Cloning, characterization, and nucleotide sequence of a gene encoding Microbispora bispora BglB, a thermostable beta-glucosidase expressed in Escherichia coli.
Genomic DNA fragments encoding beta-glucosidase activities of the thermophilic actinomycete Microbispora bispora were cloned into Escherichia coli. Transformants expressing beta-glucosidase activity were selected by their ability to hydrolyze the fluorogenic substrate 4-methylumbelliferyl-beta-D-glucoside. Two genes encoding beta-glucosidase activity were isolated and distinguished by restriction analysis, Southern hybridization, and the substrate specificities of the encoded enzymes. One gene, bglB, encoded a beta-glucosidase that was expressed intracellularly in E. coli. It exhibited a molecular mass of approximately 52,000 Da by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (PAGE) and 51,280 Da by nondenaturing gradient PAGE, a pI of 4.6, and temperature and pH optima of 60 degrees C and 6.2, respectively. Cloned BglB showed greater activity against cellobiose than against aryl-beta-D-glucosides and was thermostable, retaining about 70% of its activity after 48 h at 60 degrees C. BglB activity is activated two- to threefold in the presence of 2 to 5% (0.1 to 0.3 M) glucose. The DNA sequence of the 2.2-kb insert carrying bglB has been determined. An open reading frame which codes for a protein of 473 amino acids with a predicted molecular mass of 52,227 Da showed significant homology (40 to 47% identity) with beta-glucosidases from glycosal hydrolase family 1.
Wright
R M
RM
Department of Biochemistry and Microbiology, Cook College, Rutgers University, New Brunswick, New Jersey 08903-0231.
Yablonsky
M D
MD
Shalita
Z P
ZP
Goyal
A K
AK
Eveleigh
D E
DE
D000192:Actinomycetales / Q000201:enzymology / Q000235:genetics*; D000595:Amino Acid Sequence; D001482:Base Composition; D001483:Base Sequence; D003001:Cloning, Molecular; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D006023:Glycoproteins / Q000032:analysis; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011994:Recombinant Proteins / Q000096:biosynthesis; D015183:Restriction Mapping; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D001617:beta-Glucosidase / Q000096:biosynthesis / Q000235:genetics*
D006023:Glycoproteins; D011994:Recombinant Proteins; D001617:beta-Glucosidase