来自感染革兰氏阴性菌的长尾噬菌体科噬菌体的N-乙酰胞壁酰-L-丙氨酸酰胺酶（LysM2）的外源生产：其结构与功能评估

Exogenous Production of N-acetylmuramyl-L Alanine Amidase (LysM2) from Siphoviridae Phage Affecting Anti-Gram-Negative Bacteria: Evaluation of Its Structure and Function.

作者信息

Miri Morteza, Yazdianpour Sepideh, Abolmaali Shamsozoha, Darvish Alipour Astaneh Shakiba

机构信息

Department of Biotechnology, Faculty of Biotechnology, Semnan University, Semnan, Iran.

Department of Biology, Faculty of Basic Sciences, Semnan University, Semnan, Iran.

出版信息

Avicenna J Med Biotechnol. 2022 Jan-Mar;14(1):46-53. doi: 10.18502/ajmb.v14i1.8169.

DOI:10.18502/ajmb.v14i1.8169

PMID:35509364

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC9017466/

Abstract

BACKGROUND

To obtain endolysin with impact(s) on gram-negative bacteria as well as gram-positive bacteria, N-acetylmuramyl L-alanine-amidase (MurNAc-LAA) from a -hosted Siphoviridae phage (SPP1 phage, Subtilis Phage Pavia 1) was exogenously expressed in .

METHODS

The sequences of genes encoding peptidoglycan hydrolases were obtained from the Virus-Host database. The sequence of MurNAc-LAA was optimized by GenScript software to generate MurNAc-LAA-MMI (LysM2) for optimal expression in . Furthermore, the structure and function of LysM2 was evaluated . The optimized gene was synthesized, subcloned in the pET28a, and expressed in BL21(DE3). The antibacterial effects of the protein on the peptidoglycan substrates were studied.

RESULTS

, on 816 gene encoding a 33 protein was confirmed as specific SPP1 phage enzyme. The enzyme is composed of 271 amino acids, with a half-life of 10 in . analyses showed 34.2% alpha-helix in the secondary structure, hydrophobic N-terminal, and lysine-rich C-terminal, and no antigenic properties in LysM2 protein. This optimized endolysin revealed impacts against (sp) by turbidity, and an antibacterial activity against , and in agar diffusion assays.

CONCLUSION

Taken together, our results confirmed that LysM2 is an inhibiting agent for gram-negative bacteria.

摘要

背景

为了获得对革兰氏阴性菌和革兰氏阳性菌均有作用的内溶素，来自宿主芽孢杆菌属的肌尾噬菌体（SPP1噬菌体，枯草芽孢杆菌噬菌体帕维亚1）的N - 乙酰胞壁酰 - L - 丙氨酸酰胺酶（MurNAc - LAA）在大肠杆菌中进行了外源表达。

方法

从病毒 - 宿主数据库中获取编码肽聚糖水解酶的基因序列。通过金斯瑞软件对MurNAc - LAA序列进行优化，生成MurNAc - LAA - MMI（LysM2）以在大肠杆菌中实现最佳表达。此外，对LysM2的结构和功能进行了评估。合成优化后的基因，亚克隆到pET28a中，并在大肠杆菌BL21(DE3)中表达。研究了该蛋白对肽聚糖底物的抗菌作用。

结果

在816个基因中，一个编码33 kDa蛋白的基因被确认为特定的SPP1噬菌体酶。该酶由271个氨基酸组成，在大肠杆菌中的半衰期为10小时。二级结构分析显示α - 螺旋占34.2%，N端疏水，C端富含赖氨酸，且LysM2蛋白无抗原性。这种优化后的内溶素通过比浊法显示对粪肠球菌（sp）有作用，并且在琼脂扩散试验中对大肠杆菌、金黄色葡萄球菌有抗菌活性。