基于密码子优化策略的厚壳贻贝几丁质酶原核重组表达及功能

Recombinant Expression and Function Analysis of Mytichitin-1 from Mytilus coruscus Based on Codon Optimization

厚壳贻贝mytichitin-1是一种特殊的,可通过裂解方式产生抗菌肽的几丁质酶。对该酶的研究有助于了解贻贝的免疫防御以及几丁质酶的功能多样性机制。为深入研究厚壳贻贝mytichitin-1的结构与功能,采取原核重组表达方式,结合密码子优化策略,获得适于大肠杆菌表达体系的目标基因。通过表达以及表达产物的分离纯化,获得重组mytichitin-1蛋白。经过SDS-PAGE,western blot以及质谱验证,表明重组mytichitin-1序列无误,表达成功,在此基础上,对重组mytichitin-1开展了几丁质催化活性,几丁质结合活性和抑菌活性研究。研究结果表明重组mytichtin-1具有明显的几丁质催化活性和几丁质结合活性;同时重组mytichitin-1表现出对细菌和真菌的抑制活性,且对真菌的抑制活性相对较强。上述研究不仅成功表达出厚壳贻贝mytichitin-1,同时也为后续研究几丁质酶的功能多样性及其分子机制,以及基于厚壳贻贝几丁质酶的蛋白质工程和应用研究奠定了基础。

Chitinases have been implicated to be an important enzyme for digestion,immune,molting,and other physiological processes in the life cycle.Mytichitin-1 is a novel chitinase isolated from Mytilus coruscus serum with a special function that can be lysed into an antimicrobial peptide in vivo.The study of mytichitin-1 shed a light on the mechanism of mussel immune biology and functional diversity of chitinase.For further understanding the relationship between structure and function of this enzyme,mytichitin-1 was recombinant expressed in Escherichia coli BL21 with codon optimization.The SDS-PAGE,western blot and mass spectrometry were used for verifying the quality of recombinant mytichitin-1 and the results indicated that mytichitin-1 had been successfully recombinant expressed in E.coli with optimal expression yielding of 40 mg purified enzyme per L culture.After Nickle column isolation,HPLC purification and oxidation,recombinant mytichitin-1 was analyzed with function of chitin degradation,chitin binding,and antimicrobial activity.The results showed that recombinant mytichitin-1 can catalyze chitin as substrate with the best pH of 4.0 and the best temperature of 40°C;furthermore,recombinant mytichitin-1 showed a thermo stability at 60°C with 70%activity.The results of chitin-binding tests demonstrated that recombinant mytichitin-1 also has ability of combination with chitin.Antimicrobial function of recombinant mytichitin-1 was tested using four microorganisms as indicator,including one Gram-positive,one Gram-negative and two fungus,by method of growth inhibition.The results showed that recombinant mytichitin-1 has significant inhibition on microorganism’s growth with relative stronger activity on fungus,followed by Gram-positive and Gram-negative bacteria.Taken together,the chitinase mytichitin-1 has been successfully recombinant expressed in E.coli with codon optimization and the production has specific activities of chitin degradation,chitin binding,and antimicrobial.These results indicated that mytichitin-1 is a promising chitinase for further application research.