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Title 

Characterization and substrate specificity of an endo-β-1,4-D-glucanase I(Avicelase I) from an extracellular multienzyme complex of Bacillus circulans

Authors 

Chul Ho Kim

Publisher 

American Society for Microbiology

Issue Date 

1995

Citation 

Applied and Environmental Microbiology, vol. 61, no. 3, pp. 959-965

Keywords 

amino acid sequencebacillus circulansenzyme activityenzyme analysisenzyme purificationenzyme specificityhydrolysisbacillusdextrinshydrogen-ion concentration

Abstract 

An endo-1,4-β-D-glucanase I (Avicelase I; EC 3.2.1.4) was purified to homogeneity from an extracellular celluloxylanosome of Bacillus circulans F- 2. The purification in the presence of 6 M urea yielded homogeneous enzyme. The enzyme had a monomeric structure, its relative molecular mass being 75 kDa as determined by gel filtration and 82 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The pI was 5.4, and the N-terminal amino acid sequence was ASNIGGWVGGNESGFEFG. The optimal pH was 4.5, and the enzyme was stable at pH 4 to 10. The enzyme has a temperature optimum of 50°C, it was stable at 55°C for 46 h, and it retains approximately 20% of its activity after 30 min at 80°C. It showed high- level activity towards carboxymethyl cellulose (CMC) as well as p- nitrophenyl-β-D-cellobioside, 4-methylumbelliferyl cellobioside, xylan, Avicel, filter paper, and some cello-oligosaccharides. K(m) values for birch xylan, CMC, and Avicel were 4.8, 7.2, and 87.0 mg/ml, respectively, while V(max) values were 256, 210, and 8.6 μmol · min-1 · mg-1, respectively. Cellotetraose was preferentially cleaved into cellobiose (G2) plus G2, and cellopentaose was cleaved into G2 plus cellotriose (G3), while cellohexaose was cleaved into cellotetraose plus G2 and to a lesser extent into G3 plus G3. G3 was not cleaved at all. G2 was the main product of Avicel hydrolysis. Xylotetraose (X4) and xylobiose (X2) were mainly produced by the enzyme hydrolysis of xylan. G2 inhibited the activity of carboxymethyl cellulase and Avicelase, whereas Mg2+ stimulated it. The enzyme was completely inactivated by Hg2+, and it was inhibited by a thiol-blocking reagent. Hydrolysis of CMC took place, with a rapid decrease in viscosity but a slow liberation of reducing sugars. On the basis of these results, it appeared that the cellulase should be regarded as endo-type cellulase, although it hydrolyzed Avicel.

ISSN 

0099-2240

Appears in Collections

1. Journal Articles > Journal Articles

Registered Date

2017-04-19


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