CHAPTER 3 MATERIALS AND METHODS

CHAPTER 3 MATERIALS AND METHODS 3.1 Materials 3.1.1 Chemicals All the chemicals used in the present investigation were of analytical grade and were purchased from CDH, Glaxo, Merck and HiMedia. The substrate calcium phytate was purchased from HiMedia Company, Mumbai, India. 3.1.2 Composition of media All the media were prepared in distilled water and sterilized by autoclaving at 15 lbs/sq inch pressure, 121 o C for 20 minutes. Composition of growth medium (g/l): Sabouraud s dextrose agar (SDA) medium was used for the growth and maintenance of fungal cultures, the composition of which is as follows: Sabouraud s Dextrose Agar medium (SDA) (g/l) Peptone - 10.0 g Dextrose - 40.0 g Agar - 15.0 g ph - 5.6 138

Composition of growth medium (g/l): Nutrient agar medium (NAM) was used for the growth and maintenance of bacterial cultures, the composition of which is as follows: Nutrient agar medium (NAM) (g/l) Beef extract - 3.0 g Peptone - 5.0 g NaCl - 5.0 g Agar - 15.0 g ph - 6.8 Composition of growth medium (g/l): Malt yeast extract glucose peptone agar (MYGP) medium was used for the growth and maintenance yeast cultures, the composition of which is as follows: Malt Yeast Extract Glucose Peptone Agar (MYGP) (g/l) Glucose - 10.0 g Peptone - 5.0 g Yeast extract - 3.0 g Malt extract - 3.0 g Agar - 15.0 g ph - 5.6 Composition of solid agar medium for screening of phytase producing cultures (g/l): Phytase screening medium (PSM) containing agar was used for isolation and screening of phytase producing fungi and yeasts, the composition of which is as follows: 139

Phytase screening medium (PSM) containing agar and calcium phytate (Howson and Davis, 1983) (g/l) Glucose - 15.0 g Ammonium nitrate - 5.0 g Magnesium sulphate - 0.5 g Potassium chloride - 0.5 g Ferrous sulphate - 0.01 g Manganese sulphate - 0.01 g Calcium phytate - 5.0 g Agar - 15.0 g ph - 5.5-6.0 Composition of media used for phytase production (g/l): Various production media were analyzed for screening of fungal and yeast strains with maximum phytase producing potential. The following media were tested: Medium A: Phytase screening (PS) broth (Vohra and Satyanarayana, 2001) (g/l) Glucose - 10.0 g Ammonium sulphate - 3.0 g Magnesium sulphate - 0.5 g Potassium chloride - 0.5 g Calcium chloride - 0.1 g Calcium phytate - 1.0 g ph - 5.6 140

Medium B: Phytase screening medium (PSM broth) (Howson and Davis, 1983) (g/l) Glucose - 15.0 g Ammonium nitrate - 5.0 g Magnesium sulphate - 0.5 g Potassium chloride - 0.5 g Ferrous sulphate - 0.01 g Manganese sulphate - 0.01 g Calcium phytate - 5.0 g ph - 5.5 Medium C: Corn starch medium (Volfova et al., 1994) (g/l) Corn starch - 80.0 g Glucose - 30.0 g Sodium nitrate - 8.6 g KH 2 PO 4-0.017g Potassium chloride - 0.5 g Magnesium sulphate - 0.5 g Ferrous sulphate - 0.1 g ph - 5.0 Medium D: Glucose phosphate (GP) broth (Vohra and Satyanarayana, 2001) (g/l) Glucose - 10.0 g Ammonium sulphate - 3.0 g Magnesium sulphate - 0.5g 141

Potassium chloride - 0.5g Calcium chloride - 0.1g KH 2 PO 4-3.0 g ph - 5.6 Medium E: Peptone dextrose (PD) broth (Vohra and Satyanarayana, 2001) (g/l) Peptone - 10.0 g Dextrose - 40.0 g ph - 5.6 Medium F: Malt Yeast Extract Glucose Peptone (MYGP) broth (In et al., 2007) (g/l) Glucose - 10.0 g Peptone - 5.0 g Yeast extract - 3.0 g Malt extract - 3.0 g Calcium phytate - 1.0 g ph - 5.6 3.2 Methods 3.2.1 Sample collection Seven soil samples were collected from Meerut district (Uttar Pradesh) to carry out isolation of microbial phytase producers. The soil samples were collected from garden of Meerut Institute of Engineering & Technology (MIET) campus and different sites of poultry farms. Later five soil samples 142

were collected from dumps of distillery wastes of Meerut city (UP) and garden soil, for isolation of yeast phytase producers. These soil samples were collected in sterile polythene bags and stored at 4 o C in a refrigerator for further use. Sites with high amount of organic contents were preferred for soil sample collection. Twenty microbial cultures were procured from MIET stock culture collection and were screened for phytase production. 3.2.2 Isolation and maintenance of phytase producing microorganisms Extracellular phytase producing microorganisms were isolated from different soil samples collected from various locations. 3.2.2.1 Isolation and maintenance of fungi A. Isolation of fungi Various soil samples were subjected to serial dilution. Thus, ten gram of soil sample was suspended in 90 ml of sterile distilled water or saline solution. The suspension was shaken well at 200 rpm for 10 min and 1 ml of the suspension was transferred to 9 ml saline blank and this procedure was repeated to obtain a dilution of 1/10,000,000 times (10-7 ). 0.1 ml of the appropriate dilutions were spread on the Sabouraud s dextrose agar (SDA) plates and incubated at 30 o C for 5-7 days. Different fungal colonies appeared on the plates after incubation. All morphological contrasting colonies were purified by repeated streaking. A loop of the isolated colony was then streaked onto SDA medium and incubated at 30ºC for 96 hrs. These fungal isolates were screened for their phytase producing capability by qualitative and quantitative methods. 143

