Expression Of Endo-(1-4)-β-D-glucanase Gene CelD From Clostridium Thermocellum In Escherichia Coli And Pichia Pastoris

Clostridium thermocellum is an anaerobic thermophilic bacterium. The Endo-(1-4)-β-D-glucanase found in Clostridium thermocellum has shown the extremely thermostablity. In this study the endo-(1-4)-β-D-glucanase gene celD from Clostridium thermocellum was cloned and expressed in Escherichia coli BL21 and Pichia pastoris GS115.Then the biochemical properties of two expressed endo-(1-4)-β-D-glucanase were investigated. The main results obtained are as follows:Clostridium thermocellum could produce Endo-(1-4)-β-D-glucanase. Its genome was used as template for gene cloning.The DNA fragment obtained was cloned into pGEM-T Easy Vector, generating pGEM-T Easy TCE Vector. Sequencing result showed that its nucleotide sequence was 2058 bp, containing an Open Reading Frame(ORF) 1947bp,encoding a peptide of 649 amino acids, with a signal peptide of 41 amino acids. The deduced molecular weight of enzyme was nearly 72.4 kDa.Based on two primers 5TCE containing a EcoR I site and 3TCE containing a Not I site, the pGEM-T Easy TCE Vector was double digested with EcoR I and Not I, and then cloned into the expression vector pET-30a(+) previously digested with the same enzymes. The resulting plasmid pET30a(+)-TCE Vector was introduced into E.coli BL21(DE3). The expressed enzyme has a molecular mass of 77 kDa detected by SDS-PAGE.The expression endo-(1-4)-β-D-glucanase has normal bioactivity, which was up to 22 U/ml.After the pGEM-T Easy TCE Vector was double digested with EcoR I and Not I, it was cloned to pPIC9K, an expression vector double digested with the same restriction enzymes, and the recombinant plasmid pPIC9K-TCE was obtained. The plasmid was linearized with BglⅡ,then introduced into the host Pichia pastoris GSll5 by electroporation. The Pichia pastoris recombinants for endo-(1-4)-β-D-glucanase overexpression were screened by enzyme activity analysis and SDS-PAGE. It had an apparent molecular weight of 72 kDa as determined by SDS-PAGE.The result revealed that the endo-(1-4)-β-D-glucanase was expressed and secreted into the medium supernatant, its activity was up to 8U/ml.The biochemical properties of the endo-(1-4)-β-D-glucanase produced by E.coli TCE and Pichia pastoris TCE9K showed that both of them had different temperature of 60℃and 50℃. The results showed that the expressed endo-(1-4)-β-D-glucanase had a high thermostability, still 67% of remaining enzyme activity after 2min at 70℃. The pH for optimal activity of the enzyme was pH 6 and its pH stability is excellent.