Screening and characterization of an enzyme with beta-glucosidase activity from environmental DNA.
A novel beta-glucosidase gene, bglA, was isolated from uncultured soil bacteria and characterized. Using genomic libraries constructed from soil DNA, a gene encoding a protein that hydrolyzes a fluorogenic analog of cellulose, 4-methylumbelliferyl beta-D-cellobioside (MUC), was isolated using a microtiter plate assay. The gene, bglA, was sequenced using a shotgun approach, and expressed in E. coli. The deduced 55-kDa amino acid sequence for bglA showed a 56% identity with the family 1 glycosyl hydrolase Chloroflexus aurantiacus. Bg1A included two conserved family 1 glycosyl hydrolase regions. When using p-nitrophenyl-beta-D-glucoside (pNPG) as the substrate, the maximum activity of the purified beta-glucosidase exhibited at pH 6.5 and 55 degrees C, and was enhanced in the presence of Mn2+. The Km and Vmax values for the purified enzyme with pNPG were 0.16 mM and 19.10 micromol/min, respectively. The purified BglA enzyme hydrolyzed both pNPG and p-nitrophenyl-beta-D-fucoside. The enzyme also exhibited substantial glycosyl hydrolase activities with natural glycosyl substrates, such as sophorose, cellobiose, cellotriose, cellotetraose, and cellopentaose, yet low hydrolytic activities with gentiobiose, salicin, and arbutin. Moreover, Bg1A was able to convert the major ginsenoside Rb1 into the pharmaceutically active minor ginsenoside Rd within 24 h.
Kim
Soo-Jin
SJ
Microbial Genetics Division, National Institute of Agricultural Biotechnology, Rural Development Administration, Suwon 441-707, Korea.
Lee
Chang-Muk
CM
Kim
Min-Young
MY
Yeo
Yun-Soo
YS
Yoon
Sang-Hong
SH
Kang
Han-Cheol
HC
Koo
Bon-Sung
BS
D000595:Amino Acid Sequence; D004247:DNA / Q000302:isolation & purification*; D015723:Gene Library; D036145:Ginsenosides / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D012988:Soil Microbiology; D013379:Substrate Specificity; D013696:Temperature; D001617:beta-Glucosidase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism
D036145:Ginsenosides; C442759:ginsenoside Rb1; D004247:DNA; D001617:beta-Glucosidase