Properties of a metagenome-derived beta-glucosidase from the contents of rabbit cecum.
In this study, a previously cloned beta-glucosidase gene, umbgl3B, was heterologously expressed in Escherichia coli, and the biochemical properties of the purified enzyme were characterized. The recombinant enzyme was stable over a wide range of pH values (5.0-9.0) and below 30 degrees C. It displayed optimum enzymatic activity at pH 6.5 at 40 degrees C, under condition similar to that in the rabbit cecum, suggesting an active role of the native enzyme in vivo. The recombinant beta-glucosidase Umbgl3B showed high activity to aryl beta-D-glucosides and low activity to cellooligosaccharides, with a polymerization degree of less than 5. The enzyme had no activity toward long cellooligosaccharides or polysaccharides. The aspartic acid residue, D772, of the wild-type Umbgl3B was predicted as a nucleophile. Mutant D772A was constructed. It showed less than 1/10,000 activity of the wild-type enzyme, but had the same properties, suggesting that residue D772 plays a key role in the enzyme's activity.
Feng
Yi
Y
Guangxi Key Laboratory of Subtropical Bioresource Conservation and Utilization, College of Life Science and Technology, Guangxi University, Nanning, Guangxi, Peoples Republic of China.
Duan
Cheng-Jie
CJ
Liu
Li
L
Tang
Ji-Liang
JL
Feng
Jia-Xun
JX
D000818:Animals; D002417:Cattle; D002432:Cecum / Q000201:enzymology*; D002614:Chelating Agents / Q000494:pharmacology; D004926:Escherichia coli / Q000235:genetics; D015698:Genomic Library; D006863:Hydrogen-Ion Concentration; D008670:Metals / Q000737:chemistry / Q000494:pharmacology; D016297:Mutagenesis, Site-Directed; D011817:Rabbits; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity; D013696:Temperature; D001617:beta-Glucosidase / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*
D002614:Chelating Agents; D008670:Metals; D011994:Recombinant Proteins; D001617:beta-Glucosidase