April Journal-2009.p65 - Association of Biotechnology and Pharmacy

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Current Trends in Biotechnology and Pharmacy Vol. 3 (2) 172-180, April 2009. ISSN 0973-8916 100 g/l of NaCl, purified on the same medium and maintained on 1% malt extract agar slant at 4 0 C. Preparation of substrates Different agricultural byproducts, wheat bran (WB), wheat straw (WS), rye straw (RS) and corncob leaf (CL) were obtained from local market. These waste materials were washed first with tap water followed by distilled water to remove the adhered surface dust particles. Then bleaching operation was carried out by immersing them in hot water (75-80 0 C) for 20 minutes followed by oven drying at 45 0 C. The dried material was grinded in a mixer grinder (Remi) then sterilized at 121 0 C, 15 psi pressure for 15 minutes and stored at 4 0 C before further use. Inoculum preparation Actively growing and heavily sporulating (ten days) old malt agar slant culture was added to 10 ml sterile distilled water. The spores were gently scraped off with the help of a sterile needle and contents were passed through glass wool so as to obtain spore inoculums free from mycelia bits (31-33). A volume of one ml of spore suspension contained more than 10 6 spores. They were cultured in a medium containing soluble starch 5 g., yeast extract 2 g., KH 2 PO 4 1 g., MgSO 4 . 7H 2 O 0.5 g., distilled water 1000 ml. The medium was autoclaved (121 0 C for 15 minutes), allowed to cool, then aseptically added to sterile 500 ml Erlenmeyer flasks (100 ml added per flask).These flasks with 100 ml liquid medium were incubated with 2 ml spore suspension with autoclaved distilled water and incubated at 50 ± 2 0 C and 120 rpm for 2-3 days in the preliminary experiments and only two days in all subsequent experiments. All chemicals used were of reagent grade. Solid state fermentation Four agriculture byproducts; wheat bran, corncob leaf, rye straw, wheat straw was 174 considered as substrate. Five gm of each substrate was taken into 250 ml conical flasks. To adjust percentage moisture levels (w/v), 0.1M acetate buffer, pH = 6.0 was added. The content of the flasks were mixed thoroughly and sterilized in the autoclave at 121 0 C temperature and 1 atmospheric pressure for 20 min and then cooled at room temperature. Each flask was incubated with one ml of inoculum and subsequently rotated in a rotary incubator shaker at 50 0 C. Optimization of process parameters Various process parameters affecting á- amylase production in solid state fermentation (SSF) are optimized. The strategy was to optimize each parameter independently and subsequently optimum conditions were employed in each experimental run. The best solid substrate was selected for optimum production of á-amylase production and the suitable solid substrate was used in subsequent experiments. The tested process parameters in this study were initial moisture content (20, 30, 40, 50, 60, 70, 80, 90, 100, 110%, w/v), inoculum concentration (5, 10, 15, 20, 25, 30%, v/w), incubation time (24, 48, 72, 96, 120, 144, 168, 192 h), incubation temperature (30, 40, 50, 60, 70, 80 0 C), initial pH (4, 5, 6, 7, 8), and supplementary carbon sources (soluble starch, sucrose, maltose, glucose). On the basis of experimental data wheat bran was found to be the best solid substrate in solid state fermentation (SSF) process (details are mentioned in result and discussion section). Determination of dry weight of substrate All four substrates are not available in completely dried form. They generally contain moisture. Prior to utilize them in bioprocess, it is necessary to dry these solid substrates. Therefore, in the present study the amount of wet solid substrate was kept in the oven at 70 0 C for 24 h to remove the moisture from the solid substrate. After drying, the mass of solid substrate was measured. Production of á-amylase
Current Trends in Biotechnology and Pharmacy Vol. 3 (2) 172-180, April 2009. ISSN 0973-8916 Enzyme extraction At the end of solid state fermentation, the solid substrates were mixed thoroughly with acetate buffer (pH 6, 50 ml) containing 0.1 % tween 80 surfactant (14). The contents were mixed by shaking for two hours at 50 0 C on a rotary shaker at 200 rpm. The slurry was squeezed by muslin cloths. The extract was filtered with a Whatman No. 1 filter paper and the filtrate was used as a crude á-amylase. Enzyme assay The α-amylase enzyme was assayed accordingly to the method described by Miller (34). The reaction mixture contained 200 µl soluble starch in phosphate buffer (0.1M, pH = 6); 200 µl of diluted enzyme and 300 µl phosphate buffer. The reaction was incubated for 15 minutes at 30 0 C, 300 µl dinitrosalicylic acid (DNS) solution were added and boiled for 15 minutes. Before cooling 100 µl Rochelle salt (40 % sodium potassium tartarate) was added and colour was measured at 575 nm. One unit of á-amylase activity was defined as the amount of enzyme that releases 1 mg of reducing sugar as glucose per ml per minute under the assay conditions. All data points correspond to triplicates of independent experiments. Estimation of moisture content The moisture content of the wheat bran was estimated by drying 10 g of wheat bran to a constant weight at 70 0 C for 24 h and the dry weight was recorded. To fix the initial moisture content of the solid medium, wheat bran was soaked with the desired quantity of water. After soaking, the sample was again dried as described above and the percent moisture content was calculated by the following formula (35). % moisture content (initial) of solid medium = (wt. of the wheat bran – dry wt.) x 100/ dry wt. 175 Result and Discussion Screening of agriculture byproducts as substrates for SSF The selection of a suitable solid substrate in solid state fermentation (SSF) is a critical factor. In this study, four solid substrates are taken for growth and enzyme fermentation by the selected culture. In literature different solid substrates were found to affect the production of enzymes (35- 36). In view of this the amylase activity in U/g was measured for all four substrates at temperature of 50 0 C and pH 6. These amylase activities are illustrated in Figure 1. It is evident from this figure that the maximum amount of amylase activity (267 U/g) is obtained in presence of wheat bran alone. The activity decreases in order of wheat bran (WB)> wheat straw (WS)> rye straw (RS)> corncob leaf (CL). In previous studies also (36,37) wheat bran was found to be best substrate for glucoamylase production by an a - amylase activity (V/g) on different agricultural by products Wheat bran Corncod leaf rye straw Wheat straw Fig. 1. Effect of solid substrates on á-amylase activity Ravi et al Fig. 2. Effect of initial pH of medium on á-amylase activity
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April Journal-2009.p65 - Association of Biotechnology and Pharmacy

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