B. Maintenance of fungal isolates The fungal colonies were transferred to SDA slants and incubated at 30 o C. After 3 to 5 days of incubation, the slants with luxuriant growth were stored at 4 o C. The fungal strains were sub-cultured periodically and stock cultures were maintained in sterile paraffin oil at room temperature. 3.2.2.2 Isolation and maintenance of bacteria A. Isolation of bacteria The different soil samples were subjected to serial dilution. As in section 3.2.2.1, 1 ml of soil suspension was transferred to 9 ml saline blank and this procedure was repeated to obtain a dilution of 1/10,000,00 times (10-6 ). 0.1 ml of the appropriate dilutions were spread on the nutrient agar medium (NAM) plates and incubated at 37ºC for 24-48 hrs. Different bacterial colonies appeared on the plates after incubation. All morphological contrasting colonies were purified by repeated streaking. A loop of the isolated colony was then streaked onto NAM medium and incubated at 37ºC for 24-48 hours. These bacterial isolates were screened for their phytase producing capability on PSM solid agar plates by qualitative method. B. Maintenance of bacterial isolates The bacterial colonies were transferred to NAM slants and incubated at 37 o C. After incubation, the fully grown bacterial slants were stored at 4 o C and were sub-cultured periodically. 3.2.2.3 Isolation and maintenance of yeast A. Isolation of yeast Ten grams of soil sample was suspended in 90 ml of sterile distilled water or saline solution. The suspension was shaken well at 200 rpm for 10 minutes and then allowed soil particles to settle down. The suspension was 144

serially diluted through a series of dilutions to obtain a final concentration of 1/10,000,00 times (10-6 ) by serial dilution technique. From each dilution, 0.1 ml was pipetted out and spread on the surface of malt yeast extract glucose peptone (MYGP) agar plates and incubated at 30 o C for 2-5 days. All morphological contrasting colonies were purified by repeated streaking. A loop of the isolated colony was then streaked onto MYGP slants and incubated at 30ºC for 72 hrs. These isolates were screened for their phytase producing capability both qualitatively and quantitatively. B. Maintenance of yeast isolates After 2-5 days of growth on soild plate of MYGP medium, when luxuriant growth was observed in different yeast cultures, the yeast colonies were subcultured on MYGP slants and incubated for 2-5 days at 30 o C. The slants with luxuriant growth were stored at 4 o C in refrigerator for further use. Yeasts strains were subcultured periodically on MYGP slants and stock cultures were maintained in sterile paraffin oil at room temperature. 3.2.3 Qualitative screening of fungal, bacterial and yeast isolates by agar plate assay The qualitative screening of phytase producing isolates was carried out by plate assay procedure on phytase screening medium (PSM) (Howson and Davis, 1983). The isolated fungal, bacterial and yeast cultures were tested qualitatively and screened by observing zone of clearance or halos around phytase producing colonies. The isolates producing zone around its growth on phytase screening medium (PSM) agar plates (supplemented with 0.5% calcium phytate) were selected as phytase producers and maintained on slants for further studies. 145

Acid producing colonies also solubilize calcium phytate. A counterstaining technique was used to identify the phytate-hydrolysing ability of the microbes (Bae et al., 1999). Briefly, the technique involved flooding of the plate with 2% cobalt chloride for 5 min, after which the solution was replaced and incubated for 5 min at room temperature with freshly prepared colouring reagent containing equal volumes of 6.25% (w/v) aqueous ammonium molybdate solution and 0.42% (w/v) ammonium vanadate solution. Cultures exhibiting zone of clearance after removal of the chromogen were confirmed as phytase producers. These isolates were maintained on slants for further studies. 3.2.4 Quantitative screening of phytase producing isolates 3.2.4.1 Quantitative screening of fungal isolates A. Inoculum preparation for fermentation Spore inoculum was prepared from freshly raised 7 days old SDA slant culture. To fully sporulated agar slope culture, 20 ml of sterile distilled water with 0.01% (v/v) Tween-80 was added. The spores were scrapped using an inoculation needle under strict aseptic conditions. The spore suspension obtained was used as the inoculum. Viable spores in the fully sporulated slants were determined by the plate-count (colony count) technique. The number of spores was determined in a Neubauer counting chamber and the inoculum of 1x10 7 spores/ml was used for fermentative production of phytase. One ml of spore suspension (2%) was used as inoculum in 50 ml production medium. B. Shake flask fermentation for phytase production Fungi exhibiting the largest zones of clearance on the agar plates were selected and further subjected to quantitative screening by estimating 146

extracellular phytase production in phytase screening (PS) broth. 250 ml Erlenmeyer flasks containing 50 ml PS broth were inoculated with 1.0 ml of spore suspension (1 10 7 spores/ml) and incubated at 30 o C for 5 days at 200 rpm in triplicate. After incubation, the culture was filtered through Whatman no. 1 filter paper. The phytase activity from the culture supernatant was determined by performing phytase assay using phytic acid as substrate. The fungal isolate showing highest phytase activity was selected for further studies. 3.2.4.2 Quantitative screening of phytase producing yeasts A. Inoculum preparation for fermentation Inoculum was prepared by suspending the loopful culture in 25 ml of MYGP broth, incubated overnight at 30 o C and 180 rpm (O.D. 600 =0.87). The cell suspension (1%) was used to inoculate the production medium. B. Shake flask fermentation for phytase production Yeasts cultures exhibiting the largest zone of clearance on the agar plates were selected and further subjected to quantitative screening. For the extracellular phytase production by yeast culture, 250 ml Erlenmeyer flasks containing 50 ml malt yeast extract glucose peptone (MYGP) broth (supplemented with 0.1% calcium phytate) were inoculated with 1% of inoculum, incubated at 30 o C and 180 rpm for 2 days in triplicate. Samples were centrifuged at 10,000 rpm for 10 min and the supernatant was used for phytase analysis. The phytase activity from the culture supernatant was determined by performing phytase assay using phytic acid as substrate. The yeast isolate showing highest phytase activity was selected for further studies. 147

