Characterization of an i-type lysozyme gene from the sea cucumber Stichopus japonicus, and enzymatic and nonenzymatic antimicrobial activities of its recombinant protein
Characterization of an i-type lysozyme gene from the sea cucumber Stichopus japonicus, and enzymatic and nonenzymatic antimicrobial activities of its recombinant protein
复制标题
DOI：
10.1016/j.jbiosc.2009.01.016
复制
发表时间：
2009-06-01
影响因子：
2.8
通讯作者：
Zhu, Beiwei
中科院分区：
文献类型：
--
作者：
Cong, Lina;Yang, Xijian;Zhu, Beiwei
由于海参缺乏发达的免疫系统，可以与食物一起摄入致病菌，因此体内必须存在某种形式的活性抗菌物质进行防御。本研究采用RT-PCR和RACE PCR技术克隆了海参（Stichopus Stichopus）i型溶菌酶（SjLys）的cDNA。SjLys的cDNA全长为713 bp，开放阅读框为438，编码145个氨基酸。在SjLys中检测到两个在i型溶菌酶中保守的催化残基（Glu 34和Asp 47）和一个在活性位点附近高度保守的区域MDVGSLSCG（P/Y）（Y/F）Q1 K。此外，结构域分析表明，它是高度相似的药用水蛭去稳定酶，这属于一个新的系统发育的家庭无脊椎动物溶菌酶同时具有糖苷酶和异肽酶的活动。为了深入了解SjLys的体外抗菌活性，在大肠杆菌中异源表达成熟肽编码区。重组SjLys蛋白对革兰氏阳性菌和革兰氏阴性菌的生长均有抑制作用。结果表明，重组SjLys在100 ℃热处理50 min后，对所有供试菌株的抗菌活性均有所提高。溶菌酶是一种具有酶（糖苷酶）和非酶抗菌作用的酶。(C)2009年，生物技术协会。日本All rights reserved.
Because sea cucumbers lack a well-developed immune system and can ingest pathogenic bacteria together with food, some form of active antibacterial substances must be present in the body for defense. In this study, the cDNA of an i-type lysozyme from the sea cucumber Stichopus japonicus (designated SjLys) was cloned by RT-PCR and RACE PCR techniques. The full length cDNA of SjLys was 713 by with an open reading frame of 438 by coding for 145 amino acids. Two catalytic residues (Glu34 and Asp47), conserved in i-type lysozymes, and a highly conserved region near the active site, MDVGSLSCG(P/Y)(Y/F)Q1K, were detected in SjLys. In addition, the domain structure analysis of SjLys showed that it is highly similar to the medicinal leech destabilase, which belongs to a new phylogenetic family of invertebrate lysozymes possessing both glycosidase and isopeptidase activities. To gain insight into the in vitro antimicrobial activities of SjLys, the mature peptide coding region was heterologously expressed in Escherichia coli. The recombinant SjLys protein displayed an inhibitive effect on the growth of the tested Gram-positive and Gram-negative bacteria. A remarkable finding is that the recombinant SjLys exhibited more potent activities against all tested bacterial strains after heat-treating at 100 degrees C for 50 min. These results indicated that the S. japonicus lysozyme is an enzyme with combined enzymatic (glycosidase) and nonenzymatic antibacterial action. (C) 2009, The Society for Biotechnology. Japan. All rights reserved.