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

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.