Cloning of the cellulase gene from Ruminococcus albus and its expression in Escherichia coli.
The gene for cellulase from Ruminococcus albus F-40 was cloned in Escherichia coli HB101 with pBR322. A 3.4-kilobase-pair HindIII fragment encoding cellulase hybridized with the chromosomal DNA of R. albus. The Ouchterlony double-fusion test gave a single precipitation line between the cloned enzyme and the cellulase from R. albus. The size of the cloned fragment was reduced by using HindIII and EcoRI. The resulting active fragment had a size of 1.9 kilobase pairs; and the restriction sites EcoRI, BamHI, PvuII, EcoRI, PvuII, and HindIII, in that order, were ligated into pUC19 at the EcoRI and HindIII sites (pURA1). Cellulase production by E. coli JM103(pURA1) in Luria-Bertani broth was remarkably enhanced, up to approximately 80 times, by controlling the pH at 6.5 and by reducing the concentration of NaCl in the broth to 80 mM.
Ohmiya
K
K
Department of Food Science and Technology, School of Agriculture, Nagoya University, Japan.
Nagashima
K
K
Kajino
T
T
Goto
E
E
Tsukada
A
A
Shimizu
S
S
D000332:Aerobiosis; D000818:Animals; D002480:Cellulase / Q000096:biosynthesis / Q000235:genetics*; D003001:Cloning, Molecular; D003470:Culture Media; D004262:DNA Restriction Enzymes; D004269:DNA, Bacterial / Q000235:genetics; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics* / Q000254:growth & development; D005786:Gene Expression Regulation; D006863:Hydrogen-Ion Concentration; D005779:Immunodiffusion; D009693:Nucleic Acid Hybridization; D010459:Peptococcaceae / Q000201:enzymology / Q000235:genetics*; D012417:Rumen / Q000382:microbiology; D014169:Transformation, Bacterial
D003470:Culture Media; D004269:DNA, Bacterial; D004262:DNA Restriction Enzymes; D002480:Cellulase