Isolation and characterization of a halotolerant and protease-resistant α-galactosidase from the gut metagenome of Hermetia illucens
•We isolated a novel α-galactosidase from a gut metagenome of fly larvae.•It featured high-level resistance to solvent, salts, detergents, and proteases.•The enzyme provides good application potential for harsh bioprocesses. Hermetia illucens is a voracious insect scavenger, decomposing food waste e...
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Veröffentlicht in: | Journal of biotechnology 2018-08, Vol.279, p.47-54 |
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Zusammenfassung: | •We isolated a novel α-galactosidase from a gut metagenome of fly larvae.•It featured high-level resistance to solvent, salts, detergents, and proteases.•The enzyme provides good application potential for harsh bioprocesses.
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 α-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 α-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 α-galactosidase. The recombinant protein, rAgas2, was successfully expressed in E. coli. rAgas2 showed the highest activity at 40 °C and pH 7.0. It displayed great pH stability within a pH range of 5–11 for 15 h at 4 °C. rAgas2 was highly stable under stringent conditions, including polar organic solvents, non-ionic detergents, salt, and proteases. rAgas2 hydrolyzed α-d-galactose substrates, showing the maximum enzymatic activity toward p-nitrophenyl α-d-galactopyranoside (specific activity 128.37 U/mg). However, rAgas2 did not hydrolyze substrates linked with β-glucose moieties. Overall, Agas2 may be an attractive candidate for the degradation of α-galactose family oligosaccharides in high-salt, protease-rich and high-organic-solvent processes. |
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ISSN: | 0168-1656 1873-4863 |
DOI: | 10.1016/j.jbiotec.2018.05.003 |