Isolation of a Novel Low-Temperature-Active and Organic-Solvent-Stable Mannanase from the Intestinal Metagenome of Hermetia illucens

The black soldier fly, <i>Hermetia illucens</i>, is a voracious scavenger of various organic materials; therefore, it could be exploited as a biological system for processing daily food waste. In order to survey novel hydrolytic enzymes, we constructed a fosmid metagenome library using unculturable intestinal microorganisms from <i>H. illucens</i>. Through functional screening of the library on carboxymethyl cellulose plates, we identified a fosmid clone, the product of which displayed hydrolytic activity. Sequence analysis of the fosmid revealed a novel mannan-degrading gene, <i>ManEM6</i>, composed of 1185 base pairs encoding 394 amino acids, with a deduced 20-amino-acid <i>N</i>-terminal signal peptide sequence. The conceptual translation of <i>ManEM6</i> exhibited the highest identity (78%) to endo-1,4-β-mannosidase from <i>Dysgonomonas mossii</i>. Phylogenetic and domain analyses indicated that <i>ManEM6</i> encodes a novel mannanase with a glycoside hydrolase family 26 domain. The recombinant protein rManEM6 showed its highest activity at 40 °C and pH 7.0, and it remained stable in the range of pH 5-10.0. rManEM6 hydrolyzed substrates with β-1,4-glycosidic mannoses, showing maximum enzymatic activity toward locust bean gum galactomannan, while it did not hydrolyze <i>p</i>-nitrophenyl-β-pyranosides, demonstrating endo-form mannosidase activity. rManEM6 was highly stable under stringent conditions, including those of polar organic solvents, as well as reducing and denaturing reagents. Therefore, ManEM6 may be an attractive candidate for the degradation of mannan under high-organic-solvent and protein-denaturing processes in the food and feed industries.