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Current Trends in Biotechnology and Pharmacy Vol. 6 (2) 190-195 April 2012, ISSN 0973-8916 (Print), 2230-7303 (Online) temperature optimization, growth as well as enzyme production was estimated at various temperatures viz. 30°C, 35°C, 37°C, 40°C, 42°C, 45°C and 50°C. For checking induction effect of xylan on xylanase production, two 500 ml capacity Erlenmeyer flasks were deployed marked as control and experimental. In each flask, 200 ml of Emerson medium was put and autoclaved. In experimental flask, 1% xylan was also added. Each flask was inoculated using 2% (4 ml) inoculum. Both the flasks were incubated in an orbital shaker at 37 o C with a speed of 200 rpm. From each flask, 20 ml suspension was withdrawn aseptically at intervals of 12 hours. Each withdrawn aliquot was centrifuged at 10,000 x g for 15 minutes at 0 to 4°C in a cooling centrifuge and the supernatant was analyzed for xylanase activity. The effect of different concentrations of xylan on enzyme production was also observed using different concentrations of xylan viz. 0.5%, 1%, 2%, 3%, 4% and 5%. One control was also run with 0 % xylan. All the flasks were incubated in an orbital shaker at 37 o C with a speed of 200 rpm. From each flask, 20 ml suspension was withdrawn aseptically at intervals of 12 hours and up to 84 hours. Each withdrawn aliquot was centrifuged at 10,000 x g for 15 minutes at 0 to 4°C in a cooling centrifuge and the supernatant was analyzed for xylanase activity. Results and Discussion Screening of the bacteria: A xylanase producing bacteria was isolated from the soil mixed with petroleum products. The xylanase production by the bacteria was screened by congo red dye staining method (16). Xylanase activity was taken as positive in the bacteria by the presence of clear zone surrounding the colony (Fig. 1).These zones were enhanced on treatment of the plate with 1 mM sodium chloride. Morphological, biochemical and physiological properties of the bacteria: The Gram staining showed it to be Gram positive bacteria. 192 Microbial Type Culture Collection and Gene Bank, Institute of Microbial Technology, Chandigarh identified it as Paenibacillus macquariensis and gave it Accession number RC1819. The biochemical and physiological properties of the bacteria are shown in Table 1. The Paenibacillus macquariensis was grown at various temperatures ranging from 10 o C to 42 o C (Table 1). It showed optimum growth at 37 o C. The growth was much poor at 42 o C. Effect of pH on the growth of the bacteria was observed in the pH range of 5.2 to 10.0. It was found to have good growth at all the pHs tested in the range of pH 5.2 to pH 10.0 (Table 1). These results showed that this bacteria has much tolerance of pH change and has growth in acidic to highly alkaline range. The effect of sodium chloride on the growth of the bacteria was also checked in the range of 2 to 10% sodium chloride and was observed to exhibit same amount of growth in the presence of various concentrations of sodium chloride (Table 1). These results showed that the bacteria also has tolerance against salinity. The bacteria is capable of hydrolyzing starch and casein. It also liquefied gelatin. However, the bacteria could not utilize citrate Fig. 1. Screening of xylanase activity by using 0.5% Congo red dye method. Optimization of Xylanase Secretion from Paenibacillus macquariensis
Current Trends in Biotechnology and Pharmacy Vol. 6 (2) 190-195 April 2012, ISSN 0973-8916 (Print), 2230-7303 (Online) Table 1. Biochemical and physiological characteristics of the bacteria Tests RC1819 Gram Staining + Spore staining + Motility + Growth at 10°C - Growth at 15°C - Growth at 25°C + Growth at 37°C + Growth at 42°C - Growth at pH 5.2 + Growth at pH 8.0 + Growth at pH 9.0 + Growth at pH 10.0 + Growth at NaCl 2% + Growth at NaCl 5% + Growth at NaCl 7% + Growth at NaCl 10% + Starch hydrolysis + Casein hydrolysis + Citrate utilization - Gelatin liquefaction + H S Production 2 - MR - VP + Nitrate reduction + Indole - Catalase + Oxidase + Urea Acid production from - Arabinose + Galactose - Mannitol + Raffinose - Salicin + Xylose - Sucrose - Rhamnose - Meso-inositol + Fructose + Meeta Sharma and Anil Kumar 193 added in the growth medium. The bacteria could not produce hydrogen sulfide gas and also could not reduce nitrate. The bacteria showed catalase and oxidase activities. The bacteria showed negative tests for indole and urea hydrolysis (Table 1). The bacteria produced acid from glucose, arabinose, mannitol, salicin, fructose and mesoinositol. However, it could not produce acid from galactose, raffinose, xylose, sucrose and rhamnose indicating that these are not metabolized the same way (Table 1). Earlier, authors isolated a bacteria from dung, Bacillus halodurans, MTCC 9512 which showed optimum growth at 55oC and at pH 10 (9). Production of xylanase: Based on the growth of the bacteria and production of xylanase, Emerson medium was selected for the growth of the bacteria. Time optimization for xylanase production: The bacterial growth and xylanase production were observed up to 96 hours. It was found that the bacteria grew logarithmically up to 84 hours as observed by measuring absorbance at 600 nm. Xylanase production was observed to increase up to 48 hours and thereafter, there was decrease in production as observed by measuring enzyme activity. The culture medium showed nearly 4.5 units/ ml of xylanase activity at 48 hours in the presence of 2 % xylan. Effect of xylan on xylanase production: The effect of xylan on the production of xylanase by the bacteria was observed by adding 1 % xylan in the growth medium. A control was also prepared simultaneously under identical conditions having no xylan in the growth medium. The xylanase production was observed at 12 hours intervals. There was little xylanase production in the control flask whereas bacteria grown in the growth medium having 1% xylan showed significant production of xylanase. It was
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6th Annual Convention of Associatio
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