Chitinases From Ridgetail White Prawn Exopalaemon Carinicauda: Purification, Enzymatic Characterization And Function Analysis

In this study, two chitinases（named EcChi1 and EcChi2） were purified from the hepatopancreas of ridgetail white prawn Exopalaemon carinicauda. The enzymatic characterization and kinetic parameters of the higher activity chitinase EcChi1 were carried out. Then the chitinases EcChi1 and EcChi2 were identified by LC-ESI-MS-MS, and the nucleotide sequences of chitinases were obtained after in conjunction with the transcriptome data of E. carinicauda. Based on the techniques of molecular cloning and recombination, the chitinase genes were cloned and subcloned into expression vector to express the recombinant protein. Moreover, the expression pattern in different tissues and different molting stages of the chitinase genes were analyzed. RNAi was also carried out to analyze its function. The main results are introduced as follows:Two proteins with chitinolytic activity, named as EcChi1 and EcChi2, were purified from hepatopancreas of E. carinicauda by ion exchange chromatography and gel- filtration chromatography. According to the chromatogram, the amount of EcChi1 was much higher than that of EcChi2, which was consistent with chitinolytic act ivity detection. The specific activity of EcChi1 and EcChi2 was 1305.97 U mg-1 and 28.67 U mg-1. The results above showed that the purity and amount of EcChi1 was much higher than that of EcChi2. Hence, the purified EcChi1 was selected to study its enzymatic characteristic, including the optimal pH and temperature, effects of metal ions and enzyme stabilities. The optimal temperature and pH of EcChi1 were 37 oC and p H 4.0, respectively. Co2+, Fe3+, Zn2+, Cd2+, and Cu2+ had an obvious promoting effect upon chitinase activity of EcChi1. For colloidal chitin, the Km and Vmax values of EcChi1 were 2.09 mg mL-1 and 31.15 U mL-1 h-1.Based on the results of LC-ESI-MS-MS, the data of transcriptome and RACE technology, 4 chitinase genes, named EcChi1、EcChi2、EcChi3 and EcChi4, were obtained. EcChi1 codes EcChi1 and EcChi2 codes EcChi2. The ORF of EcChi1, EcChi2, EcChi3 and EcChi4 encoded 410, 480, 384, and 389 amino acids with a predicted molecular weight about 46346.44 Da, 53918.87 Da, 44442.25 Da, and 43849.35 Da, respectively. The theoretical isoelectric point（PI） was 4.83, 5.00, 4.76, and 4.93 respectively. All of the deduced amino acid sequences of EcChis contain a signal peptide and a glycosyl hydrolases family 18（Glyco₁8） domain. In addition, there is also one type 2 chitin-binding domain in the amino acid sequence of EcChi2 following Glyco₁8 domain. The ORFs were amplified by PCR and cloned into the pDHsp70FLAG-His expression vector. And then EcChi1, EcChi2 and EcChi3 was transformed into host insect cells. Western blot revealed the production of recombinant EcChi1, EcChi2 and EcChi3.After that, the differential expression of the four chitinases in mRNA level was analyzed in different tissues and different molting periods. The semi-quantitative RT-PCR results showed that the four chitinase genes all express at the highest level in hepatopancreas, and then stomach, but poor in other tissues. And Real-time PCR results showed that the four chitinase genes all express at the highest level in intermolt（stage C）and molt（stage E）, but poor in other stage. So it implicits that these four chitinase genes were not participating in the molting stage. And then, the dsRNA for special chitinase was synthesized and injected into shrimp. The results of the interference were valued by the observation of the molting, mortality, and the ultrastructure of the uropod of the injected shrimp. The results showed that the mortality and molting percentage of EcChi ds RNA treated shrimps were in accordance with that of control group. And the endopodite morphology of the survival and death individuals in experimental group are all normal. These suggest that the 4 chitinase genes may be not related to molting process of E. carinicauda.