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Objective: Hermetia illucens is a voracious insect scavenger that efficiently decomposes food waste. To exploit novel hydrolytic enzymes from this insect, we constructed a fosmid metagenome library using unculturable H. illucens intestinal microorganisms., Results: Functional screening of the library on carboxymethyl cellulose plates identified a fosmid clone with a product displaying hydrolytic activity. Fosmid sequence analysis revealed a novel mannan-degrading gene (ManEM17) composed of 1371 base pairs, encoding 456 amino acids with a deduced 54 amino acid N-terminal signal peptide sequence. Conceptual translation and domain analysis revealed that sequence homology was highest (46%) with endo-1,4-β-mannosidase of Anaerophaga thermohalophila. Phylogenetic and domain analysis indicated that ManEM17 belongs to a novel β-mannanase containing a glycoside hydrolase family 26 domain. The recombinant protein (rManEM17) was expressed in Escherichia coli, exhibiting the highest activity at 55 °C and pH 6.5. The protein hydrolyzed substrates with β-1,4-glycosidic mannoses; maximum specific activity (5467 U mg -1 ) occurred toward locust bean gum galactomannan. However, rManEM17 did not hydrolyze p-Nitrophenyl-β-pyranosides, demonstrating endo-form mannanase activity. Furthermore, rManEM17 was highly stable under stringent conditions, including polar organic solvents as well as chemical reducing and denaturing reagents., Conclusions: ManEM17 is an attractive candidate for mannan degradation under the high-organic-solvent and protein-denaturing processes in food and feed industries.

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., (Copyright © 2018 Elsevier B.V. All rights reserved.)

A novel pullulanase gene, PulSS4, was identified from the gut microflora of Hermetia illucens by a function-based metagenome screening. The PulSS4 gene had an open reading frame of 4455 base pairs, and encoded a mature protein of 1484 amino acids, with a signal peptide sequence of 44 amino acids. The deduced amino acid sequence of PulSS4 gene showed 51% identity with that of the amylopullulanase of Amphibacillus xylanus, exhibiting no significant sequence homology to already known pullulanases. A conserved domain analysis revealed it to be a pullulanase type II with respective active sites at the N-terminal pullulanase and C-terminal amylase domain. PulSS4 was active in the temperature range of 10-50°C, with an optimum activity at 40°C. It was active in the pH range of 6.5-10.5, with optimum pH at 9.0, and retained more than 80% of its original activity in a broad pH range of 5-11 for 24h at 30°C. Also, PulSS4 was highly stable against many different chemical reagents, including 10% polar organic solvents and 1% non-ionic detergents. Overall, PulSS4 is expected to have the strong potential for application in biotechnological industries that require high activity at moderate temperature and alkaline conditions., (Copyright © 2015 Elsevier B.V. All rights reserved.)