Cloning,Expression And Characterization Of Gene Encoding β-xylosidase From Aspergillus Niger

The hemicelluloses consist of xylan is one of the most abundant natural resources and widely existed in the paper, textile and agricultural wastes. Its hydrolysis products can be used as a carbon source for the production of various kinds of fermentation products. Xylan can be hydrolyzed into oligoxylose by the xylanase. Oligoxylose can be further hydrolyzed into two molecules of xylose by the β-xylosidase, which is the rate limiting enzyme for fermentation and utilization of hemicelluloses with the type of xylan. There are more cases for investigation of β-xylosidase in abroad than that of in domestic. It has been studied for the cloning, heterologously expression, and preliminary characterization of the gene of β-xylosidase from an Aspergillus niger strain in this paper.The conserved sequence was identified by homology comparison according to the reported Aspergillus niger β-xylosidase gene sequence. Then the specific primers were designed to amplify the target gene by PCR using an Aspergillus niger strain as material. The cloned β-xylosidase gene sequence was in the whole length of 2415 bp. The similarity was 97% between cloned DNA sequence and known Aspergillus niger β-xylosidase gene sequence, whereas the similarity of amino acid sequence was 99% for them. The 78 nucleotide sequences were predicted as signal peptides. The isoelectric point was predicted as pH 4.73. The pET32a and pPICZ A were used as expression vectors for heterologously expression in E. coli and Pichia pastoris, respectively. It has been found that the expression of the target gene in pET32a was inclusion body. The activity with β-xylosidase was detected both in the fermentation broth and the cell for the expression of pPICZαA. A strain pPICZαA-xyD11 with high β-xylosidase activity was screened out by screening the monoclonal strains expressed in the recombinant pPICZαA vector. The enzyme activity was 5.723 U/mL for the fermentation broth of strain pPICZαA-xyD11in 4 days. The conditions of expression for the strain have been optimized. The inoculation amount of OD600 0.4-0.5 seed was 20%(v/v) of the total volume of the flask. The enzyme activity was reached the highest with induction of 1.5%(v/v) methanol after4-5 days. During the process of induction, the enzyme activity could be decreased by addition of oleic acid and EDTA, whereas addition of 0.3%(v/v) Tween-80 could improve the enzyme activity. The expressed protein was primarily purified.The results of this study have provided the basis for the protein expression, enzymatic characterization, and application of the β-xylosidase gene from this Aspergillus niger strain.