild Aspergillus niger on PDA medium

In present work, ultraviolet (UV) rays were used as a physical mutagen that is potent inducers of mutations in microbial strains. Previous studies testified that physical mutagenesis is a cost effective method to produce improved enzyme producers that may be used for commercial production of the enzymes. The pyrimidines bases of DNA NA are significantly affected by UV radiations; their absorption results in GC-AT GC transitions that produce thymine dimmers and alteration in DNA structure which stops the replication process. As a result massive range of genetic variations are produced (Shin in et al., 1993). Ultraviolet radiations (200- 300 nm) are absorbed by nucleic acids specifically at 254 nm. UV rays produce alteration in DNA molecule by breaking and alteration in DNA strands and dimers nitrogen bases (Conn and Stumph, 1994). Physical mutagenesis utagenesis was carried out in dark because UV rays mutants are sensitive to light and light revert the changes, produce by UV rays due to photolytic reaction. Optimum exposure time for physical mutagenesis was achieved by drawing a 3 log kill curve which evaluates valuates the comparison of various selected exposure times in terms of microbial spore survival and incidence of positive and negative modifications. It (3 log kill

curve) determined the optimum exposure time of mutagens for killing the fungal spores at 37oC.

Fig. 4.2. Effect of Ultraviolet rays to formulate 33-log kill curve for physical mutagenesis

At this level about 90% fall in feasibility was occurred. Kill curve determination revealed that optimum exposure time of UV rays was 200 minutes, named as TS-UV-200. At this optimum exposure time 60.55% spore killing was occurred and 39.45% microbial spores were survived (Fig. 4.2). Higher proportion of rays lessens the rate of positive mutations (Petruccioliet al., 1995) and the number of sustainable colonies. olonies. Present findings are comparable with the Semashko Semashkoet al. (2000) who described that the 40.6% survival rate was attained by UV irradiation at optimum exposure time of glucose oxidase producing mutants strain of Penicillium funiculosum while 57% survival ival rate and 43% conidial killing has also been described by Singh (2006) after UV mutagenesis of Aspergillus niger. Rasulet et al al. (2011) reported that 81.41% killing and 18.58% survival rate was obtained at optimum exposure time (180 minutes) by kill curve determination for higher glucose oxidase activity from Aspergillus niger. Similar results were also reviewed by Park et al. (2002), Khattab and Bazarra (2005), Zia (2007) and Khurshid (2008). Current findings are also in agreement with the findings of Kha Khattab and Bazarra (2005), who documented that number of colonies decreased with the increase in the exposure time while number of resistant colonies rise with the increasing exposure time and then there was a sharp decline found. Khattab and Bazarra used ethyl hyl methane sulfonate (EMS) as chemical mutagen for enhanced production of glucose oxidase. Zia, (2007) investigated the effect of MNNG and EB and found 82% and 76.135% killing of microbial spores respectively using kill curve determination while Witteveen et al. (1990) claimed that the survival rate of microbial spores upto 33 33-78% by studying the enhanced production of glucose oxidase from Aspergillus niger. According to previously reviewed data, optimum survival rates studied for Aspergillus niger and Penicillium variabile mutants isolates with improved Gox production are in range 33 33-78% and 52%, respectively (Semashko (Semashkoet al., 2000).Haqet al. (2014) investigated the improved production of glucose oxidase from Aspergillus niger IIB-31 31 using EMS and nitrous acid as chemical mutagens via random mutagenesis and optimization of media components. They reported that EMS showed more positive response as its positive mutant isolates having a three threefold greater enzyme activity than the w wild strain. Colony restricted growth of Aspergillus niger at various exposure times on PDA plates containing triton X X-100 for physical mutagenesis and chemical mutagenesis are shown in Fig. 4.4 and 4.5 respectively. Among the other accessible strain

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improvement ement methods, the conventional method of mutagenesis and screening is still successfully used. In recent period, it has been consecutively used for the production of industrially significant mutant microbial strains (Haqet (Haq al., 2002).

microbial colonies growth producing yellowish color around the colonies by restricting their size and sporulation (Khattab and Bazaraa, 2005). UV optimum exposure time selected by kill curve determination was 200 minutes comprised of 13 colonies.

Fig. 4.4 Colony restricted growth of Aspergillusniger at various exposure times ontriton X-100containing 100containing PDA plates for physical mutagenesis (a. 0 minute (control), b. 40 minutes, c. 80 minutes, d. 120 minutes, e. 160 minutes, f. 200 minutes, g. 240 minutes and h. 280 minutes of UV rays exposure)

Screening Methods for Selection of Positive Mutants: Mutants After mutagenesis, positive mutant isolates having the ability of improved production of glucose oxidase were isolated and screened using following methods. Plate screening method: The colonies present on the optimum exposure time (for both mutagens) were to be reviewed using colony restrictor, triton X-100. 100. It is a detergent that has a capability to limitss the microbial growth. It produces delay in logarithmic growth phase of microorganism (Sun et al., 2008). It is a synthetic surfactant that also reduces the

