| 摘要: |
| 本研究从一株来源于西南印度洋沉积物的噬菌体BVE2基因组中得到了一条全长702 bp，编码噬菌体内溶素的基因Lysin132，编码的蛋白共含有234个氨基酸残基，预计分子量为25.74 kDa；氨基酸序列同源比对结果表明BVE2 Lysin132具有多个酰胺酶活性位点。进化树分析结果表明BVE2 Lysin132是一种N-乙酰胞壁酰-L-丙氨酸酰胺酶。将Lysin132克隆到pEASY-Blunt E2 Expression Vector中，构建了E. coli-pEASY-Lysin132表达菌株。成功用IPTG诱导表达，得到了大量带有6×His标签的重组BVE2 Lysin132。经镍柱纯化后进行SDS-PAGE电泳，得到单一目的蛋白条带。酶学性质分析结果表明，重组BVE2 Lysin132的最适反应温度为30 ℃，在中高温下热稳定性良好，在10~50 ℃下孵育90 min，仍能保持70%以上的酶活力；最适pH为7.0，在pH 6.0~8.0时均能保持80%以上的酶活力，表明该酶有较宽的pH作用范围；Na+、Mg2+可以使酶活力显著提高20%以上，Ca2+可以使酶活力提高40%以上。抑菌实验结果表明，重组BVE2 Lysin132对革兰氏阴性菌的抑制效果较好，尤其对溶藻弧菌（Vibrio alginolyticus）、坎氏弧菌（V. campbellii）、哈维氏弧菌（V. harveyi）的抑制作用显著，与市售微生物溶菌酶相比，抑制效果最高提升了40%以上。因此，本研究获得了一种拥有较宽pH作用范围且热稳定性良好的内溶素，可为开发应用于水产养殖、食品保鲜、医药等领域的新型抑菌剂研发提供基础。 |
| 关键词: 海洋生物学 噬菌体BVE2 内溶素 N-乙酰胞壁酰-L-丙氨酸酰胺酶 抑菌活性 热稳定 |
| DOI：10.3969/J.ISSN.2095-4972.20211227002 |
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| 基金项目:中国亚太经合组织合作基金 |
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| Recombinant expression and activity of endolysin from deep-sea bacteriophage BVE2 |
| ZHANG Tianyou,CHEN Yuan,YU Meishun,ZHANG Menghui,JIN Min,ZENG Runying |
| (Key Laboratory of Marine Biological Genetic Resources, Third Institute of Oceanography, MNR, Xiamen 361005, China;Marine Biological Resources Development and Utilization Engineering Technology Innovation Center, Third Institute of Oceanography, MNR, Xiamen 361005, China;School of Advanced Manufacturing, Fuzhou University, Fuzhou 362200, China) |
| Abstract: |
| In this paper, we found a 702 bp gene Lysin132 encoding phage endolysin from the genome of a bacteriophage BVE2 derived from sediments of the Southwest Indian Ocean. Lyins132 contains a total of 234 amino acid residues, and its molecular weight is estimated to be 25.74 kDa. Result of homologous amino acid sequence alignment shows that BVE2 Lysin132 has multiple amidase active sites. Phylogenetic tree analysis showed that BVE2 Lysin132 is an N-acetylmuramyl-L-alanine amidase. The BVE2 Lysin132 gene was cloned into pEASY-Blunt E2 Expression Vector to construct E. coli-pEASY-Lysin132 expression strain. Thus, it successfully induced an expression with IPTG to obtain a large number of recombinant BVE2 Lysin132 with 6×His tag. After purification by a nickel column, SDS-PAGE electrophoresis yielded a single target protein band. Results of enzymatic properties analysis show that the optimal reaction temperature of recombinant BVE2 Lysin132 is 30 ℃, which has good thermal stability at medium and high temperature. Incubating at 10-50 ℃ for 90 minutes can still maintain more than 70% of the enzyme activity; The optimum pH is 7.0, and it can maintain more than 80% of the enzyme activity at pH 6.0-8.0, indicating that the enzyme has a wide pH range. Na+, Mg2+ can significantly increase the enzyme activity by more than 20%, and Ca2+ can increase the enzyme vitality by more than 40%. Results of antibacterial experiments showed that recombinant BVE2 Lysin132 had a better inhibitory effect on Gram-negative bacteria, especially Vibrio alginolyticus, V. campbellii, V. harveyi, and had much better inhibitory effect. Compared with available microbial lysozyme, the inhibitory effect increased by more than 40%. Therefore, this article has obtained a lysozyme with a wide pH range and good thermal stability, which can provide an important theoretical basis for the development of new antibacterial agents for aquaculture, food preservation, medicine applications and so. |
| Key words: marine biology bacteriophage BVE2 Lysin N-acetylmuramyl-L-alanine amidase antibacterial activity thermostable |
