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Current Trends in Biotechnology and Pharmacy Vol. 6 (2) 190-195 April 2012, ISSN 0973-8916 (Print), 2230-7303 (Online) Optimization of Xylanase Secretion from Paenibacillus macquariensis Meeta Sharma and Anil Kumar* School of Biotechnology, Devi Ahilya University, Khandwa Rd., Indore-452001, India *For Correspondence - ak_sbt@yahoo.com Abstract A sporulating bacteria having potential to produce xylanase has been isolated from the soil mixed with petroleum products. The isolated bacteria retained violet color upon gram straining revealing its Gram positive nature. This strain was identified as Paenibacillus macquariensis based on biochemical characterization and deposited in MTCC with an accession number RC1819. The bacteria showed growth at wide variability of pH ranging from pH 5.2 to 10.0 and the temperature ranging from 25 to 37 o C. The optimum pH for xylanase production has been found to be in the range of pH 8.2 to 8.6 and the optimum temperature for xylanase production is found to be 37°C. It secreted xylanase in the culture medium (extra-cellular xylanase) in the presence of 2% xylan. It secreted nearly 4.50 units of xylanase per ml of culture medium. The data revealed that Paenibacillus macquariensis has high potential for xylanase secretion and can be used for industrial applications. Introduction Plant cell wall is mainly composed of cellulose and hemicelluloses and provides a protective barrier against the enzymatic attack of cellulases and xylanases (1). Xylan is a heterogeneous polymer composed of 1,4 linked β-D xylosyl residues. It is much abundant in nature being the major component of hemicelluloses of monocotyledonous cell walls. The complete degradation of system requires a multi-step process involving xylanases and 190 various xylan debranching enzymes viz. βxylosidase, acetylxylan-esterase, α-glucuronidase and α-arabino-furanosidase (2,3). Xylanases in conjunction with other enzymes viz. cellulases, glucanases and proteases, are widely used in animal feed, brewing, food processing and waste treatment as well as in paper and pulp industries (4-6). Combined application of xylanase, β-1,3 and 1,4glucanases can reduce the intestinal viscosity of feed for higher nutrition availability and improve the filtration rate and extraction yield in the brewing industry (7,8). Xylanase (EC 3.2.1.8) acts on β-1,4 xylan and splits β-1,4 glycosidic linkage randomly (4,9). The products are xylose, xylobiose and xylooligosaccharides and these products are used as feedstock for food and fine chemicals (10). It is an industrially important enzyme having applications in paper industry for brightening the pulp, clarification of fruit juices (9,11-12). Use of xylanase in paper industry is eco-friendly since chemicals used in paper industry cause pollution (13-15). Here, we reported isolation of a bacteria which has been identified as Paenibacillus macquariensis and has been given accession number RC-1219 by the MTCC, Chandigarh. We also optimized the conditions for xylanase production by this bacteria. Materials and Methods Collection of samples: Soil samples were collected from different places of Indore (Madhya 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) Pradesh) including one sample from petrol pump where soil was mixed with petroleum products. Screening of bacterial isolates: All soil samples were made semi-dried in the laboratory using autoclaved water. The 10 2 to 10 7 times dilutions were made from the suspension of different soil samples. Serially diluted soil samples were plated on Luria Broth (LB) nutrient agar plates and incubated at 37°C for 24 hours. The LB medium was comprised of bacto-tryptone, 10gm; yeast extract, 5gm; sodium chloride, 10gm; agar, 15gm per litre and adjusted to pH 10 using 0.1N sodium hydroxide. Few distinct colonies with distinct morphology were isolated and transferred to Petri-plates having Emersion medium (yeast extract, 5 gm; peptone, 5 gm; K 2 HPO 4 , 1 gm; MgSO 4 . 7H 2 O, 0.2 gm per litre and pH adjusted to 9 with 1N NaOH) and incubated at 37°C for 24 hours. The Emersion medium was supplemented with 1% xylan in order to isolate xylan utilizing bacteria. Screening for xylanase activity: The colonies obtained were screened for xylanase activity using congo red dye method (16). A set of replica plates was prepared and incubated at 37°C for 24 hours. Thereafter, one of the replica plate was flooded with 0.5% congo red dye and subsequently with 1 mM solution of sodium chloride. A clear zone around the colony was considered as indication of xylanase activity. The corresponding colonies from other replica plate were inoculated individually in Emersion medium broth, allowed to grow overnight at 37°C at 200 rpm in an orbital shaker. The xylanase activity was analyzed in the medium. Morphological, biochemical and physiological studies of the bacteria: Gram staining was done using Kit from Hi-media. After staining, slides were observed in a phase contrast microscope. For biochemical and physiological studies and identification of the bacteria, the culture was sent to Microbial Type Culture Collection and Gene Bank, Institute of Microbial Technology, Chandigarh. Meeta Sharma and Anil Kumar 191 Production of xylanase: The isolated bacteria was maintained on Emerson medium slants by routine sub-culturing every month and used for the production of xylanase. The growth from 24 hours old Emerson slant was scrapped into sterile Emerson medium contained in a 250 ml capacity Erlenmeyer flask and allowed to grow at 37 o C on an orbital shaker with a speed of 200 rpm. A 2 ml aliquot of this suspension was used as an inoculum for 100 ml medium. The flasks were incubated at 37 o C for 48 hours with a speed of 200 rpm. Thereafter, the bacterial cells were harvested by centrifugation of the broth at 10,000 x g for 30 minutes at 0 to 4°C in a cooling centrifuge. The supernatant contained most of the xylanase activity. Growth time optimization for xylanase production: Samples from growing broth were drawn at 4 hours intervals, centrifuged at 10,000 x g for 15 minutes in the cold condition (0 to 4°C). The resulting supernatants were analyzed for xylanase activity. Enzyme assay: Xylanase enzyme was assayed by measuring the release of the reducing sugars from birch wood xylan following the dinitrosalicylic acid (DNS) method (17). The D- xylose was used as standard during the colorimetric estimation. One unit of xylanase activity was taken as the amount of the enzyme required to release one micromole of the reducing sugar equivalent to one micromole of xylose per minute at 50 o C under the conditions of the enzyme assay. Optimization of culture conditions for xylanase production: For optimization of culture conditions, pHs ranging from pH 8.0 to 11.0 with a difference of 0.1 unit were maintained in the culture medium. The xylanase activity and bacterial growth was measured. The bacterial growth was measured using absorbance at 600 nm. The time of growth was also optimized in the range of 12 to 96 hours by withdrawing the aliquots aseptically at 12 hours intervals and analyzed for the xylanase activity. For
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6th Annual Convention of Associatio
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