期刊
JOURNAL OF BIOTECHNOLOGY
卷 279, 期 -, 页码 47-54出版社
ELSEVIER SCIENCE BV
DOI: 10.1016/j.jbiotec.2018.05.003
关键词
Alpha-galactosidase; Metagenome; Halotolerant; Hermetia illucens; Gut
资金
- National Institute of Agricultural Sciences, Rural Development Administration, Republic of Korea [PJ01086901, PJ01045703]
Hermetia illucens is a voracious insect scavenger, decomposing food waste efficiently. To survey novel hydrolytic enzymes, we constructed a fosmid metagenome library using unculturable intestinal microorganisms from H. illucens in our previous study (Lee et al., 2014). Functional screening of the library on carboxymethyl cellulose plates identified a fosmid clone the product of which displayed hydrolytic activity. Sequence analysis of the fosmid revealed a novel alpha-galactosidase gene, Agas2. The Agas2 gene is composed of 2,007 base pairs encoding 668 amino acids with a deduced 25 amino acid N-terminal signal peptide sequence. The conceptual translation and domain analysis of Agas2 showed the highest sequence identity (84%) with the putative alpha-galactosidase of Dysgonomonas sp. HGC4, exhibiting well-conserved domain homology with glycosyl hydrolase family 97. Phylogenetic analysis indicated that Agas2 may be a currently uncharacterized alpha-galactosidase. The recombinant protein, rAgas2, was successfully expressed in E. coli. rAgas2 showed the highest activity at 40 degrees C and pH 7.0. It displayed great pH stability within a pH range of 5-11 for 15 h at 4 degrees C. rAgas2 was highly stable under stringent conditions, including polar organic solvents, non-ionic detergents, salt, and proteases. rAgas2 hydrolyzed alpha-D-galactose substrates, showing the maximum enzymatic activity toward p-nitrophenyl alpha-D-galactopyranoside (specific activity 128.37 U/mg). However, rAgas2 did not hydrolyze substrates linked with beta-glucose moieties. Overall, Agas2 may be an attractive candidate for the degradation of alpha-galactose family oligosaccharides in high-salt, protease-rich and high-organic-solvent processes.
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