MOLECULAR-CLONING AND EXPRESSION OF A BETA-GLUCOSIDASE GENE FROM RUMINOCOCCUS-ALBUS IN ESCHERICHIA-COLI

A 3.4-kilobase pairs (kb) DNA fragment of an anaerobe, Ruminococcus albus, was cloned in Escherichia coli and shown to direct the synthesis of β-glucosidase. This fragment consisted of four HindIII fragments, and any derivative in which one fragment was deleted did not exhibit enzymatic activity. The activity of β-glucosidase produced by E. coli harboring the recombinant plasmid was 100-fold of that produced by R. albus. The expression of the gene was further increased when the DNA fragment was inserted into the plasmid in the opposite direction. The enzyme encoded by the recombinant plasmid showed the maximum enzymatic activity at 37°C between pH 6.0 and 6.5. The cloned enzyme could hydrolyse various synthetic substrates, cellobiose, and cellooligosaccharides. Glucose was formed from cellooligosaccharides. The majority of the enzymatic activity was detected in the cytoplasm in the E. coli transformant. These enzymatic properties were substantially the same as those of β-glucosidase prepared from R. albus.