In vitro synergistic effects of three enzymes from Bacillus subtilis CH-1 on keratin decomposition

Extracellular protease Vpr (Vpr), gamma-glutamyltranspeptidase (GGT; EC 2.3.2.2) and glyoxal/methylglyoxal reductase (YvgN; EC 1.1.1.21) are extracellular enzymes involved in feather degradation, which were identified by secretome analyses from an efficient feather-degrading strain Bacillus subtilis CH-1. The encoding sequences corresponding to the three secretory enzymes were cloned into vector pET22b for recombinant expression in Escherichia coli strain BL21 (DE3). Afterward, the proteins containing the C-terminal His-tag were purified using a Ni-IDA column. The optimal temperatures and pH values for protease activity of recombinant Vpr, GGT, and YvgN were identified as 45 °C/pH 7.0, 40 °C/pH 8.0, and 50 °C/pH 6.0 respectively when casein is the substrate. Furthermore, the synergistic effects of the three enzymes were studied using feather powder as substrate. Vpr was the core enzyme to hydrolyze keratin, while GGT and YvgN were coenzymes providing reducing activities for keratin decomposition. The keratinolytic activity was enhanced to about 1.4-folds when YvgN and Vpr applied together in comparison to Vpr alone. And the keratinolytic activity almost reached to 1.5-folds when all the three enzymes were combined to use. The study provides a novel perspective of the mechanism of keratin degradation by microorganisms, and thereby may also be of relevance for the design of an industrial process for enzymatic keratin degradation; however, additional experiments must be done to substantiate this conclusion.