title: Identification and characterization of an unusual glycosyltransferase-like enzyme with β-galactosidase activity from a soil metagenomic library.
abstract: Glycosyltransferases and glycoside hydrolases are two diversified groups of carbohydrate-active enzymes (CAZymes) in existence, they serve to build and break down the glycosidic bonds, respectively, and both categories have formed many sequence-based families. In this study, a novel gene (glyt110) conferring β-galactosidase activity was obtained from a metagenomic library of Turpan Basin soil. Sequence analysis revealed that glyt110 encoded a protein of 369 amino acids that, rather than belonging to a family typically known for β-galactosidase activity, belonged to glycosyltransferase family 4. Because of this unusual sequence information, the novel gene glyt110 was subsequently expressed in Escherichia coli BL21(DE3), and the recombinant enzyme (Glyt110) was purified and characterized. Biochemical characterization revealed that the β-galactosidase activity of Glyt110 toward o-nitrophenyl-β-D-galactopyranoside (ONPG) and lactose were identified to be 314±18.3 and 32±2.7 U/mg, correspondingly. In addition, Glyt110 can synthesize galacto-oligosaccharides (GOS) using lactose as substrate. A GOS yield of 47.2% (w/w) was achieved from 30% lactose solution at 50 °С, pH 8.0 after 10 h reaction. However, Glyt110 was unable to glycosylate either N-acetylated saccharides or lactose and galactose using UDP-gal as sugar donor, and its glycosyltransferase activity needs further investigation. These results indicated that Glyt110 is an unusual enzyme with β-galactosidase activity but phylogenetically related to glycosyltransferase. Our findings may provide opportunities to improve the insight into the relationship between glycosyltransferases and glycoside hydrolases and the sequence-based classification.
authors.affiliation: School of Life Sciences, Sun Yat-sen University, Guangzhou 510275, People's Republic of China.
authors.forename: Si-di
authors.initials: SD
authors.lastname: Wang
authors.affiliation: School of Life Sciences, Sun Yat-sen University, Guangzhou 510275, People's Republic of China.
authors.forename: Geng-shan
authors.initials: GS
authors.lastname: Guo
authors.affiliation: School of Life Sciences, Sun Yat-sen University, Guangzhou 510275, People's Republic of China.
authors.forename: Liang
authors.initials: L
authors.lastname: Li
authors.affiliation: School of Life Sciences, Sun Yat-sen University, Guangzhou 510275, People's Republic of China.
authors.forename: Li-chuang
authors.initials: LC
authors.lastname: Cao
authors.affiliation: School of Life Sciences, Sun Yat-sen University, Guangzhou 510275, People's Republic of China.
authors.forename: Ling
authors.initials: L
authors.lastname: Tong
authors.affiliation: School of Life Sciences, Sun Yat-sen University, Guangzhou 510275, People's Republic of China.
authors.forename: Guang-hui
authors.initials: GH
authors.lastname: Ren
authors.affiliation: School of Life Sciences, Sun Yat-sen University, Guangzhou 510275, People's Republic of China. Electronic address: lsslyh@mail.sysu.edu.cn.
authors.forename: Yu-huan
authors.initials: YH
authors.lastname: Liu
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000145:classification / Q000235:genetics / Q000378:metabolism; D002681:China; D003001:Cloning, Molecular; D005798:Genes, Bacterial; D015698:Genomic Library; D016695:Glycosyltransferases / Q000145:classification / Q000235:genetics* / Q000378:metabolism*; D007700:Kinetics; D054892:Metagenome; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000096:biosynthesis; D010802:Phylogeny; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D012988:Soil Microbiology; D013379:Substrate Specificity; D001616:beta-Galactosidase / Q000145:classification / Q000235:genetics* / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D009844:Oligosaccharides; D011994:Recombinant Proteins; D016695:Glycosyltransferases; D001616:beta-Galactosidase
keywords: Enzyme characterization; Glycosyltransferase; Metagenomic library; Sequence-based classification; β-Galactosidase