CHAPTER 4 EXPERIMENTAL INVESTIGATION

51 CHAPTER 4 EXPERIMENTAL INVESTIGATION

52 MATERIALS CHEMICALS Potato Dextrose Agar (PDA), skim milk powder, nutrient agar medium, Sabouraud broth, Luria broth, Czapeck Dox broth were purchased from Himedia Laboratories Pvt. Ltd., Mumbai, India. Dipotassium hydrogen phosphate, potassium dihydrogen phosphate, zinc chloride, magnesium sulphate, calcium chloride, manganese sulphate, manganese chloride,ferric chloride, sodium carbonate, sodium chloride, potassium nitrate, ammonium sulphate, glucose, fructose, mannitol, galactose, dextrose, lactose, maltose, tyrosine, BSA, cellulose, HCl, NaOH, acetic acid, ammonium nitrate, sodium nitrate, peptone, yeast extract, beef extract, tryptone, Tween-20, Tween-80, Triton X-100, methanol, ethanol, amyl alcohol, EMS, EtBr, Tris-HCl, starch, casein, TCA, copper sulphate, FC reagent were purchased from Himedia Laboratories Pvt. Ltd, Qualigens Pvt. Std., Sdfine Chemicals Pvt. Ltd and Merck Laboraties Pvt. Ltd. Sephadex G-100 was purchased from Sigma - Aldrich, USA. All other chemicals used were of analytical grade. AGROINDUSTRIAL BASED PRODUCTS Soybean meal, wheat bran, wheat rawa, wheat grass, flours of millet, green gram husk, black gram, corn, oat, rice, wheat, chickpea meal, corn meal, lobia white meal, ground nut meal were procured from local supermarket. Molasses and cheese whey were supplied by local industries of Andhra Pradesh, India. Whatman No.1 filter papers.

53 INSTRUMENTS: Digital ph meter (Elico Pvt. Ltd.) with a Calomel glass electrode assembly, Conductivity meter (Elico Pvt. Ltd.), UV/Visible Spectrophotometer ( Schimatz UV-1700). METHODS 4.1 COLLECTION OF SOIL Soil contaminated with abattoir waste (Contains bones, patches, blood and horns) was collected from slaughterhouse located in Tirupati, Chittoor district, Andhra Pradesh, India. Soil samples were collected from 0-3 inches with increment of 1 inch and described as test soil samples. Similarly, soil sample used as control which was collected from an area devoid of butchering activities. Both soil samples were placed in sterile polythene bags and were transported to the laboratory and then air dried at a temperature of 30 to 35 o C. Both the soil samples were sieved through <2 mm sieves. The fractions <2 mm were stored in a refrigerator at 4 o C for further studies. 4.2 PHYSICO-CHEMICAL PROPERTIES OF SOIL With the aim of studying the properties of both the soils, standard procedures of American Public Health Association (APHA, 2000) were used to study the physico-chemical properties of both test and control soils. The parameters studied include ph, EC, WHC, organic carbon, phosphate and potassium contents. WHC of soil samples was evaluated by finding amount of distilled water added to both soil samples to get saturation point which was equal to 100% WHC. From this, sixty percent WHC of both test and control soils were measured. Soil ph was measured at a ratio of 1:1.25 soil to water with ph meter. Organic carbon content in soil samples was estimated by the Walkley and Black method (Walkley and Black, 1934). According to this method, One gram of soil was taken in a 500 ml. Erlenmeyer

54 flask. Then 10 ml of 1N potassium dichromate solution was added followed by 20 ml of sulphuric acid and allowed the mixture to stand for 30. The prepared solution was diluted with 200 ml of deionized method. Immediately, 10 ml. phosphoric acid, 0.2g ammonium fluoride, and 10 drops diphenylamine indicator were added and titrated against 0.5N ferrous ammonium sulfate solution until the color changed from dull green to a turbid blue and the end point was brilliant green. A blank is prepared and titrated in the same manner. Experiment was performed in triplicate for quality assessment. Another factor, EC of soil samples with addition of 100 ml distilled water to one gram soil samples was measured by conductivity meter. Phosphorous content of soil samples was also quantified. For the determination of phosphorous content of soil, Soil sample of 1.5 g was taken in a 500-ml Erlenmeyer flask and 150 ml of 0.002N sulfuric acid was added. Contents are were shaken well for 30 minutes then solution was filtered. Five ml of filtrate was transferred to test tube and one ml of sulfuric- molybdate and ascorbic acid solution was added then allowed it for 15 minutes. Developed blue color was measured at 820 ηm. 4.3 ENZYME ACTIVITIES IN SOIL WITH/WITHOUT ABATTOIR WASTE DISCHARGES 4.3.1 Soil incubation studies Five grams of soil with/without abattoir waste deposition were taken in test tubes (25 x 200 mm) for determining the soil enzyme activities. Water was added to soil to get 60% WHC. The same WHC was maintained throughout incubation at room temperature (30±2 0 C) by replacing water loss during incubation. Test and control soil samples were withdrawn after 0, 7, 14, and 21 days on incubation to determine the soil enzyme activities.