3.2.5 Quantitative estimation of enzyme activity The fermented broth of fungal culture was filtered by placing Whatman No. 1 filter paper on a funnel to separate the mycelial biomass from culture medium whereas the fermented broth of yeast strain was centrifuged at 10,000 rpm for 10 min at 4 C to remove cell debris. In both the cases, the culture filtrate or supernatant was used for phytase analysis. The phytase activity was determined by modified Fiske and Subbarow method using phytic acid as substrate (Bae et al., 1999). A. Preparation of standard curve of KH 2 PO 4 A 100 ml stock solution of KH 2 PO 4 (10 μg/ml) was prepared in distilled water. A dilution series ranging from 2.5-25 μg/2.5 ml was prepared from the stock solution. A calibration graph (standard curve) was prepared with different phosphate levels according to the method of Fiske and Subbarow (1925) (Figure 3.1). This standard curve was used for the detection of the amount of inorganic phosphate (Pi) liberated due to the action of phytase enzyme by modified Fiske and Subbarow method. The procedure involved the addition of 750 µl of 5% TCA to 1 ml of dilution. This was followed by the addition of colour reagent (750 µl), prepared daily by mixing four volumes of 1.5% (w/v) ammonium molybdate in a 5.5% (v/v) sulfuric acid solution and one volume of a 2.7% (w/v) ferrous sulfate solution. The reaction involved the production of phosphomolybdate, which was measured spectrophotometrically at 700 nm. 148

Absorbance 700 nm 1.2 1 0.8 0.6 0.4 y = 0.042x R² = 0.995 0.2 0 0 10 20 30 Pi concentration (µg) Figure 3.1 Standard curve of inorganic phosphate 149

B. Phytase assay (a) Reagents (i) 0.1 M Sodium acetate buffer (ph 5.0) (ii) Substrate solution: 0.2% Calcium phytate in 0.1 M buffer (ph 5.0) (iii) Stopping reagent: 5% Trichloroacetic acid (iv) Colour reagent: Colour reagent was freshly prepared by mixing four volumes of 1.5% (w/v) ammonium molybdate in a 5.5% (v/v) sulfuric acid solution and one volume of a 2.7% (w/v) ferrous sulfate solution. (b) Procedure Phytase activity was estimated by colorimetric method using calcium phytate as substrate. The reaction mixture was prepared by the addition of 600 µl substrate with 150 µl of the crude enzyme and incubated at 35ºC for 20 min. The enzymatic reaction was stopped by adding 750 µl of a 5% (w/v) trichloroacetic acid solution and the released free orthophosphate (Pi) in the reaction was measured by a modification of the method of Fiske and Subbarow (1925). Colour reagent (750 µl), was added to the sample solution (750 µl) and the production of phosphomolybdate was measured spectrophotometrically at 700 nm. Results were compared to a standard curve prepared with inorganic phosphate (K 2 HPO 4 ) (Bae et al., 1999). One unit of phytase is defined as the amount of enzyme that liberates 1µg inorganic phosphate/ml/min under the assay conditions. Protein content determination Protein content in the phytase enzyme preparation was determined by the Lowry method using bovine serum albumin (BSA) as a standard. 150

C. Estimation of total protein content Quantitative estimation of protein was done by following the method of Lowry et al. (1951). (a) Reagents: Reagent A- 2% Na 2 CO 3 in 0.1N NaOH Reagent B- 1% solution of CuSO 4.5H 2 O in distilled water Reagent C- 2% solution of Sodium Potassium Tartarate Reagent D-Mix reagent A, B and C in 100:1:1 ratio. Prepare fresh before use. Reagent E-1N Folin s reagent. Prepare fresh before use. (b) Procedure To 1ml protein sample, 5 ml of reagent D was added and kept at room temperature for 10 min. Then, 0.5 ml reagent E was added and contents were mixed well by vortexing. After 30 min, the color intensity was read at 660 nm against the reagent blank. The amount of protein in the sample was calculated from standard curve prepared by using BSA solution (500 µg/ml). 3.2.6 Identification of phytase producers 3.2.6.1 Identification of phytase producing fungi The phytase producing fungi were identified using following techniques. A. Cello tape method Tape mounts of selected fungi were prepared for studying their morphological features. For preparing tape mount, a drop of lactophenol cotton blue was placed at the center of a clean glass slide. A strip of tape about 4 5 cm long were gently applied to the sporulating surface of fungal colony and removed. Further, the slide was subjected for microscopic examination. 151

B. Slide culture technique Slide cultures were made by setting up a small petridish moist chamber containing a V-shaped piece of glass rod resting on several layers of moistened filter-paper. A sterile block of agar medium about 1 cm 2 is placed on a flamesterilized glass slide and the slide was then set in the moist chamber on the pre placed V-shaped glass rod. The fungus was inoculated near the four edges of the agar block and a sterile cover-slip was placed. After 2-4 days of incubation, the slide was mounted on microscope and undisturbed mould structures were viewed as they grew. 3.2.6.2 Identification of phytase producing yeasts The best three yeast phytase producers based on qualitative plate assay were characterized using standardized conventional methods described by Yarrow (2000). 3.2.6.2.1 Morphological characteristics A. Cell and colony morphology The yeast isolates were grown on malt yeast extract glucose peptone solid agar (MYGP) plates and were incubated for 3 to 5 days at 30ºC. Colonies and the cells were studied using a stereomicroscope and phase contrast microscope, respectively (Yarrow, 2000). On the basis of phenotypic characterization of cultures, the selected fungal and yeast cultures were identified as Aspergillus heteromorphus and Zygosaccharomyces bailii, respectively. The identification was done by Microbial Type Culture Collection (MTCC), Institute of Microbial Technology (IMTECH), Chandigarh. 152

