杀鱼假交替单胞菌C923几丁质酶基因PpchiC的克隆表达与酶学性质

Cloning expression and enzymatic properties of chitinase gene PpchiC from Pseudoalteromonas piscicida C923

摘要:

【背景】某些假交替单胞菌可分泌几丁质酶，在降解利用几丁质为水产动物提供营养、免疫、抗病等方面有着重要潜力。【目的】克隆杀鱼假交替单胞菌(Pseudoalteromonas piscicida)C923的一个几丁质酶基因，实现其在大肠杆菌中的异源表达，并对重组几丁质酶的酶学性质进行研究。【方法】从菌株C923测序的基因组中注释到一个几丁质酶家族基因PpchiC，设计引物克隆该基因后进行生物信息学分析；构建载体进行异源表达并从温度、时间与诱导剂浓度进行表达优化；对表达蛋白进行最适温度与pH等酶学性质研究，同时比较了重组菌破碎后上清与沉淀及纯化的酶蛋白对几丁质的降解效应。【结果】基因PpchiC长1350bp，编码450个氨基酸，PpchiC蛋白理论分子量为48.76kDa，等电点为4.78，不稳定系数为29.08。结构域分析发现该蛋白含有一个类型Ⅲ几丁质结合域和一个糖苷水解酶18家族(glycosyl hydrolase 18,GH18)的催化域；PpchiC蛋白含有GH18家族几丁质酶的保守催化基序DxxDxDxE、YxR和[E/D]xx[V/I]。16℃、0.25mmol/L IPTG、诱导12h为其最优化表达条件，PpchiC在50℃、pH8.0时表现出最大酶活性；以胶体几丁质为底物时，PpchiC的K_m值为2.58mg/mL、V_max值为5.04mg/(mL·min)。降解结果表明，菌体的沉淀与上清及从上清中纯化的酶蛋白均有着较好的几丁质降解效应。【结论】杀鱼假交替单胞菌C923基因PpchiC编码GH18家族的几丁质酶，能被大肠杆菌高效表达且降解几丁质效应明显，这为PpchiC及菌株C923的应用提供了参考依据。

Abstract:

[Background] Some members of the genus Pseudoalteromonas can secrete a variety of chitinases, which play important roles in degrading chitin to provide nutrition, immunity, and disease prevention for aquatic animals. [Objective] To clone a chitinase gene of Pseudoalteromonas piscicida C923 and realize its heterologous expression in Escherichia coli, thereby exploring the enzymatic properties of the recombinant chitinase. [Methods] A potential gene PpchiC, identified and annotated as chitinase based on the analysis of the genome sequence of strain C923, was cloned by designing primers and subsequently subjected to bioinformatic analysis. Then an expression vector was constructed to conduct heterologous expression, and the expression was optimized from the temperature, time, and concentration of the inducer. The optimum temperature, pH of the expressed protein, and other enzymatic properties were studied. Finally, the degradation of chitin by the supernatant and precipitation of recombinant E. coli cells and the purified enzyme protein were compared. [Results] PpchiC was 1 350 bp in length and encoded 450 amino acids. The theoretical molecular mass of protein PpchiC was 48.76 kDa, and its isoelectric point and instability coefficient were 4.78 and 29.08, respectively. Structural analysis revealed that PpchiC contained a type III chitin-binding domain, a catalytic domain of glycosyl hydrolase 18 (GH18), and conserved motifs DxxDxDxE, YxR, and [E/D]xx[V/I]. The optimized expression conditions were 16 ℃ of temperature, induction time of 12 h, and IPTG concentration of 0.25 mmol/L. PpchiC showed maximum enzymatic activity at 50 ℃ and pH 8.0. When colloidal chitin was used as the substrate, the kinetic parameters K _m and V _max were 2.58 mg/mL and 5.04 mg/(mL·min), respectively. The results of degradation showed that the supernatant and precipitation of recombinant E. coli cells and the purified enzyme protein from the supernatant all exhibited excellent chitin degradation effects. [Conclusion] The gene PpchiC from P. piscicida C923 encodes a GH18 family chitinase, which can be highly expressed in E. coli with obvious chitin degradation effects. This study provides references for the application of Ppchic and C923 strain.