55 4.3.2 Determination of soil protease activity Protease activities of both soil samples were determined by placing five grams of each soil sample in boiling test tubes with 60% WHC at 30±2 0 C. Triplicates of both test and control soil samples were drawn after 0, 7, 14, and 21 days of incubation to calculate protease activity by the method of Speir and Ross (1975). Then 10ml of 2% w/v casein in 0.1 M Tris buffer of ph 7.5 was added and were incubated for one day. To these samples, 4ml of 17.5% TCA was added after incubation and then the suspension was filtered through Whatman No.1 filter paper. Protein content is determined in the filtrate (Lowry et al., 1951). Finally, protease activity was expressed in terms of liberating micrograms of tyrosine per g of soil per hour. 4.3.3 Estimation of soil amylase activity Amylase activities of both soil samples were determined by placing five grams of each soil sample in boiling test tubes with 60% WHC at 30±2 0 C. Triplicates of both test and control soil samples were drawn at regular interval of week days to determine amylase activity (Pancholy and Rice, 1973; Tu, 1982). After incubation, soil samples were transferred to 250 ml Erlenmeyer flasks. After 15 min, 6 ml of 0.2 M acetate-phosphate buffer (ph 5.5) containing 1% starch was added to soil samples and flasks were plugged with cotton and held for 24 h at 30±2 0 C for amylase activity. After desired incubation, soil extracts were passed through Whatman No.1 filter paper and maltose content (amylase) in the filtrate was assayed by dinitrosalicyclic acid reagent. 4.4 ENUMERATION OF MICROORGANISMS FROM SOIL 4.4.1 Isolation of fungi The fungal population enumerated from test and control soil by serial dilution and spread plate techniques. The medium used for growth of the molds was Potato

56 Dextrose Agar (PDA). The medium was sterilized by autoclaving at 15 psi for 15 min at 121 0 C. Then, the sterilized medium was cooled to 40 to 50 0 C. Approximately 20 ml of warm medium was poured into the sterile petriplates and allowed to solidify at room temperature. The soil samples were suspended in water by vigorous mixing and were serially diluted. Appropriate dilutions (10-3 to 10-5 ) were plated into PDA plates. Then, the plates were incubated at temperature of 30±2 0 C for seven days. After incubation, colonies developed on the solid medium were repeatedly streaked on PDA plates till the well separated and isolated colonies were obtained. These cultures were periodically subcultured and maintained on PDA slants at 4 0 C for further studies. 4.4.2 Isolation of bacteria Nutrient agar medium was used for the isolation of bacteria from both the test and control soils by using techniques such as serial dilution, spread plate and streak plate methods. Dilutions of 10-6 to 10-9 were used for bacterial isolation from soil samples and agar plates were incubated at room temperature for 24 h and developed bacterial colonies were counted in both test and control soils. 4.4.3 Morphology of fungal isolates Sizes of isolated fungi, color of colonies and shapes were noted with naked eyes. The structure and different dimensions of high yield isolated fungal strain were examined on freshly prepared wet mounts by light microscopy of exponentially growing liquid culture. The fungal isolates were further identified based on their macroscopic and microscopic characteristics. 4.5 SCREENING OF FUNGAL ISOLATES FOR ACID PROTEASE PRODUCTION In order to select the suitable strains for acid protease production, fungal isolates from soil were screened for proteolytic activity. Separate screening

57 procedures (Plate method and SmF) were carried out for the selection of strains for protease. 4.5.1 Plate screening method For this method, casein - agar medium was prepared by dissolving one gram of casein and two grams of agar in 100 ml of distilled water and sterilized in autoclave at 15 psi for 15 min. Then the sterilized mixture, approximately 20 ml, was poured into the sterile petriplates and allowed to solidify at room temperature. An agar well of 0.5 mm diameter was made by borer in the center of casein - agar plate and then it was filled with 100 µl of spore suspension of Aspergillus spp. The plates were incubated at temperature of 30±2 0 C for seven days. Then the formation of zone of casein clearance was measured at the end of 72 hour. 4.5.2 Submerged fermentation The composition of basal media such as Luria broth, Sabouraud broth, and a referred medium (Haq et al., 2006) were mentioned in Table 4.5.2.1-3. Total of five fungal isolates (Isolate 1 5) were screened for protease production among five different media viz. Luria broth, Sabouraud broth, diluted molasses (Dilution factor: 5), dairy industry effluents, and a referred medium (Haq et al., 2006). All the above said media were steam sterilized. The fungal inoculum was prepared by addition of 10ml of 0.1% Triton X-100 solution to the 7 th day old slant and was shaken well to obtain homogeneous spore suspension. Table 4.5.2.1 Composition of Luria broth (ph 7.0±0.2) Ingredients Quantity Peptic digest 5( g l -1 ) Beef extract 3( g l -1 ) Lactose 5( g l -1 ) Distilled water 1000 ml

