A novel metagenome-derived beta-galactosidase: gene cloning, overexpression, purification and characterization.
A novel beta-galactosidase gene, zd410, was isolated by screening a soil metagenomic library. Sequence analysis revealed that zd410 encodes a protein of 672 amino acids with a predicted molecular weight of 78.6 kDa. The recombinant ZD410 was expressed and purified in Pichia pastoris, with a yield of ca. 300 mg from 1 L culture. The purified enzyme displayed optimal activity at 38 degrees C and pH 7.0. Given that the enzyme had 54% of the maximal activity at 20 degrees C and 11% of the maximal activity at close to 0 degrees C, ZD410 was regarded as a cold-adapted beta-galactosidase. ZD410 displays high enzymatic activity for its synthetic substrate-ONPG (o-nitrophenyl-beta-D-galactopyranoside, 243 U/mg) and its natural substrate-lactose (25.4 U/mg), while its activity was slightly stimulated by addition of Na(+), K(+), or Ca(2+) at low concentrations. ZD410 is a good candidate of beta-galactosidases for food industry after further study.
Wang
Kui
K
State Key Laboratory of Biocontrol, Sun Yat-sen University, Guangzhou, 510275, People's Republic of China.
Li
Gang
G
Yu
Shi Qin
SQ
Zhang
Chen Ting
CT
Liu
Yu Huan
YH
D002118:Calcium / Q000494:pharmacology; D003001:Cloning, Molecular; D016000:Cluster Analysis; D003067:Coenzymes / Q000494:pharmacology; D004795:Enzyme Stability; D015870:Gene Expression; D006863:Hydrogen-Ion Concentration; D007785:Lactose / Q000378:metabolism; D054892:Metagenome; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009598:Nitrophenylgalactosides / Q000378:metabolism; D010802:Phylogeny; D010843:Pichia / Q000235:genetics; D011188:Potassium / Q000494:pharmacology; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D012964:Sodium / Q000494:pharmacology; D012988:Soil Microbiology; D013696:Temperature; D001616:beta-Galactosidase / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*
D003067:Coenzymes; D011994:Recombinant Proteins; C055012:2-nitrophenylgalactoside; D009598:Nitrophenylgalactosides; D012964:Sodium; D001616:beta-Galactosidase; D007785:Lactose; D011188:Potassium; D002118:Calcium