Engineering diacetylchitobiose deacetylase from Pyrococcus horikoshii towards an efficient glucosamine production

In this study, semi-rational design based on site-directed saturation mutagenesis and surface charge modification was used to improve the catalytic efficiency of the diacetylchitobiose deacetylase derived from Pyrococcus horikoshii (PhDac). PhDac mutant M14, which was screened by site-directed saturation mutagenesis, showed a ~ 2.21 -fold enhanced catalytic efficiency (k_cat/K_m) and the specific activity was improved by 70.02%. To keep the stability of glucosamine (GlcN), we reduced the optimal pH of M14 by modifying the surface charge from -35 to -59 to obtain mutant M20, whose specific activity reached 2 -fold of the wild-type. The conversion rate of N-acetylglucosamine (GlcNAc) to GlcN catalyzed by M20 reached 94.3%. Moreover, the decline of GlcN production was slowed down by the reduction of pH when temperature was higher than 50 ℃. Our results would accelerate the process of industrial production of GlcN by biocatalysis.