Cloning and expression of β-glucosidase from Flavobacterium meningosepticum: A new member of family B β-glucosidase

摘要

A β-glucosidase gene from Flavobacterium meningosepticum has been cloned and expressed in Escherichia coli. The entire nucleotide sequence was determined and analyzed. An open-reading frame of 2,178 bp encoding a polypeptide of 726 amino acids with a calculated M_r of 79,952 was derived from the sequence. The coding region is flanked by a putative promoter and transcription terminator sequences. The nucleotide sequence accession number of the complete gene sequence in Genbank is AF015915. Based on the comparison of amino acid sequences, this enzyme is classified as a family B β-glucosidase and is highly homologous with sequences from Clostridium thermocellum, Kluyvermyces fragilis, and Agrobacterium tumefaciens. The cloned enzyme was purified to near homogeneity by ammonium sulfate fractionation (35 to approximately 75%) and chromatography on a cation-exchanged column at pH 6.9. This expressed β-glucosidase has optimum activity at pH 4.2 to approximately 5.0 and 50 °C and is stable in the pH range of 5.0 to approximately 8.1 at 25 °C. It showed a high specificity on the glycone portion of aryl-β-D-glycosides. All of these characteristics are highly consistent with those of the native β-glucosidase.