Molecular cloning of two genes for beta-D-glucosidase in Bacillus sp. GL1 and identification of one as a gellan-degrading enzyme.
In the bacterium Bacillus sp. GL1, gellan is depolymerized to give a tetrasaccharide by extracellular gellan lyase and then the tetrasaccharide is converted to constituent monosaccharides by intracellular glycosidases. Two genes encoding one of the glycosidases, beta-D-glucosidase (Bgl), were cloned in a genomic DNA library of the bacterium constructed in Escherichia coli and nucleotide sequences of the genes were determined. One of the genes, termed bglA, contained an open reading frame (ORF) consisting of 1344 base pairs coding a polypeptide (BglA) with a molecular mass of 51 kDa and the other, termed bglB, 2268 base pairs coding a protein (BglB) with a molecular mass of 82 kDa. By homology analyses of the ORFs against protein sequence databases, beta-D-glucosidase A (BglA) and beta-D-glucosidase B (BglB) were found to be classified into subfamilies BGA and BGB of cellulase family BG, respectively. BglA and BglB purified from E. coli were monomeric enzymes with molecular masses of 50 and 82 kDa and most active at pH 6.0 and 8.0, respectively. BglA showed broader substrate specificity than BglB. Only BglA acted on the tetrasaccharide produced from gellan by gellan lyase and released glucose from the molecule.
Hashimoto
W
W
Research Institute for Food Science, Kyoto University, Uji, 611-0011, Japan. hasimoto@food2.kyoto-u.ac.jp
Miki
H
H
Nankai
H
H
Sato
N
N
Kawai
S
S
Murata
K
K
D000595:Amino Acid Sequence; D001407:Bacillus / Q000201:enzymology* / Q000235:genetics*; D001483:Base Sequence; D002240:Carbohydrate Sequence; D002413:Cations, Divalent / Q000737:chemistry; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D005798:Genes, Bacterial / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011135:Polysaccharides, Bacterial / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis; D017422:Sequence Analysis, DNA; D012689:Sequence Homology, Nucleic Acid; D013379:Substrate Specificity; D013696:Temperature; D001617:beta-Glucosidase / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification
D002413:Cations, Divalent; D011135:Polysaccharides, Bacterial; D011994:Recombinant Proteins; C048288:gellan gum; D001617:beta-Glucosidase