[Cloning and characterization of a beta-glucosidase from marine metagenome].
In the present study, through a functional strategy, a metagenome library of the marine microbes from the surface water of the South China Sea was screened for beta-glucosidase and six positive clones were obtained. One of these clones, pSB47B2, was subcloned and further analysed in sequence. The result showed that there was an open reading frame for a novel beta-glucosidase, which was nominated as bgl1B. Using pET22b(+) as vector and Escherichia coli BL21(DE3) as host, Bgl1B was overexpressed recombinantly with high yield obtained and substantial enzymatic activity detected. The recombinant protein (rBgllB) was purified by Ni-NTA affinity chromatography and further biochemically characterized. The results indicated that, with pNPG as substrate, the optimum pH and temperature for the hydrolytic activity of rBgl1B were about 6.5 and 40 degrees C respectively. Under the optimum conditions, rBgl1B hydrolyzed pNPG with an activity up to 39.7 U/mg, Km and Vmax being 0.288 mmol/L and 36.9 micromol/min respectively. In addition, rBgl1B could also hydrolyze cellobiose, with a Km of 0.173 mmol/L and a Vmax of 35 micromol/min. However, we did not detect evident hydrolytic activity of rBgl1B to lactose, maltose, sucrose, and CMC. The enzymatic activity of rBgl1B to pNPG was stimulated to certain degrees by low concentration of Ca2+ or Mn2+, whereas it exhibited significant tolerance against high Na+. Distinguished from most of the beta-glucosidases derived from fungi, which display the highest activities under acidic conditions, rBgl1B exhibited relatively higher activity and stability at pH between 7.0 and 9.0.
Fang
Wei
W
Biotechnology Center, School of Life Science, Anhui University, Hefei 230039, China.
Fang
Zemin
Z
Liu
Juanjuan
J
Hong
Yuzhi
Y
Peng
Hui
H
Zhang
Xuecheng
X
Sun
Baolin
B
Xiao
Yazhong
Y
D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D054892:Metagenome / Q000235:genetics*; D056186:Metagenomics / Q000379:methods; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D012623:Seawater / Q000382:microbiology*; D001617:beta-Glucosidase / Q000096:biosynthesis / Q000235:genetics*
D011994:Recombinant Proteins; D001617:beta-Glucosidase