Molecular cloning and expression in Escherichia coli of a cellodextrinase gene from Bacteroides succinogenes S85.
A DNA fragment coding for a cellodextrinase of Bacteroides succinogenes S85 was isolated by screening of a pBR322 gene library in Escherichia coli HB101. Of 100,000 colonies screened on a complex medium with methylumbelliferyl-beta-D-cellobioside as the indicator substrate, two cellodextrinase-positive clones (CB1 and CB2) were isolated. The DNA inserts from the two recombinant plasmids were 7.7 kilobase pairs in size and had similar restriction maps. After subcloning from pCB2, a 2.5-kilobase-pair insert which coded for cellodextrinase activity was isolated. The enzyme was located in the cytoplasm of the E. coli host. It exhibited no activity on carboxymethyl cellulose, Avicel microcrystalline cellulose, acid-swollen cellulose, or cellobiose but hydrolyzed p-nitrophenyl-beta-D-cellobioside and p-nitrophenyl-beta-D-lactoside. The Km (0.1 mM) for the hydrolysis of p-nitrophenyl-cellobioside by the enzyme expressed in E. coli was similar to that reported for the purified enzyme from B. succinogenes. Expression of the cellodextrinase gene was subjected to catabolite repression by glucose and was not induced by cellobiose. The origin of the DNA insert from B. succinogenes was confirmed by Southern blot analysis. Western blotting (immunoblotting) using antibodies raised against the purified B. succinogenes cellodextrinase revealed a protein with a molecular weight of approximately 50,000 in E. coli clones which comigrated with the native enzyme isolated from B. succinogenes. These data indicate that the cellodextrinase gene expressed in E. coli is fully functional and codes for an enzyme with properties similar to those of the native enzyme.
Gong
J H
JH
Department of Microbiology, University of Guelph, Ontario, Canada.
Lo
R Y
RY
Forsberg
C W
CW
D001439:Bacteroides / Q000201:enzymology / Q000235:genetics*; D015139:Blotting, Southern; D015153:Blotting, Western; D002475:Cellobiose / Q000378:metabolism; D002480:Cellulase / Q000235:genetics* / Q000378:metabolism; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D005786:Gene Expression Regulation; D005947:Glucose / Q000378:metabolism; D005990:Glycerol / Q000378:metabolism; D010957:Plasmids; D015183:Restriction Mapping
D004269:DNA, Bacterial; D002475:Cellobiose; D002480:Cellulase; D005947:Glucose; D005990:Glycerol