Molecular cloning and characterization of endoxylanase gene and its expression in B.subtilis for mass production of xylobiose

Abstract

A gene encoding the endoxylanase from Bacillus sp. was cloned and expressed in Bacillus subtilis. Genomic DNA fragments of Bacillus sp. derived from partial digestion by Sau3AI were ligated into BamHI site of pUC19, and the resulting plasmids were transformed into E.coli HB101. Of 2,000 transformants screened, one (pKJX1) formed a clear zone on a xylan plate and it was identified to carry 3.1 kb Sau3AI fragment. The insert in the pKJX1 recombinant was subsequently subjected to extensive mapping and a series of subclonings into pUC19. A 750bp Sau3AI subfragment was found to code for the xylanase and its entire nucleotide sequence was determined. The xylanase gene was 642 base pairs long and encoded 213 amino acids. Promoter and SD sequence were located on the upstream regions of the start codon. To be expressed in B. subtilis, a 1.63 kb SmaI fragment was subcloned into a Bacillus expression vector (pJH27U△88) and xylanase activity up to 105.5 Units/ml was obtained with B. subtilis (pJHKJ4). The xylanase produced in B. subtilis (pJHKJ4) were purified by an ion exchange chromatography and products of xylanase reaction were analyzed by HPLC, using a carbohydrate analysis column. When the reactions were carried out at 40℃, xylobiose ($X_2$) was produced from xylan as a major product and its portion in the hydrolysates was increased as time went.