3.2.7 Biomass determination To determine the fungal biomass, the culture was filtered through Whatman No. 1 filter paper. The filtered mycelium was dried at 80 o C for 24 hrs on a preweighed filter paper to constant weight and expressed as grams dry weight/l medium (g dry wt/l). For estimation of yeast biomass, the culture was centrifuged at 10,000 rpm for 10 min at 4 o C and the pellet was washed twice with distilled water. The washed pellet was inverted on tissue paper and dried at room temperature for 10-15 min in a preweighed centrifuged tube to constant weight and expressed as grams wet weight/l medium (g wet wt/l). 3.2.8 Optimization of culture conditions for maximum phytase production from Aspergillus heteromorphus under submerged conditions Various media were studied for fermentative production of phytase under submerged conditions. The medium with maximum enzyme production potential was selected for further optimization. Thus the effects of incubation period, agitation, temperature, ph of culture medium, substrate concentration, inoculum size, carbon sources, nitrogen sources, metal ions and various additives including detergents, chelating agents and solvents on enzyme production were analyzed. In the optimization of the parameters studied before the study of effect of inoculum size, 1% inoculum was used. 3.2.8.1 Effect of different media To obtain maximum phytase production, different production media (ph 5.6), namely, media A, B, C, D and E were tested under submerged conditions. All the media were inoculated with Aspergillus heteromorphus and incubated 153

at 30ºC for 5 days and phytase activity as well as growth (in terms of biomass) were determined. 3.2.8.2 Effect of incubation period To study the effect of incubation period, ten Erlenmeyer flasks containing 50 ml medium A were inoculated with seed culture of Aspergillus heteromorphus and incubated at 30ºC. The phytase activity and growth were estimated by harvesting one flask at regular intervals of 24 hrs up to 7 days. 3.2.8.3 Effect of agitation The effect of different shaker speed (such as 100, 120, 150, 180, 200 and 220 rpm) on phytase production by Aspergillus heteromorphus was studied. Each 250 ml flask containing 50 ml medium A was inoculated with fungal spores and incubated on orbital shaker at 30 o C for 6 days. The growth and phytase activity were determined. 3.2.8.4 Effect of temperature In order to analyze the effect of different incubation temperatures (25 o C, 30 o C, 35 o C, 40 o C, 45 o C and 50 o C) on phytase production by Aspergillus heteromorphus, 50 ml medium A contained in 250 ml flask was inoculated and incubated at different temperatures for 5 days. Fungal growth and phytase activity were determined after 5 days. 3.2.8.5 Effect of ph of culture medium The effect of different ph (ranging from 3.0-10.0) on phytase production by Aspergillus heteromorphus was studied. Each 250 ml flask containing 50 ml medium A of different ph was inoculated with fungal strain and incubated at 30 o C for 5 days. The growth and phytase activity were determined. 154

3.2.8.6 Effect of substrate concentration The effect of different substrate concentration (0.1, 0.2, 0.3, 0.4, 0.5 and 0.6%) on phytase production by Aspergillus heteromorphus was analyzed. The production of extracellular fungal phytase was induced by limiting concentration of inorganic phosphate in the growth medium (Shieh et al., 1968). In the case, phytase was induced by using a synthetic medium (PS broth) containing phytate as the sole source of phosphate and fulfill the requirement of phosphorus for their growth. Each 250 ml flask containing 50 ml production medium (supplemented with calcium phytate as substrate) inoculated with fungal strain was incubated at 30ºC for 5 days and growth as well as phytase activity were determined. 3.2.8.7 Effect of inoculum size In order to investigate the effect of different inoculum size on phytase production by Aspergillus heteromorphus, Erlenmeyer flasks (250 ml) containing 50 ml medium A were inoculated with different inoculum percentage (1, 2, 3, 4 and 5%, v/v) and incubated at 30 o C for 5 days. The growth and phytase activity were determined as mentioned earlier. 3.2.8.8 Effect of carbon sources To study the effect of different carbon sources on phytase production, the medium was supplemented (at 1% w/v concentration) with different sugars (glucose, sucrose, maltose, lactose, galactose, cellulose, cellobiose, soluble starch, myo-inositol, glycerol and fructose). The production medium with 0.1% calcium phytate and glucose was kept as control. These flasks were inoculated with 2% spore suspension of Aspergillus heteromorphus and incubated at 30 o C for 120 hrs and phytase activity was estimated. 155

3.2.8.9 Effect of nitrogen sources Effect of various nitrogen supplements like inorganic sources (0.3%): ammonium sulphate [(NH 4 ) 2 SO 4 ], ammonium nitrate (NH 4 NO 3 ), ammonium chloride (NH 4 Cl), ammonium acetate (CH 3 COONH 4 ), ammonium oxalate [(NH 4 ) 2 C 2 O 4.H 2 O], ammonium ferrous sulphate [(NH 4 ) 2 SO 4. FeSO 4.6H 2 O)] and sodium nitrate (NaNO 3 ) and organic nitrogen sources (0.3%) such as casein, urea, peptone, tryptone, yeast extract, beef extract, malt extract, and soybean meal on phytase production from Aspergillus heteromorphus was studied. The production medium A was supplemented with different organic and inorganic nitrogen sources and inoculated with freshly prepared inoculum (2%) of Aspergillus heteromorphus. Phytase activity was determined after 5 days. 3.2.8.10 Effect of metal ions The effect of metal ions (0.1% w/v) on enzyme production was studied by adding different salts, viz. NaCl, KCl, CaCl 2, MgCl 2, MnCl 2, ZnSO 4, NiCl 2, CoCl 2, PbCl 2, BaCl 2, HgCl 2 and AgCl 2 in the production medium. After incubation of the culture in these conditions, phytase activity was determined as described earlier. 3.2.8.11 Effect of additives The effect of additives (detergents and solvents) (0.1% w/v) on enzyme production was studied by adding Tween-20, Tween-40, Tween-60, Tween- 80, Triton X-100, sodium dodecyl sulphate (SDS), and solvents like β- mercaptoethanol, dithiothreitol (DTT), toluene, ethylenediaminetetra-acetic acid (EDTA) and glycerol in the production medium. After incubation of the 156

