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.

在这项研究中，基于定点饱和诱变和表面电荷修饰的半理性设计被用来提高源自火球菌堀越（PhDac）的二乙酰壳二糖脱乙酰酶的催化效率。通过定点饱和诱变筛选出的PhDac突变体M14的催化效率（ k _cat /K _m ）提高了约2.21倍，比活性提高了70.02%。为了保持葡萄糖胺（GlcN）的稳定性，我们通过将表面电荷从-35修改为-59来降低M14的最适pH，以获得突变体M20，其比活性达到野生型的2倍。 M20催化N-乙酰氨基葡萄糖(GlcNAc)向GlcN的转化率达到94.3%。此外，当温度高于50 ℃时，pH的降低减缓了GlcN产量的下降。我们的研究结果将加速生物催化工业生产 GlcN 的进程。