Molecular cloning of endo-β-1,4-glucanase gene of clostridium thermocellum in escherichia coli

Abstract

Endo-$\beta$-1,4-glucanse gene of Clostridium thermocellum ATCC 27405 was cloned in Escherichi coli JM 83 using pUC 9 plasmid as a cloning vector. Chromosomal DNA of C. thermocellum was isolated and digested with EcoRI. pUC 9 DNA was also digested with EcoRI and dephosphorylated by treating with calf intestinal phosphatase. Both digested DNA preparations were mixed and ligated with T4 DNA ligase. With the ligation mixture E. coli JM 83 was transformed to make a gene library. Approximately 1750 ampicillin resistant E. coli transformants were obtained. The transformants were grown on LB agar plates containing carboxymethylcellulose (CMC) and screened for CMCase-positive colonies using Congo Red dye method. Three colonies formed halos indicating hydrolysis of CMC. From a colony showed the largest halo, recombinant plasmids were isolated, retransferred into E. coli JM 83, and through the Congo Red plate-assay insertion of a CMCase directing gene in the recombinant plasmid was confirmed. The new recombinant plasmid contained a 4 kb chromosomal DNA fragment in which a CMCase gene is contained. Restriction sites on the 6.7 Kb pKH 40 were mapped. The crude enzyme produced by E. coli JM 83 (pKH 40) hydrolyzed CMC, pNPC, filter paper and Avicel. The hydrolytic ratios of CMC/pNPC and CMC/filter paper were 200 and 74, respectively. From there, the enzyme encoded by the C. thermocellum gene was identified as endo-$\beta$-1,4glucanase. Further characterization of this enzyme differs from that of other C. thermocellum cellulases reported previously. We have cloned a new endo-$\beta$-1,4-glucanase gene from C. thermocellum.