| In nature, xylanases catalyze the hydrolysis of xylans and play an important role in the circulation of materials; and they have many applications on the textile industry, paper pulp, animal feed, production of fruit wine and juice, etc. In papermaking industry, using xylanolytic enzymes as pulp bleaching auxiliary can increase paper whiteness and strength, reduce the dosage of chlorine, and thus reduce the production cost, and environmental pollution. In the feed industry, hydrolysis of xylan performed by the xylanolytic enzymes added into the feed can alleviate the anti-nutrition functionality of xylan. The hydrolysis product, xylan of low functionality, can promote the growth of bifidobacterium, inhibit the proliferation of putrefactionbacteria, enhance mineral absorption, as well as strengthening immunity. In the food industry, for instances, application of xylanase in bread processing, xylanase can hydrolyze xylan into xy-looligosaccharides inside flour so as to improve the texture, structure and softness of the bread. In addition, xylanolytic enzymes can be used in the brewing industry. Reports also noted the usage of xylanolytic enzymes in the biofuel production in the hope of alleviating the problem of energy deprivation.To date, many of the xylanase genes have been cloned and expressed, and the three-dimental crystal structure has been analyzed. However, Alternaria xylanase has yet to be studied. Plant pathogenic fungi can infect plant tissue, therefore they can produce a complex enzymatic system to degrade lignocellulose. In a previous study Alternaria xylanase gene xyn186was cloned which is the first xylanase gene from genus Alternaria. xyn186gene using its own signal peptide process heterologous expression in Pichia pastoris and the enzymatic properties of XYN186were preliminarily investigated.In this study, the new xylanase gene xyn186’s nucleotide sequences was analyzed. The gene contains a696bp open reading frame, coding19amino acid signal peptide and213amino acids11family glycosyl hydrolysis enzyme. XYN186has two highly conservative amino acid residues of11family glycosyl hydrolysis enzyme (E106, E197), containing two potential N-glycosylation sites. Comparisons of the deduced amino acid sequence of the endoxylanase gene with those of other endoxylanase compiled in the GenBank/EMBL Data Bank revealed high identity to the xylanase B of Aspergillus niger (60%). The mature cDNA was cloned into vector pHBM905A, and expressed in P. pastor is GS115. Xylanase-secreting transformants were selected on plate containing RBB-xylan. Xylanase activity was indicated by the formation of clear halos around the individual clones. Transfromant which have largest halos was selected to induce the xylanase expression. The purified enzyme showed one band with a molecular mass of23kDa, even though it has two putative N-glycosylation sites. After deglycosylation by endoH, the one band remained unchanged, which implies that the recombinant XYN186from the P. pastoris was not glycosylated. Enzymology character study shows that the optimum temperature for the enzyme is50℃, the optimal pH is6, in pH4.5-6.5has more than80%of the activity, is a acidic xylanolytic enzymes. Under the optimal condition used in this study, the enzyme had a specific activity of81.56U·mg-1to substrate birchwood xylan with a Km of1.404mg·ml-1and a Vmax of0.2748mmol·min-1·mg-1. Enzyme in40℃with good thermal stability, but in50℃processing15min almost completely lose activity. Cu2+and Hg2+almost completely inhibited the enzyme activity, other experiment metal ions have little effect on the enzyme. One of the P. pastoris transformants, which was examined, had two copy quantities as was expected. |
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