Cloning of the thermostable cellulase gene from newly isolated Bacillus subtilis and its expression in Escherichia coli.
A bacterial strain with high cellulase activity (0.26 U/ml culture medium) was isolated from hot spring, and classified and named as B. subtilis DR by morphological and 16SrDNA gene sequence analysis. A thermostable endocellulase, CelDR, was purified from the isolated strain. The optimum temperature of the enzyme reaction was 50 degrees C, and CelDR retained 70% of its maximum activity at 75 degrees C after incubation for 30 min. The putative gene celDR, consisting an open reading frame (ORF) of 1,524 nucleotides and encoding a protein of 508 amino acids with a molecular weight of 55 kDa, was purified from B. subtilis DR and cloned into pET-28a for expression. The cellulase production in E. coli BL21 (DE3) was enhanced to approximately three times that of the wild-type strain.
Li
Wang
W
College of Animal Sciences, Northwest A&F University, Yangling, 712100, People's Republic of China.
Zhang
Wei-Wei
WW
Yang
Ming-Ming
MM
Chen
Yu-Lin
YL
D000595:Amino Acid Sequence; D001412:Bacillus subtilis / Q000201:enzymology* / Q000235:genetics / Q000302:isolation & purification*; D002480:Cellulase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D003001:Cloning, Molecular; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D015870:Gene Expression; D008969:Molecular Sequence Data; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013696:Temperature
D002480:Cellulase