58 Table 4.5.2.2 Composition of Sabouraud broth (ph 5.6±0.2) Ingredients Quantity peptone 10( g l -1 ) Glucose 40( g l -1 ) Distilled water 1000 ml Table 4.5.2.3 Referred medium composition (Haq et al., 2006) Ingredients (ph 7.0) Soybean meal Glucose Polypeptone Yeast extract KH 2 PO 4 NaCl Distilled water Quantity 2.00 g 2.00 g 1.00 g 0.20 g 0.20 g 0.20 g 100 ml Each flask was inoculated with 2 ml of spore suspension of each fungal strain. Flasks were agitated with 150 rpm at 30±2 0 C for seven days. At the end of fermentation, the contents of flasks were filtered and then analyzed for extracellular acid protease activity. 4.6 ANALYTICAL TECHNIQUES Selection of suitable fungal strain was depending on the it s growth and the secretion of large amounts of enzyme. Hence, the protease enzyme was assayed according the method of Sinha and Sinha (2009). The procedure followed for all the

59 enzyme assays throughout this study was same. In addition to enzyme activity, fungal biomass and protein content were estimated. 4.6.1 Protease activity assay For protease assay, three ml of 0.65% w/v casein solution in 0.05M Tris - HCl (ph 6.5), 0.5 ml of the crude enzyme solution was added and the mixture was incubated for 10 min at 32±2 o C. Then three ml of 0.44M TriCholoro Acetic acid (TCA) was admixed and the precipitate was removed by filtration through Whatman No.1 filter medium, after standing for 30 min. Then, one ml of the filtrate was taken in a test tube, three ml of 0.55M Na 2 CO 3 and one ml FC reagent (diluted 2 times) were added. The color was developed for 30 min and absorbance was measured at 660 ηm using spectrophotometer. The control was run in a similar manner except that TCA was added before the addition of enzyme solution. A reagent blank was prepared by adding three ml of Na 2 CO 3 and 1 ml of FC reagent to one ml distilled water (Sinha and Sinha, 2009). 4.6.2 Protein estimation Protein content in the fungal filtrate was determined by using BSA as standard (Lowry et al., 1951). Alkaline solution of 5.5 ml was added to protein solution, mixed well and was incubated at room temperature for 10 minutes. Double diluted FC reagent was added to tube was allowed to stand for 30 minutes and the blue colored formed was measured at 660 ηm. Blank solution was prepared without protein. From the standard graph, protein concentration was estimated. 4.6.3 Mycelial dry weight For the determination of fungal dry weight, the fermented broth was filtered using preweighed Whatman No.1 filter paper. It was washed with water thrice and

60 then dried at 105 0 C over night in a hot air oven and weighed using an electronic balance (Haq et al., 2006). 4.7 PRODUCTION AND OPTIMIZATION OF ACID PROTEASE THROUGH SUBMERGED FERMENTATION To obtain higher yield of acid protease, factors influencing the production by Aspergillus spp. were studied in SmF through conventional method. In this methodology, the factors influencing the production were studied by examining one factor at a time, keeping the other factors constant. Once the optimization has been done with respect to a factor it was incorporated in the experiment for the optimization of the next factor. 4.7.1 Submerged fermentation For SmF, diluted molasses was used as the main carbon source for enzyme production. The initial ph of diluted molasses was 5.00. Fermentation was performed in 250 ml Erlenmeyer flask containing 50 ml of sterilized diluted molasses. The medium was cooled to room temperature and was inoculated with 2ml of fungal spore suspension. The flasks after inoculation were placed in the orbital shaker rotating at 150 rpm and at 30±2 0 C for 7 days. Later, the contents of the flasks were filtered using Whatman No.1 filter paper and the filtrate was used for the assay of protease enzyme. One unit of protease (U) can be defined as the amount of enzyme that releases 1 μ mole tyrosine/min under the reaction conditions. Protease activity was represented as Protease unit per ml of enzyme (U ml -1 ). All the experiments were conducted in triplicates in 250 ml Erlenmeyer flasks and the results were the mean and standard deviation of three trials.

