A beta-glucosidase gene (bgl3) from Streptomyces sp. strain QM-B814. Molecular cloning, nucleotide sequence, purification and characterization of the encoded enzyme, a new member of family 1 glycosyl hydrolases.
A beta-glucosidase gene (bgl3) from Streptomyces sp. QM-B814 (American Type Culture Collection 11238) has been cloned by functional complementation of a beta-glucosidase-negative mutant of Streptomyces lividans. An open-reading frame of 1440 nucleotides encoding a polypeptide of 479 amino acids was found by sequencing. The encoded protein (Bgl3) shows extensive similarity (over 45% identity) with beta-glycosidases from family-1 glycosyl hydrolases. The cloned enzyme, purified following ammonium sulphate precipitation and two chromatographic steps, is monomeric with molecular mass 52.6 kDa, as determined by mass spectrometry, and an isoelectric point of pI 4.4. The enzyme appears to be a beta-glucosidase with broad substrate specificity, is active on cellooligomers, and performs transglycosylation reactions. The estimated apparent Km values for p-nitrophenyl-beta-D-glucopyranoside and cellobiose are 0.27 mM and 7.9 mM, respectively. The Ki values for glucose and delta-gluconolactone, using p-nitrophenyl-beta-D-glucopyranoside as a substrate, are 65 mM and 0.08 mM, respectively. The purified enzyme has a pH optimum of pH 6.5 and the temperature optimum for activity is 50 degrees C.
Perez-Pons
J A
JA
Institut de Biologia Fonamental, Universitat Autònoma de Barcelona, Spain.
Cayetano
A
A
Rebordosa
X
X
Lloberas
J
J
Guasch
A
A
Querol
E
E
D000595:Amino Acid Sequence; D001483:Base Sequence; D002475:Cellobiose / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D004274:DNA, Recombinant; D005798:Genes, Bacterial; D005965:Glucuronates / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D009154:Mutation; D013302:Streptomyces / Q000201:enzymology* / Q000235:genetics; D001617:beta-Glucosidase / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism
D004274:DNA, Recombinant; D005965:Glucuronates; C002730:4-nitrophenylglucuronide; D002475:Cellobiose; D001617:beta-Glucosidase