When colonies present on UV optimum exposure time were again exposed to PDA plates containing 1% triton X X-100, 5 of them designated as TS-UV-2, 2, TS TS-UV-3, TS-UV-5, TS-UV-6 and TS-UV-77 displayed more growth resistance against triton X-100 100 as compared to the rrest of the others colonies/ as compared to others colonies present (Fig. 4.6). For chemical mutagenesis, 13 colonies present at optimal exposure time were subjected to 1% triton X X-100 containing PDA plates for further screening. Khattab and Bazaraa (2005) (2005), Zia (2007) and Rasulet al. (2011) also used Triton X X-100 as colony restrictor for the selection and screening of highly potential mutant

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colonies having the overproducing capacity of glucose oxide by hindering the growth of low potential Aspergillus niger mutant colonies.

potential against selective marker w were further screened using enzyme diffusion zone test and well test. It was found that, Aspergillus niger TS-UV-22 is best positive mutant colonies

Fig. 4.6 Plate screening of selected dose by UV rays mutagenesis

Fig. 4.8. Selection of positive mutants by selective marker for UV rays mutagenesis

Screening of mutant by Selective Marker: The colonies selected from colony restrictor were then subjected to 22 Deoxy-D-glucose (2-hydroxyl hydroxyl group replaced by hydrogen atom) at 1mg/mL level which is used as selective marker for precise screening and selection. In case of UV mutagenesis, 3 colonies named as TS-UV-2, TS-UV-55 and TS-UV-6 TS displayed stronger growth than others colonies on β-D-glucose β analogues compound containing PDA plates (Fig. 4.8). Glucose oxidase substrate (glucose) analogues compound namely 2-deoxy-D-glucose glucose is employed as a criterion to screen the highly resistant positive mutant isolates (Azin and Noroozi, 2001). Khattab and Bazaraa (2005) also examined growth resistance of EMS and UV mutant on the 2-deoxy-D-glucose 2 and screened the colonies having capability of enhanced glucose oxidase production. The 2-Deoxy--D-glucose that is used selective marker in this study is up taken by the glucose transporters of the cell but it cannot undergo further glycolysis. The cell growth is inhibited because of slow glycolysis and loss of energy production (Pelicanoet al., 2006). There were some colonies that appeared in larger size and darker colour than the other colonies were selected for further screening process. Enzyme Diffusion zone test and Well test for selection: selection These methods are specific for the identification of positive mutants established on enzymatic reaction (Fiedureket (Fiedurek al., 1986, Petruccioliet al., 1995, Park et al., 2000 and Melherbeet Melherbe al., ., 2003). The mutant colonies that displayed greater growth

with increased diffusion zones of glucose oxidase with 1.38 fold increase in enzyme activity as compared to wild strain. While results obtained by enzyme diffusion zone test was re reconformed by employing well test. From well test, it was conformed that Aspergillusniger AspergillusnigerTS-UV-2 mutant colonies have great potential otential for glucose oxidase production with 1.46 fold increase in enzyme activity as compared to wild strain. Well test has some advantages as compared to diffusion zone test i.e. in case of diffusion zone test, there is mixture of microbial spores or colonies onies may be present and reaction mixture may not completely absorbed by the microbial spores due to solid agar medium while in case of well test, only desired microbial colony spores are placed in well and reaction mixture is easily absorbed by the spores in the well. Finally, in the current study results showed that TS TS-UV-2 (Fig. 4.10 and 4.12) have greater capacity to yield glucose oxidase as compared to other mutants and corresponding wild strain (Table 4.1). As previously cited by Fiedurek et al. (1986), enzyme activity was improved from 1.5% to 18% after ultraviolet irradiation of A. niger as compared to wild strain, assessed by diffusion zone test. Semashko Semashkoet al. (2000) adopted a diffusion zone analysis to determine the positive mutants of NMU and UV light. Park et al al. (2000) also select potential mutants glucose oxidase producers by using the enzyme diffusion zone procedure. Khattab and Bazaraa (2005) and Rasulet al. (2011) also exhibited an enzyme diffusion zone test for isolation of screening of potentially otentially overproducing glucose

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oxidase mutants by measuring the size of dark brown zone formed around the colony.

Melherbeet al., 2003, Khattab and Bazaraa, 2005; Rasulet al., 2011 and Haqet al., 2014).

Analytical test: The mutant colonies that shown greater growth potential against glucose analogue 2-Deoxy-D-glucose was further analyzed by analytical test for optical and quantifiable investigation using spectrophotometer. Results shown in Table (4.1) indicated that A. niger mutant colonies namely TS-UV-2 have greater ability to produce glucose oxidase as compared to other mutants colonies and corresponding wild Aspergillus niger that was used as control.