61 4.7.2 Protease production on combinations of cheese whey and molasses Cheese whey was rich in lactose and protein amount. To improve the production medium for higher protease activity, fermentation was performed with various combinations of cheese whey and diluted molasses as main cheap sources. Fermentation was detailed in section 4.7.1. Optimum combination of molasses and cheese whey was fixed as main cheap sources in further experiments. 4.7.3 Effect of incubation time The incubation time was determined by carrying out the fermentation for 3 to 7 days with medium consisting of equal volumes of molasses and cheese whey with initial ph of 6.2 at room temperature. At regular intervals of 24 h the samples were analyzed. 4.7.4 Effect of inoculum size on protease production in submerged fermentation The optimum fungal spore density was investigating by performing SmF with the size range of inoculum from 0.5 to 20 % v/v and protease enzyme was assayed after 120 h of incubation at room temperature. 4.7.5 Effect of temperature on protease activity Temperature plays an important role in bioprocess as it affects the growth of microorganisms. Hence, temperature for growth of Aspergillus spp. and the production of protease was optimized by incubating shake flasks at various temperatures viz. 25±2 0 C, 32±2 0 C and 55±2 0 C. The enzyme activity was determined at the end of fifth day of incubation. 4.7.6 Effect of ph on protease production To determine the impact of ph the medium was prepared by using 1:1 ratio of diluted molasses and whey with different ph range of 3 to 9.

62 4.7.7 Effect of nitrogen source on acid protease production The production medium was supplemented with 1.0% w/v of various inorganic, organic and agroindustrial based nitrogen sources viz. potassium nitrate, sodium nitrate, ammonium nitrate, casein, beef extract, peptone, yeast extract, and soybean meal. The nitrogen content by mass percent of beef extract, potassium nitrate, sodium nitrate and yeast extract was in the range of 10 to 16%. Soybean meal has 48% crude protein and that of ammonium nitrate was 35%. Casein has 2% of nitrogen by weight. Further investigation was performed with different concentrations of best protein source in the range of 0.5 to 6.0 percent. 4.7.8 Effect of carbon source on acid protease production Acid protease production was enhanced with the supplementation of 1.0% w/v various carbon sources viz. monosaccharides (glucose and mannitol), disaccharides (sucrose and lactose) and polysaccharides (cellulose and starch). The mass percent of carbon for above mentioned monosaccharides and disaccharides was 41±1%. Further, the effect of best carbon source was investigated with its concentration range of 0.5 to 3.0 percentages. 4.7.9 Effect of lignocellulosic flours on protease production In order to design cost effective fermentation, the basal medium was amended with 1% w/v of different flours of cereals like rice, wheat, oat, corn, millet, pearl millet, black gram, green gram for protease production by Aspergillus spp. 4.7.10 Effect of salts on protease production To further increase the protein amount in fermentation, the experiment was performed with the addition of different salts (0.05% w/v) like sodium chloride, calcium chloride, magnesium sulphate, manganese chloride, zinc sulphate, potassium

63 chloride and ferric chloride to fermentation medium. Control was the medium without addition of above mentioned salts. 4.7.11 Effect of surfactants on protease production Surfactant such as Tween-20, Tween-80, and Triton X-100 at a level of 100 µl was added in order to investigate the effect of surfactants on acid protease production. 4.8 PRODUCTION AND OPTIMIZATION OF ACID PROTEASE UNDER SOLID STATE FERMENTATION Various nutritional, physical and chemical factors influencing the production of acid protease by Aspergillus spp. under SSF were studied by using conventional approach. 4.8.1 Solid state fermentation The wheat based substrates (10 g) viz. wheat bran, wheat rawa, wheat grass powder, and wheat flour were used as main carbon substrates in the present study. It was moistened with 20 ml of salt mineral solution (ph 6.0) comprised (g l -1 ): KH 2 PO 4 1, K 2 HPO 4 2, Mg 2 SO 4 1, CaCl 2 0.1 and ZnSO 4 0.01 and then sterilized at 15 psi for 15 min. Then each flask was inoculated with 20% v/w spore suspension of Aspergillus spp. and incubated at room temperature for five days. Crude protease was extracted by the addition of 50 ml of various solvents viz. water, and 50% aqueous mixtures of various solvents such as ethanol, methanol and amyl alcohol to fermented solids and then, contents were mixed at speed 150 rpm at room temperature for an hour. Later, the contents of the flasks were filtered using Whatman No.1 filter paper and the filtrate was used for the assay of crude protease and protein as mentioned in section 4.6. A unit of protease activity (U) was defined as the amount of enzyme liberating micrograms of tyrosine per ml of enzyme per min of incubation time. The protease activity was reported enzyme unit per gram of solid substrate, (U g -1 ). All the

