Bioremediation-related Enzyme Gene Recombination And High-efficiency Expression Technology Research

Cellulose and hemicellulose are the most abundant renewable resource on earth. This bioconversion plays an important role in global carbon cycle. The enzymes from thermophil,show high thermostability and strong catalytic efficiency, these potential can satisfy the demands of industrial production. Escherichia coli is often the first choice for the expression of recombinant enzymes because it's easy, fast and inexpensive cultivation, and its vector systems have been well developed, it is extravagant for using existing expression systems of E.coli to produce large amount of industrial enzymes. But it is a common problem for foreign gene form inclusion bodies in the cytoplasm and/or periplasm upon gene over-expression in E.coli pHsh is an expression plasmid that uses a synthetic heat-shock (Hsh) promoter, in which gene expression is regulated by an alternative sigma factor (?32). Gene expression was induced was induced by a temperature upshift. The gene xynA of Thermomyces lanuginosus DSM 5826 was mainly found in inclusion bodies, but using pHsh-ex, high levels of active soluble enzymes was obtained. On this foundation, the gene celD from Clostridium thermocellum ATCC 27405 encoding cellulase D was cloned, CelD was over-produced in E.coli by using plasmid pHsh in soluble formation After site-directed mutagenesis the target protein was over-produced in E.coli, this strategy can be used in industrial production. The fused enzyme associating the Thermoanaerobacter ethanolicus JW200 (xarB) with the Thermomyces lanuginosus DSM 5826 (XynA) was produced in E. coli. Fusion enzyme(XarB-XynA) was compared to the free enzymes (XarB and XynA ) for reducing sugar (RS)release efficiency. But the fusion enzyme (XarB-XynA) was mainly found in inclusion bodies during high-level expression, using plasmid pHsh we tried several approaches t to solve the formation of inclusion bodies.1. The gene celD from C. thermocellum ATCC 27405 encoding cellulase D was cloned into expression vectors pET-20b(+) and pHsh to produce recombinant enzyme, CelD. In comparison, CelD was over-produced in E. coli by using plasmid pHsh in soluble formation.By reducing mRNA secondary structure and replacing the rare codons for N-terminal amino acids of the target protein, the expression level of CelD was increased from 4.1 ±0.3 Uml-1 to 6.4±0.4 U ml-1 in LB medium. CelD was purified by heat treatment and a Ni-NTA affinity. The purified CelD exhibited the highest activity at pH 5.4 and 60?, and retained more than 50% activity after incubated at 70? for 1 h. The cellulase activity of CelD was significantly enhanced by Ca2+ and inhibited by EDTA.2. The trifunctional enzyme (XarB-XynA) associating the T. ethanolicus xylosidasearabinosidase (XarB ) with the T. lanuginosus xylanase (XynA) was produced in E.coli to study the effect of the physical association of the fusion partners on the enzymatic efficiency. Recombinant XarB, XynA and XarB-XynA were purified to homogeneity and characterized. The optimal pH and temperature of the XarB-XynA were found to be similar to those of the XarB and XynA, except for less temperature optimum of ?-arabinosidase activity.Its pH and xylanase activity exhibited more stable than those of the XarB and XynA. Finally,the XarB-XynA was tested for degradation of oat spelt xylan and wheat bran, the XarB-XynA was found to be more facile than the corresponding free enzyme degradation of wheat bran but provided little or no advantage on purified xylan. Furthermore cooperation within a trifunctional enzyme containing linker SAGSSAAGSGSG between each partner was achieved, leading to a trifunctional enzyme with enhanced enzymatic efficiency on arabinoxylan.3. The trifunctional enzyme (XarB-XynA) associating the T. ethanolicus xylosidasearabinosidase (XarB) with the T. lanuginosus xylanase (XynA) was produced in E.coli, but the majority of the fusion protein (XarB-XynA) was found in inclusion bodies .To determine the motifs that caused inclusion body formation, we truncated xynA2 to the fragments encoding only 99 amino acids of N-terminus or 97 amino acids of C-terminus, and fused each fragment to the 3' end of xarB. SDS-PAGE analysis showed that the fusion of either N-terminal or C-terminal polypeptide to the C-terminus of XarB could lead to inclusion body formation. The fused gene xarB-xynA was also cloned into pHsh-ex to generate pHsh-ex-xarB-xynA. SDS-PAGE analysis revealed that the level of soluble XarB-XynA protein of 110 kDa was significantly increased by using pHsh-ex to export the recombinant protein to periplasm. N-acetyl-L-ornithine deacetylase from E. coli K12 and pai2-egfp from human were also cloned into pHsh to improve the soluble expression.