CHARACTERIZATION AND IMPROVED PRODUCTION OF XYLANOLYTIC ENZYMES OF THERMOPHILIC MICROORGANISMS

Interest in xylanase production and applications is due to its importance in the bioconversion of hemicellulose and pulp and paper industry.\ud \ud An active xylanolytic Bacillus subtilis IBB strain was isolated by enrichment culture and serial dilution methods after screening a large number of isolates. Culture conditions were optimized for maximal cell growth and enzyme production. Significant level of xylanase was secreted when B. subtilis IBB was grown in the presence of glucose as carbon source suggesting that enzyme is synthesized constitutively. Maximum cell growth and xylanase production were obtained when B. subtilis IBB was grown on xylan as a carbon source and ammonium sulphate as a nitrogen source at pH 6.0 after 10 hours of fermentation at 50oC. When the medium was supplemented with .0.25% sucrose, in addition to 0.5% xylan. Xylanase activity increased by more than 2 fold. Xylanase activity was enhanced by another 20% when 0.2% Tween 80 was added while 0.2% Triton-X 100 increased the activity by 10%. The maximal xylanase activity obtained after optimizing fermentation conditions was 12.8 U/ml of culture supernatant. Amongst the natural substrates used, 0.5% pretreated bagasse was found to be the best carbon source for xylanase production giving 4.0 U of xylanase per ml of culture supernatant. About 1.7 fold increase in xylanase activity97.0 U/ml) was observed when the medium was supplemented with 0.25% sucrose.\ud \ud The enzyme was purified 7.5 fold by chromatography on Q-Sepharose chromatofcusing and gel filtration on Sephadex G-75. The molecular weight of the purified xylanase was found to be 22 kDa by SDS-PAGE. The purified enzyme showed a high specific activity of 1200 units/mg protein. The optimum pH and temperature for xylanase 25 ml increased from 15.1 to 28.0 and 14.8 to 28.2 % respectively when amount of the enzyme was increased from 10U to 20U. On further increasing the enzyme concentration upto 40U, the degree hydrolysis of rice straw and bagasse increased from 28.2 to 36.0 and 28.2 to 35.5% respectively.\ud \ud Xylanase treatment to Kraft pulp resulted increased percentage loss in weight with increasing incubation period Kappa number decreased from 70.0 to 56.0 when the incubation period was increased upto 6 hours in 25 ml reaction mixture containing 1.0 g of pulp sample at 50oC . 2.0-6.8% weight losses of pulp samples were observed when the amount of xylanase was increased from 20 to 100U under the same reaction conditions. Kappa number decreased from 70.0 to 45.0 with the loss of lignin contents from 10.0 to 6.4% when the amount of xylanase was increased from 20 to 100U. the amount of reducing sugars in the hydrolysates increased with the increasing enzyme concentration.\ud \ud When the amount of pulp was increased from 1.0 to 3.0 g in 25 ml reaction mixture, the percentage weight losses decreased from 5.2 to 4.0. An increase in reducing sugars was observed in the hydrolysates. Lignin contents were reduced accompanied by decrease in kappa number from 70.0 to 41.0\ud \ud The gene coding for xylanase activity in B. subtilis IBB was cloned E. coli by using pUC 19 as a vector. The transformants were screened for xylanase activity on the basis of clearing haloes around the colonies grown on agar plates containing xylan as detected by staining with Congo red. A single xylanase positive clone was identified containing 1.8 kb genomic insert. Xylanase activity secreted in the culture medium by E. coli clone was 1.5 fold higher than that produced by the parent B. subtilis strain and supplementation of LB medium with xylan resulted in the further increase in xylanase activity. This showed that the expression of xylanase gene was under the control of the xylanase promoter carried by the insert. Southern hybridization analysis showed that Sau3AI fragment of the chromosomal DNA was cloned in recombinant plasmid.\ud \ud Xylanase from E. coli clone was purified by Q-Sepharose chromatography and chromatofocusing. The purified enzyme had a MW of 32 kDa. The pH and temperature optima were 6.0 and 60oC, respectively. The enzyme was stable upto 60oC and between the pH range of 5.0-8.0. The enzymatic properties were almost identical to those of purified xylanase of parent strain i.e. B. subtilis IBB except the molecular weight. Insertion of xylanase gene into cloning vector may be performed in such a way so as to fuse each other, producing a hybrid gene, so the product of gene expression is a hybrid protein with increased molecular weight.\ud \ud Production of xylanases from xylanase genes cloned into a non cellulolytic host is one means of achieving culluluase free preparation. So crude culture supernatant from E. coli xylanase positive clone holds a greater potential for mass production and applications in pulp, paper and jute processing industries