64 solid state fermentations were conducted in triplicate in 250 ml Erlenmeyer flasks and the results were the mean and standard deviation of three samples. Wheat rawa and distilled water were fixed as main substrate and extraction solvent in further experiments. 4.8.2 Effect of process parameters for protease production The effect of various parameters on enzyme production was studied. The impact of fungal inoculum density was performed by inoculating production media with a range of 0.5 5.0 ml of spore suspension. The effects of temperature and incubation time on protease production were studied by conducting the bioprocess at temperature range 25-40 0 C and incubation period range of 3 9 days. To investigate the influence of initial moisture content, the step was performed with the range of initial moisture content (50 to 90 % v/w). The influence of initial ph of medium was conducted on enzyme production with ph range 3 11. 4.8.3 Effect of various carbon and nitrogen sources To enhance the acid protease production, wheat rawa was supplemented with one gram of various carbon sources viz. glucose, galactose, fructose, dextrose, sucrose, lactose, cellulose, starch. Further fermentation medium was designed with one gram (10% w/w) of various nitrogen sources such as soybean meal, corn meal, skim milk powder, lobia white meal, chickpea meal, yeast extract, tryptone, peptone, sodium nitrate, potassium nitrate and ammonium sulphate. To utilize the waste in order to lower the cost of fermentation, the study was performed using one gram of fruit and vegetable waste such as pomegranate fruit peel, mango fruit peel, carrot peel and potato peel instead of fructose.

65 4.8.4 Polynomial models for protease activity The experimental data on moisture content, inoculum size, incubation period, temperature and ph were used to formulate the following polynomial models using MATLAB (R2008b) 7.7.0471 (Jyoti and Rita, 2006): PA (ph) = a1 (ph) 5 + a2 (ph) 4 + a3 (ph) 3 + a4 (ph) 2 +a5 (ph) + a6 --- (4.8.1) PA (T) = b1 (T) 3 + b2 (T) 2 +b3 (T) + b4 --- (4.8.2) PA (t) = c1 (t) 3 + c2 (t) 2 + c3 (t) + c4 --- (4.8.3) PA (I) = d1 (I 5 ) + d2 (I 4 ) + d3 (I 3 ) +d4 (I 2 ) + d5 (I) + d6 --- (4.8.4) PA (M) = e1 (M 2 ) + e2 (M) + e3 --- (4.8.5) 4.9 STATISTICAL APPROACH FOR OPTIMIZATION OF ACID PROTEASE PRODUCTION BY ASPERGILLUS SPP. A suitable medium was also formulated through statistical optimization methodology since it has various advantages of being rapid and reliable in short listing of nutrients at varying concentrations leading to significant reduction in the total number of experiments (Akolkar et al., 2009). 4.9.1 Fermentation In the present approach, the production medium was moistened with 60% v/w salt mineral solution contained (g l -1 ) K 2 HPO 4 1, KH 2 PO 4 3, Mg 2 SO 4 1, CaCl 2 0.1 and ZnSO 4 0.01 in 250ml Erlenmeyer flasks. The sterilized and cooled medium was inoculated with 10% v/w fungal spore suspension. Then the contents of the flasks were mixed thoroughly and incubated at 32±2 0 C for five days. Crude protease was extracted by adding 50 ml of distilled water to fermented solids and the contents were mixed with 150 rpm at 20±2 0 C for one hour. Later, the contents of the flasks were filtered using Whatman No.1 filter paper and the filtrate was used for the assay of

66 protease enzyme. Fermentation was performed in three trials and the response was the mean of three trials. 4.9.2 Statistical optimization Two sequential steps of statistical approach such as PBD and RSM were performed to design the optimized production medium for acid protease from Aspergillus spp. For this study, air dried potato peel was cut into pieces of size of one centimeter approximately. 4.9.2.1 Plackett - Burman design for screening of media components In this approach, seven solid substrates including carbon and nitrogen sources were screened for acid protease production in eight combinations with two test levels and experiments were performed according to design matrix (Table 4.9.2.1.1 and 5.4.1.1). Higher and lower levels of variables were based on the results of SSF through OVAT which were detailed in section 4.8. Table 4.9.2.1 High and low levels of solid substrates S. No. Substrates High(+) (g) Low(-) (g) 1 Wheat bran 10.0 5.0 2 Wheat rawa 10.0 5.0 3 Groundnut meal 1.0 0.5 4 Soybean meal 5.0 2.5 5 Corn flour 5.0 2.5 6 Dried potato peel 5.0 2.5 7 Rice flour 1.0 0.5

