CLONING OF THE CELB GENE ENCODING ENDO-1,4-BETA-GLUCANASE-2 FROM CLOSTRIDIUM-JOSUI IN ESCHERICHIA-COLI AND THE PROPERTIES OF THE TRANSLATED PRODUCT

The gene celB on a 3.9-kilobase-pair (kbp) EcoRI fragment encoding endo-1,4-beta-glucanase of Clostridium josui was cloned into Escherichia coli. The structural gene located on the 1.6 kbp Sau3AI fragment excised from the EcoRI fragment was expressed by the lac promoter in the transformant E. coli JM103 (pUCJ2) in modified Luria-Bertani broth with an activity 1,000 times (1120 U/l) higher than that on the EcoRI fragment. The translation product of celB in pUCJ2 was purified by CM Bio-Gel A column chromatography. The homogeneous enzyme was 42 kD of the molecular weight by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The optima for the temperature and pH of the enzyme were 60-degrees-C and 5.5, respectively. The enzyme hydrolyzed cellotetraose, cellopentaose and cellohexaose but not cellobiose and cellotriose. The mode of degradation of cellooligomers (G4 --> 2G2, G5 --> G2 + G3, G6 --> 2G3) of the enzyme suggested that it acts as an endo-1,4-beta-glucanase. This endoglucanase is distinguishable from those characterized by us previously with respect to its pH optimum and cellobiose-transferring activity.