Cloning and characterization of two β-glucosidase/xylosidase enzymes from yak rumen metagenome.
Two β-glucosidase/xylosidase genes, Rubg3A and Rubg3B, were cloned from yak rumen uncultured microorganisms by metagenome method and function-based screening. Recombinant RuBG3A and RuBG3B purified from Escherichia coli were characterized for enzymatic properties, and they exhibited activity against 4-nitrophenyl-β-D: -glucopyranoside and 4-nitrophenyl-β-D: -xylopyranoside, suggesting bifunctional β-glucosidase/xylosidase activity. Chromatography analysis showed that they could effectively hydrolyze cellooligosaccharide substrates, indicating the facilitation in saccharification of cellulose. RuBG3A and RuBG3B can also increase the reducing sugar released in xylan hydrolysis to 218% and 169%, respectively, through synergism with xylanase, suggesting their application in hemicellulose saccharification. Molecular modeling and substrate docking showed that there should be one active center responsible for the bifunctional activity in each enzyme, since the active site pocket is substantially wide to allow the entry of both β-glucosidic or β-xylosidic substrates, which elucidated the structure-function relationship in substrate specificities. Therefore, the enzymatic properties, the participation in hydrolysis of cellooligosaccharides, and the synergism with xylanase make RuBG3A and RuBG3B very interesting candidates for saccharification of both cellulose and hemicellulose.
State Key Laboratory of Genetic Engineering, School of Life Sciences, Fudan University, No. 220 Handan Road, Shanghai, China.
Lei
L
Bao
Qiang
Q
Huang
Lei
L
Chang
Qingwen
Q
Sun
Jungang
J
Zhou
Hong
H
Lu
D000117:Acetylglucosamine / Q000031:analogs & derivatives / Q000737:chemistry; D000595:Amino Acid Sequence; D000818:Animals; D020134:Catalytic Domain; D002417:Cattle / Q000235:genetics* / Q000382:microbiology; D044602:Cellulases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification*; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D006026:Glycoside Hydrolases / Q000378:metabolism; D006868:Hydrolysis; D007700:Kinetics; D054892:Metagenome / Q000235:genetics; D008969:Molecular Sequence Data; D010802:Phylogeny; D012417:Rumen / Q000201:enzymology* / Q000382:microbiology; D014990:Xylans / Q000737:chemistry; D014995:Xylosidases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*
D014990:Xylans; D044602:Cellulases; D006026:Glycoside Hydrolases; D014995:Xylosidases; C023305:hemicellulase; C026579:exo-1,4-beta-D-xylosidase; D000117:Acetylglucosamine