67 This statistical design was described by equation (3.4.2.1). Based on the main effects and p-values, wheat bran, soybean meal and dried potato peel have profound effect on the protease enzyme production and were further optimized by RSM. 4.9.2.2 Response surface methodology RSM is the process of adjusting variables towards the optimum response of fermentation. BBD was adopted to optimize the levels of three identified substrates. The individual and interactive effects were noticed at three levels (-1, 0, +1) of solid substrates (Table 4.9.2.2). Table 4.9.2.2 Three levels of substrates with actual and coded values used in RSM Substrates(g) Levels -1 0 +1 Wheat bran 5.0 7.5 10.0 Soybean meal 2.5 3.75 5.0 Dried potato peel 2.5 3.75 5.0 Detailed design was given in Table 5.4.2.1. A second order polynomial equation, fitted to data by multiple regression procedure, resulted in quadratic model and it was given by equation 3.4.2.2. 4.10 STRAIN IMPROVEMENT FOR ENHANCED PRODUCTION OF ACID PROTEASE To enhance the acid protease production, wild Aspergillus spp. was mutated genetically by conventional approach which was performed through physical and chemical methods. Further, the production of enzyme was investigated by both wild type and UV treated physical and EMS treated chemical mutant strains in both SmF and SSF with optimized media designed through 4.6 and 4.7 sections.

68 4.10.1 Physical mutation by UV irradiation The fungal cultures Aspergillus spp. was subjected to physical mutation (Dutta and Banerjee, 2006). Various serial dilutions of fungal suspension were prepared and dilution 10-8, 10-9 and 10-10 were distributed into sterilized petriplates (2 ml in each plate). These were exposed to UV radiations for varying time periods ranging from 5 to 120 min in UV chamber (UVC 260ηm) keeping the distance of UV source at 15 cm. UV treated fungal suspensions of 0.1 ml was inoculated in 25 ml petriplate containing 1% casein and 2% agar. 4.10.2 Chemical mutation by EMS According to the procedures of Vasudeo et al. (2011) and Nadeem et al. (2010), chemical mutation was performed. In order to determine the effective EMS concentration, eight test tubes with two ml of cell suspension (10 6 ) each were taken and one of them was kept aside as control and rest of them were incubated with concentrations of EMS ranges from 2 to 10 mg per two ml of fungal suspension for 30 and 60 min time periods at room temperature (32±2 0 C). After required period of treatment, the cells were centrifuged at 3000 rpm for 10 min at 4 0 C, washed with sterilized phosphate buffer (ph 7.0) twice. Chemical treated fungal suspension of 0.1 ml was poured into sterilized petriplates containing Czapeck Dox agar medium. Morphological changes of fungal suspension were continuously monitored. Significant morphological changes were found in spore suspension treated with 6 mg of EMS for one hour incubation and then suspension was diluted. The plates having less than or equal to 1.00% of survival rate (approximately) over the control (without treatment) were selected for screening of hyper-proteolytic mutants on (1% w/v) casein (2% w/v ) agar plates.

69 4.11 PRODUCTION AND COMPARISON OF ACID PROTEASE BY WILD TYPE AND MUTANT STRAINS 4.11.1 Submerged fermentation Acid protease production was carried out with both wild and mutant strains in SmF with molasses and cheese whey as the substrates in fermentation medium as explained in section 4.7. 4.11.2 Solid state fermentation Batch SSF was performed with optimized medium as detailed the above section (4.9). The medium was moistened with 60% salt mineral solution. Later the flasks were autoclaved, inoculated and incubated at room temp for five days. Then, protease was extracted with 50ml distilled water from the fermented solids and the contents were filtered with Whatman No.1 filter paper. The culture filtrate was used for enzyme assay. 4.12 PURIFICATION OF ACID PROTEASE Extracellular acid protease from chemical mutant of Aspergillus spp was produced by SSF under optimum conditions as mentioned in section 4.9. The culture supernatant was obtained by two sequential unit operations as filtration and centrifugation. Further, the enzyme was purified by ammonium sulphate precipitate followed by dialysis and gel filtration. All the purification steps were carried out at temperature 0 to 4 0 C. After each step in the purification procedure, specific activities (U mg -1 protein) were determined. Protocols of acid protease and protein estimation were detailed in section 4.6. 4.12.1 Precipitation of acid protease by Ammonium sulphate Enzyme acid protease was recovered from the filtrate by salt precipitation. For this, ammonium sulphate was added slowly to the 25 ml of crude enzyme source with

