Characterization of a beta-glucosidase produced by a high-specific growth-rate mutant of Cellulomonas flavigena.
The mutant strain PN-120 of Cellulomonas flavigena produces a ss-glucosidase that is 10-fold more active than the corresponding enzyme isolated from the parental strain. These enzymes were partially purified through Q Sepharose and Bio-Gel filtration. A single protein band was detected on polyacrylamide-gel electrophoresis/zymogram using 4-methylumbelliferyl-beta-D-glucoside. On sodium dodecyl sulfate-PAGE, the enzyme displayed three protein bands, suggesting that in C. flavigena the enzyme is oligomeric with a molecular mass of 210 kDa. On purification, the specific activity of ss-glucosidase isolated from PN-120 was increased 16-fold and showed three times more affinity for cellobiose than the enzyme of the parental strain; nevertheless, the optimum pH and temperature were similar for both enzymes. The kinetic parameters suggested that the increase in the activity of the enzyme, from the mutant strain, was caused by a mutation that affects the catalytic site of the enzyme. The partial amino-acid sequence of the isolated enzyme confirmed that it is a beta-glucosidase because of its homology with other beta-glucosidases produced by cellulolytic bacteria and fungi.
Barrera-Islas
Gaspar A
GA
Departamento de Biotecnología y Bioingeniería, CINVESTAV-IPN, Avenida IPN 2508, Zacatenco, México, 07000 DF, México.
Ramos-Valdivia
Ana C
AC
Salgado
Luis M
LM
Ponce-Noyola
Teresa
T
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D040143:Cellulomonas / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D004591:Electrophoresis, Polyacrylamide Gel; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D009154:Mutation; D020539:Sequence Analysis, Protein; D017386:Sequence Homology, Amino Acid; D013696:Temperature; D001617:beta-Glucosidase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D001426:Bacterial Proteins; D001617:beta-Glucosidase