culture in these conditions, phytase activity was determined from the culture filtrate. 3.2.8.12 Phytase production under optimized culture conditions The medium optimized at this stage for phytase production by Aspergillus heteromorphus was termed as Phytase production medium and was used in further studies. The composition or production medium and optimized conditions for phytase production by Aspergillus heteromorphus were as follows: Phytase Production Medium (g/l) Glucose - 5.0 g Yeast extract - 3.0 g Magnesium sulphate - 0.5 g Potassium chloride - 0.5 g Calcium chloride - 0.1 g Calcium phytate - 1.0 g ph - 6.0 Temperature - 30 o C Incubation time - 120 hrs 3.2.9 Optimization of culture conditions for maximum phytase production from Zygosaccharomyces bailii under submerged conditions Various media were studied for fermentative production of phytase under submerged conditions. The medium with maximum enzyme production potential was selected for further optimization. Thus the effects of incubation period, agitation, temperature, ph of culture medium, substrate concentration, inoculum size, carbon sources, nitrogen sources, metal ions and various 157

additives including detergents, chelating agents and solvents on enzyme production were analyzed. For all the studies, 1% v/v inoculum from overnight grown culture of Zygosaccharomyces bailii was used, except in the case where the effect of inoculum size was studied. 3.2.9.1 Effect of different media To obtain maximum phytase production, different production medium (ph 5.5) such as medium A, B, C, D, E and F were tested under submerged conditions. All the media were inoculated and incubated at 30ºC for 60 hrs and phytase activity as well as growth (in terms of biomass) was determined. 3.2.9.2 Effect of incubation period To study the effect of incubation period, ten Erlenmeyer flasks containing 50 ml medium F were inoculated with 1% (v/v) seed culture of Zygosaccharomyces bailii and incubated at 30ºC. The phytase activity and growth were estimated by harvesting a flask at regular intervals of 6 hrs up to 3 days. 3.2.9.3 Effect of agitation The effect of different shaker speed (such as 100, 120, 150, 180, 200 and 220 rpm) on phytase production by Zygosaccharomyces bailii was studied. Each 250 ml flask containing 50 ml medium F was inoculated and incubated on orbital shaker at 30 o C for 2 days. The growth and phytase activity were determined. 3.2.9.4 Effect of temperature on enzyme production The effect of different incubation temperatures (25 o C, 30 o C, 35 o C, 40 o C, 45 o C and 50 o C) on phytase production by Zygosaccharomyces bailii was studied. Each 250 ml flask containing 50 ml production medium F was 158

inoculated and incubated at different temperatures for 2 days and growth as well as phytase activity was determined. 3.2.9.5 Effect of ph of phytase production The effect of different ph (ranging from 3.0 to 9.0) on phytase production by Zygosaccharomyces bailii was studied. Each 250 ml flask containing 50 ml medium F of different ph was inoculated and incubated at 30 o C for 2 days. The growth and phytase activity were determined. 3.2.9.6 Effect of substrate concentration The effect of different substrate concentration (0.1, 0.2, 0.3, 0.4, 0.5 and 0.6%) on phytase production by Zygosaccharomyces bailii was studied. The production of extracellular yeast phytase was induced by limiting concentration of inorganic phosphate in the growth medium (Lambrechts et al., 1992). In this case, yeast phytase was induced by using MYGP broth containing phytate as the sole source of phosphate and fulfill the requirement of phosphorus for their growth. Each 250 ml flask containing 50 ml production medium (supplemented with calcium phytate as substrate) was inoculated and incubated at 30ºC for 2 days and growth as well as phytase activity were determined. 3.2.9.7 Effect of inoculum size In order to investigate the effect of different inoculum size on phytase production by Zygosaccharomyces bailii, Erlenmeyer flasks (250 ml) containing 50 ml medium F were inoculated with different inoculum percentage (1, 2, 3, 4 and 5%, v/v) and incubated at 30 o C for 2 days. The growth and phytase activity were determined as mentioned earlier. 159

3.2.9.8 Effect of carbon source To study the effect of different carbon sources on phytase production, the medium was supplemented (at 1% w/v concentration) with different sugars (glucose, sucrose, maltose, lactose, galactose, cellulose, cellobiose, soluble starch, myo-inositol, glycerol and fructose). The production medium with 0.1% calcium phytate and glucose was kept as control. These flasks were inoculated and incubated at 30 o C for 42 hrs and phytase activity was estimated. 3.2.9.9 Effect of nitrogen source Effect of nitrogen supplements like organic nitrogen sources (0.3%) such as casein, urea, peptone, tryptone, yeast extract, beef extract, malt extract, and soybean meal and inorganic sources (0.3%) such as ammonium sulphate [(NH 4 ) 2 SO 4 ], ammonium nitrate (NH 4 NO 3 ), ammonium chloride (NH 4 Cl), ammonium acetate (CH 3 COONH 4 ), sodium nitrate (NaNO 3 ) on phytase production from Zygosaccharomyces bailii was studied. The production medium F was supplemented with different organic and inorganic nitrogen sources and inoculated with Z. bailii. These flasks were incubated at 30 o C for 42 hrs, after which phytase activity was determined. 3.2.9.10 Effect of metal ions The effect of metal ions (0.1% w/v) on enzyme production was studied by adding different salts viz. NaCl, KCl, CaCl 2, MgCl 2, MnCl 2, ZnSO 4, NiCl 2, CoCl 2, PbCl 2, BaCl 2, HgCl 2 and AgCl 2 in the production medium. After incubation of the culture in these conditions, phytase activity was determined. 3.2.9.11 Effect of additives The effect of additives (detergents, chelating agent and solvents) (0.1% w/v) on enzyme production was studied by adding the following; Tween-20, Tween-40, Tween-60, Tween-80, Triton X-100, sodium dodecyl sulphate 160