70 constant stirring. Saturation table of ammonium sulphate was presented in Appendix A. The concentration of salt was increased in 10% increment from 20% to 90% saturation in order to precipitate the enzyme (Kumar et al., 2008). A total amount of 453 g l -1 of ammonium sulphate was added to precipitate protein of crude enzyme source. The resulted enzyme precipitate was separated by centrifugation at 3000 rpm for 30 min at 4 0 C. The solid pellet was dissolved in 50 mm Tris-HCl (ph 5.5) and kept in refrigerator for further purification. 4.12.2 Dialysis The enzyme solution to be desalted was taken inside a dialysis bag (Nitrocellulose membrane with cutoff < 10 KDA) and the two ends were secured tightly to prevent leakage. The bag was now suspended in a large vessel containing about 100 fold excess of 50 mm Tris-HCl buffer (ph 5.5) and the contents were kept stirred in cold. Salt molecules pass freely and get diluted by the large volume of fluid in the external medium. During the course of dialysis the buffer was frequently changed with the fresh lot until no traces of ammonium were found in the buffer upon testing with Nessler s reagent. Nearly process time of 24 h was required to remove the salt completely from the crude enzyme source. The dialyzed fraction was considered as ammonium sulphate- precipitated fraction. 4.12.3 Gel filtration chromatography The dialysed and concentrated fraction was subjected to gel filtration on Sephadex G-100 column. Five grams of Sephadex G-100 was suspended in 100 ml of 50mM Tris-HCl buffer (ph 5.5) and kept for swelling overnight with intermittent stirring at shorter intervals to prevent formation of lumps. The swelling gel bead solution was poured into a column tube (2.0 x 15 cm) which was previously inserted with glass wool at the bottom. The gel beads were allowed to settle gently without

71 trapping of air bubbles. In this fashion, the prepared column was pre-equilibrated with 0.05 M Tris-HCl buffer (ph 5.5). The dialyzed and concentrated fraction (Ammonium sulphate precipitated fraction) was loaded on Sephadex G-100 column. The loaded column was eluted with the same buffer. One ml fractions of eluent were collected in test tubes and were maintained at 4 0 C. The optical densities of collected fractions were read at 280 ηm in a spectrophotometer for protein content. Proteins in the sample were resolved into peaks on the column. One ml fractions corresponding to each protein peak was pooled together. 4.12.4 Electrophoresis (SDS-PAGE) Sodium dodecyl sulphate Polyacyalmide gel electrophoresis (SDS-PAGE) as described by Laemmli (1970), is a frequently applied method for the determination of the purity of the enzyme. The enzyme preparation was denatured by boiling in the presence of 1% SDS and 1% 2-mercaptoethanol and subjected to SDS-PAGE on slab gel with 1% stacking gel (ph 6.8) over layered on 12% separating gel (ph 8.8). Stock solution of acrylamide (30 g), N, N-methylene -bis- acrylamide was prepared. Separating gel was prepared with acryalmide solution, 1.5 M Tris base (ph 8.8), 0.1 percent of SDS and 3.3 ml of distilled water. It was chemically polarized with 0.05% w/v APS and 0.05% v/v TEMED. The solution was cast into slabs and was over layered with butanol to exclude contact with air. The stacking gel containing 4% w/v acryalmide, 1.0 M Tris-HCl (ph 6.8), 0.01% SDS, 0.05% w/v APS and 0.05% v/v TEMED was over layered on separating gel. Samples (50-200 µg) were mixed with an equal volume of sample buffer having 0.0625 M Tris-HCl (ph 6.8), 10% v/v glycerol, 5% 2-mercaptoethanol, 2% SDS and 0.002% bromophenol blue and heated in a boiling water bath for 5 min. After cooling samples were loaded into the wells.