Glucose oxidase production Production of glucose oxidase was carried out from selected mutants colony isolates and wild stains of Aspergillus niger as control using pre-optimized fermentation media using corn steep liquor (CSL) as an economical substrate. Culture cultivation was conducted in triplicate using 2% CSL as a substrate for 36 hrs of incubation at 30°C and 150 rpm.

Fig. 4. 10 Enzyme diffusion zone test for the selection of best mutant of UV rays mutagenesis

Fig. 4.12 Well test for the selection of positive mutant of UV rays mutagenesis (central well represents the control) Table 4.1. Screening analysis of wild and mutant derived strains of Aspergillusniger S.No

Strain

Diffusion Zone test (cm)

Well test (cm)

Enzyme activity/ Analytical test (U/ml)

%age increase in Enzyme activity

1.4 2.05 1.9 1.6

10.34 15.8 11.49 12.1

100 152.8 111.12 117.02

Ultraviolet radiations (UV) Mutagenesis 1. Wild/control 1.3 2. TS-UV-2 1.8 3. TS-UV-5 1.5 4. TS-UV-6 1.6

These selected mutant strains were used for further analysis. In current study, it was found that TS-UV-2 having 152.8% increase in glucose oxidase respectively. A multistagerepeated mutagenization using Aspergillus niger resulted in 30% enhancement in Gox activity (Frederick et al., 1990). According to previously cited literature Aspergillus and Penicillium sp. mutants produced using numerous mutagens having glucose oxidase overproducing ability as compared to parental wild strains (Park et al., 2000, Semashkoet al., 2000,

Glucose oxidase activity of all wild and mutants colonies were shown in Tables (4.2) that clearly described that TS-UV-2 mutant colony have potentially highest enzyme activity corresponding to other mutant isolates and parental Aspergillus niger strain and these positive mutant isolate then named as TS-UV-200, where 200 represents the optimum exposure time of mutagen. Mutant namely TS-UV-200 had been selected in the present study displayed that there was 225.62% increase in enzyme activity as compared to wild strain as shown in tables

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(4.2). Results obtained in present investigation are in accordance with the results of Gromada and Fiedurek (1997), they described the greater than 125% increase in the glucose oxidase production after mutagenization of Aspergillus niger. Recent findings were also compared with the study of Witteveenet al. (1993), they obtained almost similar results after Aspergillus niger mutagenesis. Park et al. (2000) treated recombinant S. cerevisiae containing Gox gene with UV irradiation and presented 80% increase in enzyme activity when compared to its natural microbial strain. Table 4.2 Glucose oxidase activity of UV mutant strains of Aspergillus niger produced by liquid stat fermentation Type

Enzyme activity %age increase in (U ml-1) Enzyme activity Ultraviolet radiations (UV) Mutagenesis Wild/control 11.825 100 TS-UV-2 26.68 225.62 TS-UV-5 17.42 147.32 TS-UV-6 20.32 171.84

Khattab and Bazaraa (2005) found that UV rays and ethyl methane was most effective mutagens and all fusants showed increase in activity from 285.5 to 394.2% as compared to the original strain. Singh (2006) generated a mutant of Aspergillus niger by repetitive UV irradiation from Aspergillus niger and exhibited that the maximum frequency of positive isolates (25.5%) was achieved with 149% higher rate (3.43 fold increase) of activity than that of parental Aspergillusniger stain in LSF using sugarcane molasses as substrate. Gothoskar and Dharmadhikari, (2013) reported that EB as chemical mutagen is more affective for improved production of glucose oxidase from Aspergillus niger as compared to UV rays. UV rays produced 47% increase while EB produced 54% increase in glucose oxidase production from mutant isolates of A.niger over the parental strain.Glucose oxidase producing capability of Aspergillus niger and Penicillium funiculosum was significantly amplified after mutagenesis as described in previously cited literature (Semashkoet al., 2000; Zia et al., 2012b and Haqet al., 2014). According to the current findings, it is concluded that ultraviolet rays can be resourcefully employed for the mutagenesis of Aspergillus niger for improved glucose oxidase production…..UV rays are potent mutagen rays because it displayed significantly better response. It is also found that mutant derived strains of Aspergillus niger designated as TSUV-200 have a noteworthy capability for enhanced glucose oxidase production as compared to its wild strain and it could be valuable for further advanceresearch investigations and commercial scale production of glucose oxidase. Present results are in agreement with Semashkoet al. (2000), who documented that UV radiation and NMU can be proficiently employed for the mutagenesis of penicillium funiculosum to get augmented glucose oxidase production and also revealed that the mutagenic effectiveness of NMU was better than that of UV radiations. Conclusion Based on the above results, it is determined that UV rays can be efficiently used to produce positive mutations in A. niger for the hyper production of GOX. It is also evaluated that mutant derived Aspergillus niger strain namely TS-VU200has a highly remarkable capability for GOX production by

consuming pre-optimized media nutrients and cultivation conditions corresponding to its parent strain A. niger and it could be valuable for further advance investigation and commercial scale production of glucose oxidase (GOX).

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