(SDS), ethylene diaminetetra-acetic acid (EDTA), β-mercaptoethanol, dithiothreitol (DTT), toluene and glycerol, in the production medium. After 2 days incubation of the culture in these conditions, phytase activity was determined from the culture filtrate. 3.2.9.12 Phytase production under optimized culture conditions: The medium optimized at this stage for phytase production by Zygosaccharomyces bailii was termed as Phytase Production Medium and was used in further studies. The composition of production medium and conditions optimized for phytase production by Zygosaccharomyces bailii were as follows: Phytase production medium (g/l) Malt extract - 3.0 Yeast extract - 3.0 Glucose - 5.0 Peptone - 5.0 Calcium phytate - 1.0 ph of medium - 6.0 Temperature - 30 o C Incubation time - 42 hrs 3.2.10 Partial purification of phytase The phytase from Aspergillus heteromorphus and Zygosaccharomyces bailii was partially purified by ammonium sulphate precipitation followed with acetone precipitation method. 161

3.2.10.1 Preparation of crude enzyme The fungal culture Aspergillus heteromorphus and the yeast strain Zygosaccharomyces bailii were grown in the optimized production medium (Phytase production medium) and incubated at 30ºC for 120 hrs and 42 hrs, respectively. After incubation, the fungal culture was filtered through Whatman No. 1 filter paper whereas the yeast culture was centrifuged at 10,000 rpm for 10 min at 4 o C to obtain clear culture filtrate. The filtrate/supernatant was used as crude enzyme to estimate phytase activity (3.2.5) The crude filtrate from both the cultures was subjected to two-step precipitation method. The ammonium sulphate fractionation and acetone precipitation methods were used to purify enzyme protein by altering their solubility. The resulting samples of proteins of respective cultures were then dialyzed against 0.1 M acetate buffer (ph 5.0). The dialyzed protein solution was used as source of phytase and stored at 4 o C for further studies. 3.2.10.2 Ammonium sulfate precipitation The crude enzyme extract from both the microbial sources was subjected to ammonium sulfate precipitation at 0-90% saturation. A volume each of 30 ml of crude phytase was taken, added slowly to required quantity of ammonium sulphate to obtain various saturation levels; 0 30, 30 60 and 60 90 %. The addition of ammonium sulphate was done under constant stirring at 4 C for 30 min. The stirring was continued for another 30 min and then allowed for settlement for 3 hours at 4 C. The precipitated proteins were separated by centrifugation at 8000 rpm at 4 C for 20 min. The separated proteins were then dissolved in minimum amount of 0.1 M acetate buffer (ph 5.0) and dialyzed against acetate buffer (0.01 M, ph 5.0) at 4 C. The buffer 162

was stirred gently using a magnetic stirrer to enhance solute exchange. Dialysis was conducted over night and the buffer was changed several times to increase the efficiency of the dialysis. The phytase activity and protein content of precipitated sample was determined by performing phytase assay. 3.2.10.3 Acetone precipitation The dialysed enzyme fraction of both the microbial sources was used for acetone precipitation. In this method, 40 ml of acetone was cooled to -20 o C and then, 10 ml enzyme sample was added in acetone-compatible tube. Both the tubes containing phytase from each culture were vortexed and incubated at -20 o C. After incubation for 60 min the tubes were centrifuged for 10 min at 10,000 x g and the supernatant was decanted properly without dislodging the pellet. These steps were repeated 2-3 times for removal of interfering substances. After that the acetone was allowed to evaporate from the uncapped tube at room temperature for 30 min. The resulting pellet was dissolved in acetate buffer to make the final volume to 8 ml with thorough vortex. The phytase activity and protein content of precipitated sample was determined. 3.2.11 Immobilization of phytase The partially phytase enzyme from both the microbial sources was immobilized by using entrapment and crosslinking methods. 3.2.11.1 Entrapment of phytase in different matrices For immobilization of phytase enzyme, different matrices such as sodium alginate (3%), k-carrageenan (4%) and polyacrylamide (10%) were used for entrapment of partially purified phytase enzyme. 163

3.2.11.1.1 Entrapment of phytase in alginate beads Ten ml suspension containing 3 ml of phytase and 3% sodium alginate (UV sterilized) was extruded dropwise through a 2 ml syringe into 0.2 M CaCl 2 solution at 4 C with gentle stirring and spherical and uniform sized (diameter 3 ± 0.1 mm) beads of calcium alginate were obtained. After 2 hrs, beads were washed with double distilled water. The enzyme activity of the beads was determined immediately and remaining beads were stored at 4 o C for determining the enzyme activity during storage. (a) Effect of different concentrations of sodium alginate on the activity of immobilized phytase Effect of different concentrations of sodium alginate was studied using different concentrations of sodium alginate, viz. 1%, 1.5%, 2.0%, 2.5%, 3.0%, 3.5%, 4.0%, 4.5% and 5%. The enzyme assay was performed as described in section 3.2.11.3 and phytase activity was calculated. (b) Effect of amount of enzyme entrapped in alginate beads The effect of different amounts of enzyme (Chapter 4) entrapped in sodium alginate solution used for bead formation was studied. (c) Effect of number of calcium alginate beads in reaction mixture Effect of number of beads of calcium alginate was studied using 1, 2, 3, 4, 5, 6 and 7 beads of calcium alginate in a reaction mixture. 3.2.11.1.2 Entrapment of phytase in k-carrageenan blocks The phytase enzyme (2 ml) was thoroughly mixed with 8 ml suspension of carrageenan (4%) and kept at 4 C for 1 hr to allow gelation. After gelation, the gel was soaked in cold 0.3 M KCl solution for 1 hr to give stability. After 2 hrs, the gel was cut into blocks of two sizes: (0.5 x 0.5 cm 2 ) and (1.0 x 1.0 164