72 The samples were stacked at 50 V and run at 100 V about 6 hours using 0.025 M Tris- HCl, 0.192 M glycine buffer (ph 8.3) containing 0.1% SDS as the electrode buffer. 4.13 CHARACTERIZATION OF PURIFIED ACID PROTEASE The properties of purified extracellular acid proteases of Aspergillus spp. were studied and the enzymes were characterized. Two approaches were used i.e. conventional method and RSM. Experiments were done in triplicates. 4.13.1 CONVENTIONAL APPROACH Conventional method is easily adopted to characterize acid protease. This method allows the variation of one parameter while fixing the remaining variables which influence the enzyme specific activity. 4.13.1.1 Effect of incubation time The effect of enzyme incubation time with substrate (0.65% w/v) on the enzymatic activity of acid protease was studied by measuring caseinolysis in buffer Tris-HCl (ph 6.0). Reactions were carried out at 30±2 C for reaction time range of 10 to 50 min. Control was taken as ten minutes of reaction time of casein with protease source. 4.13.1.2 Effect of ph on activity The effect of ph on the enzymatic activity of protease was studied by measuring caseinolysis in buffers (0.05 M) with different ph. The buffers used were Tris-HCl (ph 3.0 7.0) and phosphate buffer (ph 9.0-11.0). Enzymatic reactions were carried out at room temperature for 20 min with casein (0.65% w/v). Initial ph of reaction mixture of six was the control for the determination of impact of various ph on the relative activity of purified protease.

73 4.13.1.3 Effect of temperature on activity Influence of temperature on the activity of column purified enzyme was tested in temperature range of 20 to 50 0 C. In this study, the substrate, casein (ph 5.00), was incubated with protease for 20 minutes. Temperature of 30 0 C was used as the control for the effect of different temperatures on enzyme activity. 4.13.1.4 Effect of surfactants on activity The effects of surfactants on the activity of the enzymes were studied. The enzymes were incubated with various surfactants at 1% concentration for 20 min at 39±1 0 C, and the residual activities were determined. Activity of the controls not containing any surfactant was also determined. 4.13.1.5 Effect of metal ions The effect of various metal ions (Ca 2+, Mg 2+, Fe 2+, Mn 2+, Zn 2+, and Cu 2+ ) on the enzyme activity was investigated. The enzymes were incubated with various metal ion sources (5 mm) in 50 mm Tris-HCl buffer (ph 5.0) at 39±1 C for 20 min and the enzyme activity was determined. The enzyme activity in buffer without any of these metal ion sources (control) was also determined. 4.13.1.6 Effect of casein concentration To optimize the casein concentration for acid protease activity, the effect of various concentrations of casein (0.2 3.0% w/v) was performed. The acid protease enzymes were incubated with different amounts of substrate in 50 mm Tris-HCl buffer (ph 5.0) for reaction time of 20 min at 35±2 C. The enzyme activity in buffer with casein concentration of 0.65% w/v was taken as control. 4.13.2 Response surface methodology With the basic knowledge of purification steps of acid protease by conventional method, the accuracy in optimization of parameters such as incubation

74 time, ph and temperature was further investigated by one of the statistical approaches, CCCD. The optimum casein concentration (2.2% w/v) determined by conventional method and the purified enzyme load of 0.2 ml (Specific activity: 104.46 U/mg) were used in the present study. The variables that would affect the enzyme activity were studied at five different levels (Table 4.13.2). Table 4.13.2 Testing levels of parameters in statistical methodology Variable Levels -1.68-1 0 1 1.68 Ph 3 4 5 6 7 Temperature, 0 C 20 30 40 50 60 Incubation time, min 10 15 20 25 30 The detailed design was listed in Table 5.7.2.1 4.14 KINETICS OF SUBSTRATE INHIBITION ON ACID PROTEASE ACTIVITY Protease assay was performed with casein concentration range (0.2 to 1.8%) in order to study the effect of casein on reaction velocity of crude protease enzyme. The following model equations were taken from Shuler and Kargi (2003) and Reed et al. (2010). The reaction scheme for uncompetitive substrate inhibition is E K 1 k2 S E. S E + S P K N S. E. S 4 k E S P Fig. 4.14 A reaction diagram for substrate inhibition

75 With the definitions of [ S][ ES ] K N, [ ES 2 ] [ S][ E] K1 ---------- (4.14.1) [ ES ] The assumption of rapid equilibrium yields K Vmax [ S] [ S] [ S] V 2 1 K N ---------- (4.14.2) 2 [ S] At low substrate concentrations, 1, and inhibition effect is not observed. The rate is K N V Vmax K1 [ 1 ] [ S] ----------- (4.14.3) Or 1 V 1 V K V max 1 S 1 ----------- (4.14.4) max A plot of 1/V versus 1/[S] results in a line of slope dominant. The rate in this case is K V 1 max <<1, and inhibition is V Vmax [ S] [1 ] K N ----------- (4.14.5) or 1 V 1 V max [ S] V max 1 ----------- (4.14.6) K N A plot of 1/V versus [S] results in a line of slope 1/ (K N V max ) and intercept of 1/V max.

76 The substrate concentration resulting in the maximum reaction rate can be determined dv by setting 0. The [S] max is given by d[ S] [S] max = K 1 K N ------------- (4.14.7)