The Cloning And Expression Of Xylanase Encoding Gene From Bacillus Circulans BC

In this study the xylanase gene of B. circulans BC was cloned and expressed in the E. coli BL21 by using pET-30a(+) and pGEX-4T-3 as expression vectors, then the biochemical properties of expression products were analyzed. The main results are as follows:Bacilus circulans BC could produce xylanase, and its genome was used as template for xylanase gene cloning. The homology of nucleotide sequences of J3 -1,4-xylanase genes from different microbes in the GenBank were analyzed, and a pair of primers, XP51 for 5' end fragment and XP31 for 3' end fragment, were designed based on their conserved regions. The xylanase gene from Bacillus circulans BC was obtained by touchdown polymerase chain reaction (TD-PCR), then was cloned into pGEM?T easy Vector, generating pGEM?T easy BCX Vector. Sequencing results showed that the nucleotide sequence was 702bp, and it had an open reading frame(ORF) of 639bp encoding a polypeptide of 213 amino acids with a theoretical molecular weight of 23.2kDa. The accession number of the xylanase gene sequence is AF490980 in the GenBank.Strong homology of the nucleotide sequence and amino acid sequence of the xylanase was found between Bacillus circulans BC and other Bacillus sources. The nucleotide sequence and amino acid sequence identity were 98% and 97% for AF490979 (B. subtilis-2); 96% and 97% for X07723 (B. circulans); 96% and 97% for M36648(5. subtilis); 97% and 97% for X59058 (Bacillus sp.); 96% and 97% for Z34519 (B. subtilis 168); 92% and 94% for AF441773 (Bacillus sp. NBL420); 91% and 94% for U51675 (Bacillus sp.), respectively. Comparison of thesequences indicated that there is a putative 28-amino-acid signal peptide in front of the amino terminus of the mature enzyme.Based on the multiple cloning sites(MCS) of expression vectors, two expression primers, upstream primer XP52 containing a BamHI restriction site and downstream primer XP32 including a Xhol restriction site, were designed corresponding to open reading frame of the cloned xylanase gene. By using TD-PCR method a DNA fragment, which comprised of the xylanase encoding region flanked with restriction enzyme sites at both ends, was amplified using pGEM?T easy BCX Vector as template. The PCR fragment was ligated with pGEM?T easy Vector, generating subcloned vector of pGEM?T easy BCXE Vector. The subcloned vector was double digested with restriction endonucleases, and a target fragment was recovered. Then it was inserted into pET-30a(+) vector and pGEX-4T-3 vector at BamHI and Xhol restriction sites, respectively. The recombinant expression plasmids pET-BCXE and pGEX-BCXE were obtained, and then they were transformed into E. coli BL21. Two positive recombinants, E. colt BCE5 containing pET-BCXE vector and E. coli BCE1 harboring pGEX-BCXE vector, were obtained, which forming zones of clearing on RBB-xylan plates. The levels of xylanase activity expressed by E. coli BCE5 and E. coli BCE1 were up to 135.41U/ml (2.33 times as that of B. circulans BC ) and 123.5U/ml (2.13 times as that of B. circulans BC ), respectively.The biochemical properties of the xylanases produced by E.coli BCE5 and E.coli BCE1 showed that both of them had an optimum temperature of 50~60癈 and pH of 5.0, and they were stable at 40 癈 and pH3.0-9.0, these characterizations were similar to that of enzyme produced by B. circulans BC. The xylanase expressed by E. coli BCE5 was purified by ion exchange and gel permeation chromatography, it had an apparent molecular weight of 20.3kDa as determined by SDS-PAGE, which is consistent with that calculated mass from the amino acid analysis of the mature xylanase. It was suggested that the purified xylanase was mature xylanase and there was indeed a signal peptide of 28 amino acids in the xylanase from B. circulans BC.