Molecular cloning and expression of an endo-beta-1,4-D-glucanase I (Avicelase I) gene from Bacillus cellulyticus K-12 and characterization of the recombinant enzyme.
Bacillus cellulyticus K-12 Avicelase (Avicelase I; EC 3.2.1.4) gene (ace A) has been cloned in Escherichia coli by using the vector pT7T3U19 and HindIII- HindIII libraries of the chromosomal inserts. The libraries were screened for the expression of avicelase by monitoring the immunoreaction of the antiavicelase (immunoscreening). Positive clones (Ac-3, Ac-5, and Ac-7) contained the identical 3.5-kb HindIII fragment as determined by restriction mapping and Southern hybridization, and expressed avicelase efficiently and constitutively using its own promoter in the heterologous host. From the immunoblotting analysis, a polypeptide that showed a carboxymethylcellulase (CMCase) activity with an Mr of 64,000 was detected. The recombinant endo 1,4-beta-D-glucanase I was purified to homogeneity from an intracellular fraction of E. coli by DEAE-Toyopearl M650, Phenyl Toyoperal M650, and TSK gel HW50S chromatography. The enzyme had a monomeric structure, its relative molecular mass being 65 kDa by gel filtration and 64 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The pI was 5.3 and the optimal pH was 4.6, and the enzyme was stable at pH 4.0-10.5. The enzyme had a temperature optimum of 50 degrees C and was stable at 55 degrees C for 48 h, and retained approx 20% of its activity after 30 min at 70 degrees C. It showed high activity toward carboxymethylcellulose (CMC) as well as p-nitrophenyl-beta-D-cellobioside, 4-methylumbelliferyl cellobioside, Avicel, filter paper, and some cellooligosaccharides. Km values for CMC and Avicel were 7.6 and 85.2 mg/mL, respectively, whereas Vmax values were 201 and 9.2 micromol x min(-1) x mg(-1), respectively. Cellotetraose (G4) was preferentially cleaved into cellobiose (G2) and cellopentaose (G5) was cleaved into G2 + cellotriose (G3), whereas cellohexaose (G6) was cleaved into G4 + G2 and, to a lesser extent, into G3 + G3. G3 was not cleaved at all. G2 was the main product of Avicel hydrolysis. G2 inhibited whereas Mg++ stimulated the activity of CMCase and Avicelase. Hydrolysis of CMC took place with a rapid decrease in viscosity but a slow liberation of reducing sugars. Based on these results, it appeared that the cellulase should be regarded as endo type, although it hydrolyzed Avicel.
Department of Gynecology and Biochemistry and Molecular Biology, College of Oriental Medicine, Dongguk University, Kyungju-City, Kyungpook 780-714, Korea.
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