Gene Tandem And The Construction Of High-Effective Expression System Of β-Mannanase Gene

The gene expression in tandem of the key enzymes which are required in herbaceous extraction such as mannanase, xylanase etc. and the construction of high-effictive compound-enzyme genetic engineering strain for herbaceous extraction are the keys to ensure the sustainable development of herbaceous fiber industry. After the studies on gene expression in tandem of the mannanase and xylanase, three conclusions were made as follows:1, The fusion PCR is used to study the directional bi-gene splicing of Erwinia carotovora CXJZ11-01 xylanase gene (EU233656) and Erwinia carotovora CXJZ95-198 mannanase gene (DQ364440) in accordence with the permutation methord. The xylanase dimmer was inserted into the pMD18-T simple vector, and the recombinant plasmid was transformed into E.coli JM109 cells. Screened through the selective plates, approximately 70% of positive clones were obtained. Identification of positive clones showed that two xylanase genes have been connected successfully. Determination of the xylanase activity in fermentation broth did not achieve the affinity level, indicating that there are some difficulties in gene expression in tandem.2, According to the reported sequences of Erwinia carotovora CXJZ95-198β-mannase gene (man) and Bacillus thuringiensis CTC S-layer protein promoter (slp), tow pairs of specific primer were designed and synthesized to construct a gene splice of slp-man. The strong promoter of Bacillus thuringiensis CTC Surface Layer Protein and theβ-mannanase gene ORF of Erwinia carotovora CXJZ95-198 were ligated via Gene Splicing by Overlap Extension ( Gene SOEing ). The obeined directional gene splice of slp-man was then inserted into the high-effective expression vector pET-28a+. The recombinat plasmid was trasformed into Escherichia coli JM109, and a high-effective expression system ofβ-mannanase was constructed.3,By using the methords of DNS and hydrolyzed circle caused, the enzyme producing regulation of the high-effective expression system show that, theβ-mannanase activity of the recombinant strain was up to 671.3U/ml after 11 hours′fermentation, which was as 1.48 times as that in E. carotovora CXJZ95-198.