Cloning and Expression of An β-1,3-1,4-glucanase Gene (bglS) from Endophytic Bacillus amyloliquefaciens Strain TB2 in Escherichia coli and Characteristics of the Enzyme
Abstract：β-1,3-1,4-endoglucanase is a ubiquitous function enzyme in plant endophytic Bacillus. To evaluate the function of the enzyme, a β-1,3-1,4-endoglucanase gene (bglS) was cloned from endophytic B. amyloliquefaciens strain TB2 and expressed in Escherichia coli. The ORF length of the gene was 732 bp, which encoded 243 amino acids. The theoretical molecular weight of the bglS protein was 27.33 kD and pI was 6.89. The ORF sequence has been registered in GenBank (Accession No. EU368224). The nucleotide and deduced amino acid sequences of the gene shared high identities with that of B. amyloliquefaciens FZB42 β-1,3-1,4-endoglucanase which was associated with plant(The genome accession No. of FZB42 in GenBank is CP000560), and both of the identities were 97%. The ORF and its promoter of bglS were inserted into XbaⅠ/HindⅢ-digested pUC18 vector to construct expression plasmid pUC-bglS which was transformed and expressed in Escherichia coli BL 21. The result of SDS-PAGE showed that about 27 kD protein was expressed in E.coli BL 21::pUC-bglS. The optimum temperature and pH for reaction of the enzyme were 55℃ and 6.2, respectively. The enzyme maintained over 85% of the original enzyme activity between pH 4.4 and pH 6.8 after incubated at 4℃ for 30 min. When the enzyme was stored at temperature below 50℃ for 60 min, it could maintained over 80% of the original enzyme activity. Ca2+ and Fe2+ could enhance the enzyme activity, however the Cu2+, Zn2+, Mg2+ and Mn2+ had an inhibited effect on its activity. The result applies a base for further research on β-1,3-1,4-endoglucanase from endophytic B. amyloliquefaciens TB2.
