Construction And Application Of A Novel Chimeric Chitinase

Chitin is the second largest renewable resource in nature.N-acetylglucosamine,the completed degradation product of chitin,is a valuable monosaccharide used in many fields such as food,medicine,cosmetics,agriculture and industry.Enzymatic conversion of chitin is the direction of industrial development.In this study,we cloned and characterized a GH20 family ?-N-acetylhexosaminidase SaHEX from Streptomyces alfalae ACCC40021.Then we evaluated the synergistic action between SaHEX and chitinase.Based on the previous studies of our laboratory,the enzyme activity and kinetic parameters of GH19 chitinase SaChiB were further determined.The hydrolysis efficiency of crystal ?-chitin combined SaChiB with SaHEX was evaluated.Finally,a novel chimeric chitinase was constructed by fusion of SaChiB with SaHEX,which can improve the degradation activity and conversion efficiency of chitinase significantly,and degraded directly ?-chitin to produce GlcNAc with high purity(>98%).1.The SaHEX was successfully cloned and expressed in Escherichia coli BL21(DE3).The purified SaHEX exhibited the highest activity of 1149.7 U/mg towards pNP-GlcNAc at 60? and pH 5.5.It has good stability at the temperature under50? and in the pH range of 4.5-8.5.SaHEX showed a broad substrate spectrum.It can hydrolyze colloidal chitin and chitooligosaccharides with different polymerization degree.The thin layer chromatography(TLC)showed that it cleaved the substrate by a exo-type manner.The degree of synergy between SaHEX and SgCtn is 3.1.The highest conversion of 1%colloidal chitin can be achieved to 93.7%after 6 h by combined 50 ?g/mL SaHEX with 2 mg/mL SgCtn,and GlcNAc is the only product with purity over 98%.2.The multiple alignment and homologous modelling indicated that SaChiB is a classic GH19 family chitinase with two domains.Then expression levels of different induction temperature srevealed the highest level of soluble expression was achieved at 20?.SaChiB showed a broad substrate spectrum including different forms of chitin.The highest conversion of 1%?-chitin can reach to 95.2%in 8 h by combination of 2 mg/mL SaChiB with 50 ?g/mL SaHEX,and 2.88 g/L of GlcN Ac was obtained with purity of more than 98%.3.In order to further improve the efficiency of enzymatic conversion of chitin,SaChiB-HEX,a novel chimeric chitinase,was constructed by fusion of SaChiB with SaHEX.The fusion enzyme exhibited the highest activity at 30? and pH 7.0,and showed good stability under 45? and in the range of pH 4.5-8.5,respectively.SaChiB-HEX displayed a broad substrate spectrum.It can hydrolyze different forms of chitin and also can hydrolyze(GlcNAc)2 to GlcNAc.The maximum yield of 99.4%was achieved by 5 nmol/mL(423.5 ?g/mL)of SaChiB-HEX with 1%?-chitin at 8 h,and GlcNAc is the only product.The chimeric chitinase SaChiB-HEX significantly improved the degradation efficiency of chitin,and has the great potential as biocatalyst for the production of GlcNAc in industries.