Construction, purification, and properties of a truncated alkaline endoglucanase from Bacillus sp. KSM-635.
Part of a 2.4-kb DNA fragment that encoded the amino-terminal 584 residues (65 kDa) of an alkaline endoglucanase from Bacillus sp. KSM-635 (941 amino acid residues; 105 kDa) was spontaneously deleted during subcloning of the fragment. The remaining 1.1-kb insert of the deleted plasmid encoded amino acids from Ala228 to Leu584 of the enzyme. However, Escherichia coli HB101 cells harboring this plasmid produced an active endoglucanase. After addition of a termination codon, TAA, immediately downstream of the codon for Leu584, the 1.1-kb fragment was inserted into an expression vector, pHSP64. The resultant plasmid was introduced into Bacillus subtilis ISW1214 for extracellular production of the truncated endoglucanase. The enzyme was then purified to homogeneity from a culture of the recombinant B. subtilis cells. Amino-terminal sequencing of the enzyme showed that the enzyme consisted of 7 amino acid residues encoded by the vector and 357 amino acid residues encoded by the truncated gene, with a molecular mass of 40.2 kDa. The purified enzyme was very active against carboxymethylcellulose and its pH and temperature profiles were almost identical to those of the enzyme produced by Bacillus sp. KSM-635.
Tochigi Research Laboratories of Kao Corporation, Japan.
K
K
Ozaki
Y
Y
Hayashi
N
N
Sumitomo
S
S
Kawai
S
S
Ito
D000595:Amino Acid Sequence; D001407:Bacillus / Q000201:enzymology*; D001412:Bacillus subtilis / Q000201:enzymology / Q000235:genetics; D001483:Base Sequence; D002480:Cellulase / Q000737:chemistry* / Q000235:genetics*; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D005798:Genes, Bacterial; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification; D013696:Temperature; D014170:Transformation, Genetic
D011994:Recombinant Proteins; D002480:Cellulase