| Chitin is the second abundant renewable natural polymer on earth.N-acetyl-D-glucosamine(GlcNAc),the complete degradation product of chitin,has been utilized in the food,cosmetics,medicine,agriculture and biofuels fields.At present,chitin is mainly degraded by chemical methods using strong acid,resulting in low yield,poor product purity and heavy environmental pollution.Enzymatic conversion of chitin is the desirable direction of industrial development in advantage of low-cost,high production with good purity and environment-friendness.Based on the CAZy analysis and whole genome sequencing of Streptomyces alfalfae ACCC40021,a chitinase gene Sachi18e,belonging to Glycoside Hydrolase family 18(GH18),was found.In this study,we cloned,expressed and characterized the chitinase SaChi18E,and further improved its catalytic efficiency of hydrolyzing chitin to GlcNAc by protein engineering technology.1.The chitinase SaChi18E was cloned and expressed in soluble form in Escherichia coli BL21(DE3).The recombinant enzyme was purified by Ni-NTA column and the enzymatic properties of SaChi18E were determined.The optimum temperature and pH of the enzyme was 40℃ and pH 6.0,respectively.It had good stability under 40℃ and in the range of pH 5.0-11.0.Thin-layer chromatography(TLC)analysis showed its action mode is endo-cleavage type.The kinetic parameters of SaChi18E towards colloidal chitin were Km of 0.41 mg/mL,Vmax of 25.06 U/mg,Kcat of 34.09 s-1 and Kcat/Km of 82.95 mL/(mg·s),respectively.2.To improve the efficiency of hydrolysis of chitin to GlcNAc,SaHEX,the N-acetyl hexosamidase belonging to GH20 family,was successfully fused on the C-terminus of SaChi18E to construct chimeric chitinase SaChi18E-HEX by Gibson assembly.The chimeric enzyme successfully expressed in soluble form in E.coli BL21(DE3).SaChi18E-HEX exhibited maximal activity at 40℃ and pH 5.0,respectively.TLC analysis showed that SaChi18E-HEX cleaved substrates synergistically by both endo-and exo-manners.The results of the hydrolytic assay showed that the catalytic efficiency of the chimeric chitinase is 5.9 folds higher than CmChi1,which has the highest conversion rate of GlcNAc from chitin reported so far.The maximum conversion rate of 98.5%was achieved at 4 hrs.GlcNAc was released as the unique product with a purity of>98.5%confirmed by MALDI-TOF mass spectrometry analysis.Our findings above suggested that the novel fusion chitinase has the potential to be used for enzymatic conversion of chitin to GlcNAc in industries. |