cm 2 ) and washed with sterilized double distilled water. The enzyme activity of blocks was determined immediately and remaining blocks were stored at 4 o C for further analysis. (a) Effect of number of phytase entrapped carrageenan blocks in the reaction mixture Effect of number of blocks of carageenan of two sizes was studied using 2, 3, 4 and 5 blocks of carageenan in the reaction mixture. The enzyme assay was performed as described in section 3.2.11.3 and phytase activity was calculated. (b) Effect of amount of enzyme entrapped in carrageenan blocks The effect of different amounts of enzyme (Chapter 4) entrapped in carrageenan solution used for block formation was investigated. 3.2.11.1.3 Entrapment of phytase in polyacrylamide blocks The phytase enzyme (2 ml) was thoroughly mixed with 8 ml suspension of polyacrylamide (10%) and kept at 4 C for 1 hr to allow gelation. After 1 hr, the gel was cut into blocks of two sizes (0.5 x 0.5 cm 2 ) and (1.0 x 1.0 cm 2 ) and washed with sterilized double distilled water. The enzyme activity of blocks was determined immediately and remaining blocks were stored at 4 o C. (a) Effect of number of phytase entrapped polyacrylamide blocks in the reaction mixture Effect of number of blocks of polyacrylamide was studied using 2, 3, 4 and 5 blocks in a reaction mixture. The enzyme assay was performed as described in section 3.2.11.3 and phytase activity was calculated. 165

(b) Effect of amount of enzyme entrapped in polyacrylamide blocks The effect of different amounts of enzyme (Chapter 4) entrapped in polyacrylamide solution used for blocks formation was also studied. 3.2.11.2 Immobilization of phytase by crosslinking method For immobilization of phytase enzyme, gelatin particles were used as inert support material to carry out the crosslinking reaction (Kennedy et al. 1984). Preparation of crosslinked gelatin particles For the preparation of activated gelatin particles, 1.5 g gelatin particles (solid support) were taken and mixed with 2.5% aqueous solution of glutaraldehyde. The solution was mixed thoroughly by stirring for 90 min at room temperature. After the termination of reaction, glutaraldehyde solution was discarded and gelatin particles were washed with water. The crosslinked gelatin particles were stored in 15% (w/v) NaCl at 4 C. Phytase (5 ml) was coupled onto glutaraldehyde crosslinked gelatin particles at 4 C for a period of 12 hrs. Enzyme activity was determined spectrophotometrically at 700 nm. (a) Effect of different volumes of substrate on the activity of immobilized phytase Effect of different volumes (0.6 ml, 1 ml and 2 ml) of substrate solution of 0.2% concentration on the enzyme activity of cross-linked phytase was studied. The reaction mixture consisted of 0.5 g phytase coupled glutaraldehyde crosslinked gelatin particles and different volumes of substrate solution. The enzyme assay was performed as described in section 3.2.11.3 and phytase activity was calculated. 166

3.2.11.3 Enzyme assay for immobilized phytase A. Enzyme assay for immobilized phytase from Aspergillus heteromorphus The reaction mixture consisted of 600 µl substrate (0.2% calcium phytate in 0.1 M acetate buffer; ph 5.0) and phytase from Aspergillus heteromorphus immobilized in calcium alginate beads or carrageeenan/ polyacrylamide blocks. It was incubated at 35 C for 20 min. After incubation, beads or blocks were removed from the tubes for reuse and 750 µl TCA was added to stop the reaction. Further phytase assay procedure was similar as that of soluble phytase assay described in section 3.2.5. B. Enzyme assay for immobilized phytase from Zygosaccharomyces bailii The reaction mixture consisted of 600 µl substrate (0.2% calcium phytate in 0.1 M acetate buffer; ph 5.0) and phytase from Zygosaccharomyces bailii immobilized in calcium alginate beads or carrageeenan/ polyacrylamide blocks. It was incubated at 35 C for 20 min. After incubation, beads or blocks were removed from the tubes for reuse and 750 µl TCA was added to stop the reaction. Further phytase assay procedure was similar as that of soluble phytase assay described in section 3.2.5. 3.2.12 Biochemical characterization of immobilized phytase The biochemical properties of phytase immobilized by various procedures were studied and compared with that of its free enzyme. The partially purified phytase from Aspergillus heteromorphus and Zygosaccharomyces bailii were used as free enzyme. The ones showing best results have been described below for immobilized phytases from fungal and yeast strains. 167

