Amino acid sequence and stereoselective hydrolytic reaction of an endo-1,4-beta-glucanase from a Bacillus strain.
Bacillus sp. KSM-522 produces three different extracellular endo-1,4-beta-glucanases [EGs; Okoshi et al., Agric. Biol. Chem., 54, 83-89 (1990)]. Here, we report the molecular cloning and sequencing of the gene for the fourth EG (EG-IV) of the organism and the mechanism of its hydrolytic reaction. The structural gene contained an open reading frame of 1911 bp, corresponding to 636 amino acids, the amino acid sequence of which was very close to that of an EG of Clostridium cellulovorans, belonging to the cellulase family E2. The molecular mass of the extracellular mature enzyme (Ser26 through Lys636) was calculated to be 69,076 Da, a value close to the 69.2 kDa measured for the recombinant EG-IV expressed in Bacillus subtilis. The optimum pH and temperature for activity of the recombinant enzyme were pH 8.0 and 50 degrees C, respectively. By 1H-NMR spectroscopy, we demonstrated that the hydrolysis of p-nitrophenyl beta-D-cellotrioside by EG-IV proceeded with inversion of the anomeric configuration.
Hitomi
J
J
Tochigi Research Laboratories, Kao Corporation, Japan.
Hatada
Y
Y
Kawaminami
S
S
Kawai
S
S
Ito
S
S
D000595:Amino Acid Sequence; D001407:Bacillus / Q000201:enzymology*; D001412:Bacillus subtilis / Q000235:genetics; D001483:Base Sequence; D002480:Cellulase / Q000737:chemistry / Q000235:genetics*; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000201:enzymology; D005798:Genes, Bacterial; D006868:Hydrolysis; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D009578:Nitrobenzenes / Q000737:chemistry; D011994:Recombinant Proteins / Q000737:chemistry; D013237:Stereoisomerism; D014312:Trisaccharides / Q000737:chemistry
C094834:4-nitrophenyl cellotrioside; D009578:Nitrobenzenes; D011994:Recombinant Proteins; D014312:Trisaccharides; D002480:Cellulase