Purification and characterization of a Bacillus polymyxa beta-glucosidase expressed in Escherichia coli.
The beta-glucosidase encoded by the bglA gene from Bacillus polymyxa was overproduced in Escherichia coli by using a plasmid in which bglA is under control of the lacI promoter. Induction with isopropyl-beta-D-thiogalactopyranoside allowed an increase in the specific activity of the enzyme of about 100 times the basal level of gene expression. The enzyme was purified by a two-step procedure involving salting out with ammonium sulfate and ion-exchange chromatography with DEAE-cellulose. Fractions of beta-glucosidase activity recovered by this procedure, after electrophoresis in an acrylamide gel and staining with silver nitrate, yielded a single band of protein. This band was shown by a zymogram to correspond to beta-glucosidase activity. The purified protein showed an apparent molecular mass of 50 kDa and an isoelectric point of 4.6, values in agreement with those expected from the nucleotide sequence of the gene. Km values of the enzyme, with either cellobiose or p-nitrophenyl-beta-D-glucoside as the substrate, were determined. It was shown that the enzyme is competitively inhibited by glucose. The effects of different metallic ions and other agents were studied. Hg2+ was strongly inhibitory, while none of the other cations tested had any significant effect. Ethanol did not show the stimulating effect observed with other beta-glucosidases. The mechanism of enzyme action was investigated. High-pressure liquid chromatography analysis with cellobiose as the substrate confirmed previous data revealing the formation of two products, glucose and another, unidentified, compound. Results presented here indicate that this compound is cellotriose formed by transglycosylation.
Painbeni
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E
Instituto de Agroquímica y Tecnología de Alimentos, Consejo Superior de Investigaciones Científicas, Valencia, Spain.
Valles
S
S
Polaina
J
J
Flors
A
A
D001407:Bacillus / Q000201:enzymology* / Q000235:genetics; D003001:Cloning, Molecular; D004790:Enzyme Induction / Q000187:drug effects; D004926:Escherichia coli / Q000187:drug effects / Q000235:genetics; D007544:Isopropyl Thiogalactoside / Q000494:pharmacology; D007763:Lac Operon / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000235:genetics; D011994:Recombinant Proteins / Q000302:isolation & purification; D001617:beta-Glucosidase / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification
D011994:Recombinant Proteins; D007544:Isopropyl Thiogalactoside; D001617:beta-Glucosidase