3.2.12.1 Enzymatic properties of free and immobilized fungal phytase The following biochemical properties of carrageenan entrapped phytase and cross-linked phytase from Aspergillus heteromorphus were studied. As described in section 3.2.11.3 A, the substrate used in each case was 0.2% calcium phytate in acetate buffer, ph 5.0. 3.2.12.1.1 Effect of ph on phytase activity and stability The ph activity profile of free, entrapped and cross-linked phytase was studied by incubating samples with calcium phytate (600 µl) in buffers of different ph ranging from 3.0 to 6.5 at 35ºC for 20 min. The buffers (0.1 M) used were Glycine-HCl (ph 2.5-3.5), acetate buffer (ph 4.0 6.0), HEPES (N- 2-Hydroxy ethyl piperazine-n-ethane sulphonic acid) (ph 6.5). The enzyme assay was performed and phytase activity was calculated. The ph stabilities were examined by incubating the enzyme solution with the buffers of ph ranging from 3.0 to 6.5 at 4ºC for 60 min prior to performing the routine assay. 3.2.12.1.2 Influence of temperature on free and immobilized enzyme activity The optimum temperature of free and immobilized phytases (carrageenan-entrapped phytase and cross-linked phytase) was determined by incubating with substrate (600 µl) at various temperatures ranging from 30 o C to 60 o C. Phytase activity was determined by standard assay procedure. 3.2.12.1.3 Thermal stability To check thermal stability, the free and immobilized enzymes was preincubated at different temperatures ranging 30 o C to 60 o C for 60 min and then enzyme was assayed using the standard phytase assay. 168

3.2.12.2 Enzymatic properties of free and immobilized yeast phytase The following biochemical properties of carrageenan entrapped phytase from Zygosaccharomyces bailii were studied. As described in section 3.2.11.3 B, the substrate used in each case was 0.2% calcium phytate in acetate buffer (ph 5.0). 3.2.12.2.1 Effect of ph on phytase activity and stability To determine the optimum ph, the free and carragenan entrapped phytase enzyme was incubated with substrate (600 µl) in buffers of different ph ranging from 3.0 to 6.5 at 35ºC for 20 min. The buffers (0.1 M) used were Glycine-HCl (ph 2.5 3.5), acetate buffer (ph 4.0 6.0) and HEPES (N-2- Hydroxyethylpiperazine-N -2-ethane sulphonic acid) (ph 6.5). Phytase activity was estimated. The ph stabilities were examined by incubating the enzyme solution with the buffers of ph ranging from 3.0 to 6.5 at 4ºC for 60 min prior to performing the routine assay. 3.2.12.2.2 Influence of temperature on free and immobilised enzyme The optimum temperature of free and immobilized phytases (carragenanentrapped phytase) was determined by incubating the enzyme with substrate (600 µl) at various temperatures ranging from 30 o C to 60 o C for 20 min and phytase activity was calculated. 3.2.12.2.3 Thermal stability To determine the thermal stability of enzyme, the free and immobilized enzyme was pre-incubated at different temperatures ranging 30 o C to 60 o C for 60 min and then enzyme was assayed using the standard phytase assay. 169

3.2.12.3 Operational stability (Reusability) of immobilized phytase The stability of immobilized phytase on repeated use was examined by measuring the enzyme activity after each successive run. The carragenanentrapped phytase of both sources and cross-linked fungal phytase was incubated with substrate solution. After incubation, the reaction mixture was decanted. The immobilized enzyme was collected, washed with distilled water and re-suspended in 1 ml freshly prepared substrate solution (600 µl) to start a new run. The enzyme was reused upto 8 cycles and enzyme activity was estimated after each successive run. The supernatant was used to determine the phytase activity. 3.2.12.4 Storage stability of free and immobilized phytase The storage stability of immobilized enzyme was investigated by measuring the activity after storage at 4 o C for a week and the residual activity measurements were performed at regular intervals of 24 hrs. Immobilized and free fungal phytase were stored in 0.1 M of acetate buffer (ph 4.0) whereas free and immobilized yeast phytase were stored in 0.1 M of acetate buffer of ph 4.5. Result Analysis All the experiments and enzyme assay were performed in triplicate, and the mean values with standard deviation (SD) are presented. 170

References 1. Bae H.D., Yanke L.J. & Cheng K.J. (1999) A novel staining method for detecting phytase activity. Journal of Microbiological Methods, 39, 17-22. 2. Fiske C.H. & Subbrow Y. (1925) The colorimetric determination of phosphorus. Journal of Biological Chemistry, 66, 376-400. 3. Harland B.F. & Morris E.R. (1995) Phytate: A good or a bad food component. Nutrition Research, 15, 733-754. 4. Howson S.J. & Davis R.P. (1983) Production of phytate-hydrolysing enzyme by some fungi. Enzyme and Microbial Technology, 5, 377-382. 5. In M., Kim K. & Oh N. (2007) Phytate degradation by immobilized Saccharomyces cerevisiae phytase in soybean-curd whey. Biotechnology and Bioprocess Engineering, 12, 348-353. 6. Lowry O.H., Rosenbrough N.J., Farr A.L. & Randall R.J. (1951) Protein measurement with Folin-phenol reagent. Journal of Biological Chemistry, 193, 265-275. 7. Quan C.S., Zhang L.H., Wang Y.J., & Ohta Y.Y. (2001) Production of phytase in a low phosphate medium by a novel yeast Candida krusei. Journal of Bioscience and Bioengineering, 92, 154-160. 8. Shieh T.R. & Ware J.H. (1968) Survey of microorganisms for the production of extracellular phytase. Applied Microbiology, 169, 1348-1351. 9. Vats P. & Banerjee U.C. (2002) Studies on the production of phytase by a newly isolated strain of Aspergillus niger van Teighem obtained from rotten wood-logs. Process Biochemistry, 38, 211-217. 171

10. Vohra A. & Satyanarayana T. (2001) Phytase production by the yeast Pichia anomala. Biotechnology Letters, 23, 551-554. 11. Volfova O., Dvorakova J., Hanzlikova A. & Jandera A. (1994) Phytase from Aspergillus niger. Folia Microbiologica, 9, 481-484. 12. Yarrow D. (2000) Methods for the isolation, maintenance and identification of yeast. In The Yeasts: A taxonomic study (Kurtzman C.P. & Fell J.W, eds.), 4th edn., Elsevier Science, New York, pp 77-97. 172