Paenibacillus campinasensis BL11纖維素酵素基因之分子選殖、大量表現與純化

Abstract

Paenibacillus campinasensis BL11 was isolated by screening bacterial strains from thermo-alkaline black liquor (58oC, pH 9.83). The black liquor was collected from Hsinying Paper Mill of Taiwan Pulp and Paper Company. The collected sample contained large amounts of hemicellulose, lignin and small fiber fragments. Strain BL11 could exhibit many kinds of polysaccharide hydrolases. Strain BL11 exhibited strong cellulase activity and possessed three different kinds of cellulases. According to the result of SDS-PAGE and zymogram the cellulases are 42 kDa, 57 kDa and 87 kDa in molecular mass, respectively. One of the cellulases has been cloned and analyzed. The cellulase gene (PcBC56) was cloned in Escherichia coli (DH5α) with pBCKS(+). The cellulase-related plasmid, pBCCMC1, was isolated by screening a library of randomly cloned BL11 DNA fragments on carboxylmethylcellulase (CMC) indicator plates. The cloned BL11 cellulase gene is composed of 1,473 bp nucleotides that is a C-terminal-lost DNA sequence. PcBC56 was fused with 75 bp of MCS of pBCKS(+) and became a new ORF (PcBC56b) which encode a protein of 56 kDa. The N-terminal of the cellulase contains a deduced signal peptide of 29 amino acids in length and a glycosyl hydrolase domain (catalytic domain) in family 5. There is a carbohydrate binding module in family III at C-terminal of this. The cloned BL11 cellulase was fused with a His-tag at its C-terminal for purification by gene manipulation. The engineered BL11 cellulase was induced with 0.1 mM IPTG at 28oC in an E. coli host for overexpression. The induced cellulase was purified with Ni-NTA agarose by bio-affinity but without success. In the other hand, zymographic analysis of the PcBC56b gave two cellulase activities whose molecular weights were 56 kDa and 35 kDa. The apparent ca. 18 kDa different between extracellular cellulase and its precursor form minus the signal peptide (ca. 3 kDa) could be due to one of several post-translational modifications. The C-terminal segment of the cellulase might be removed so that the purification would be fail because of the loss of His-tag. The optimal temperature and pH of the induced crude cellulase were 70oC and pH 6. The induced crude cellulase activity was 170.97 IU/mg. It would be mush better after purification and have potential for further industrial application.