| Xylanase can hydrolyze B-1, 4-glycosidic linkages of the xylan backbone to produce short chain xylo-oligosacchrides of various lengths. Hence, endo-B-xylanase is the crucial enzyme components of microbial xylanolytic enzyme systems. Microbial xylanases have attracted considerable research interest in recent years because their potential application in the food, animal feed, paper and pulp industries. In the present dissertation, a thermostable xylanase from Thermomyces lanuginosus CBS288.54 were investigated.The effects of different factors on xylanase production by T.lanuginosus CBS288.54 in shake flask conditionEight improved xylanase producing strains were screened by shake flask cultivation after spores of Thermomyces lanuginosus CBS288.54 were treated with UV-irradiation and ethyl-methanesul fonate. Cultivation conditions of the thermophilic fungus Thermomyces lanuginosus CBS288.54-M18 for thermostable xylanase production were investigated. Corn-cob xylan (water -insoluble) was found to be the best inducer and carbon source. Tryptone+yeast extract was the best nitrogen sources. The optimal temperature and the initial pH of the cultures for xylanase production were 50 癈 and pH 7.0, respectively. The highest xylanase activity of 1,834 u/mL was achieved under these optimal conditions.The effects of different factors on xylanase production by T.lanuginosus CBS288.54-M18 under solid state culture (SSC)The cultivation conditions for the production of thermostable xylanase from Thermomyces lanuginosus CBS288.54-M18 under solid state culture (SSC) were investigated. The effects of carbon and nitrogen sources, carbon source to mineral solution ratio, fermentation temperature and initial pH of culture on enzyme production were studied. Results showed that, by using wheat bran and corncob at the ratio of 8:2 as the carbon source, yeast extract and tryptone at the ratio of 1:1 as the nitrogen source, amount of xylanase produced by the culture increased 200% compared to those without optimized carbon and nitrogen sources. The optimal carbon source to mineral solution ratio and the initial pH of cultures for enzyme production was 1:3 and pH7.0, respectively. Under these conditions, the culture yielded cellulose-free and protease-free xylanase with activity of 15023 U/g of carbon source after fermentation at 50C for 5 days.Purification of the culture filtrate of T.lanuginosus CBS 288.54-M18Xylanase was purified 33.6-fold to homogeneity by ammonium sulfate precipitation, gel filtration, and ion exchange chromatography with a recovery yield of 21.5%. It appeared as a single protein band on SDS-PAGE gel with a molecular mass of approx. 26.2 kDa.Characterizations of purified xylanaseThe purified xylanase had a neutral optimum pH within the range of pH 7.0-7.5, and it was stable over pH 6.5-10.0. The optimal temperature of xylanase was 70癈 and it was stable up to 60C at pH 7.5. The xylanase was highly specific towards xylan, but did not exhibit other enzyme activity. Apparent Km values of the xylanase for birchwood, beechwood, soluble oat-spelt and insoluble oat-spelt xylans were 4.0,4.7,2.0 and 23.4 mg ml-1, respectively. Based on the molecular mass, catalytic properties and amino acid composition, the xylanase is a typical endo-acting xylanase and belongs to the Family 11 xylanase.Chemical modification of purified xylanase from T.lanuginosus CBS 288.54-M18Chemicals like NBS, WRK, DTNB, PMSF, Phenylgloxal hydrate, DEPC were used to react with the thermostable xylanase from Thermomyces lanuginosus. The thermostable xylanase could be inactivated by NBS(N-bromosuccinmide) and WRK(N-ethyl-5-phenylisoxazolium-3-sulfonate), indicating that try-ptophan residues and glutamate/aspartate residues might be involved in the active site of the enzyme. Chemical modification on the xylanase with NBS and WRK revealed that trytophan and glutamate/aspartate were essential for its activity. Three-mg/mL birchwood xylan inhibited the inactivation of xylanase by NBS completely. The xylan substrate had no effect on...
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