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Abstract：
This study is an extension of our previous studies in which the lysozyme was isolated and purified from Bacillus subtilis BSN314 (Naveed et al., 2022; Naveed et al., 2023). In this study, the lysozyme genes were cloned into the E. coli BL21. For the expression of lysozyme in E. coli BL21, two target genes, Lyz-1 and Lyz-2, were ligated into the modified vector pET28a to generate pET28a-Lyz1 and pET28a-Lyz2, respectively. To increase the production rate of the enzyme, 0.5-mM concentration of IPTG was added to the culture media and incubated at 37 °C and 220 rpm for 24 h. Lyz1 was identified as N-acetylmuramoyl-L-alanine amidase and Lyz2 as D-alanyl-D-alanine carboxypeptidase. They were purified by multi-step methodology (ammonium sulfate, precipitation, dialysis, and ultrafiltration), and antimicrobial activity was determined. For Lyz1, the lowest MIC/MBC (0.25 μg/mL; with highest ZOI = 22 mm) were recorded against Micrococcus luteus, whereas the highest MIC/MBC with lowest ZOI were measured against Salmonella typhimurium (2.50 μg /mL; with ZOI = 10 mm). As compared with Aspergillus oryzae (MIC/MFC; 3.00 μg/mL), a higher concentration of lysozyme was required to control the growth of Saccharomyces cerevisiae (MIC/MFC; 50 μg/mL). Atomic force microscopy (AFM) was used to analyze the disintegrating effect of Lyz1 on the cells of selected Gram-positive bacteria, Gram-negative bacteria, and yeast. The AFM results showed that, as compared to Gram-negative bacteria, a lower concentration of lysozyme (Lyz1) was required to disintegrate the cell of Gram-positive bacteria.
摘要：
这项研究是我们先前研究的扩展，其中溶菌酶是从枯草芽孢杆菌BSN314中分离和纯化的（Naveed等人。,2022年；Naveed等人。,2023年）。在这项研究中,将溶菌酶基因克隆到大肠杆菌BL21中。对于溶菌酶在大肠杆菌BL21中的表达，两个靶基因，将Lyz-1和Lyz-2连接到修饰的载体pET28a中以分别产生pET28a-Lyz1和pET28a-Lyz2。为了提高酶的生产率，将0.5mM浓度的IPTG添加到培养基中并在37°C和220rpm下孵育24小时。将Lyz1鉴定为N-乙酰胞壁酰-L-丙氨酸酰胺酶,将Lyz2鉴定为D-丙氨酰-D-丙氨酸羧肽酶。它们通过多步方法纯化(硫酸铵，降水,透析,和超滤），并测定抗菌活性。对于Lyz1，最低MIC/MBC（0.25μg/mL；最高ZOI=22mm）记录为针对黄体微球菌，而最高的MIC/MBC和最低的ZOI是针对鼠伤寒沙门氏菌（2.50μg/mL；ZOI=10mm）测量的。与米曲霉(MIC/MFC;3.00μg/mL)相比，需要较高浓度的溶菌酶来控制酿酒酵母的生长(MIC/MFC;50μg/mL)。原子力显微镜（AFM）用于分析Lyz1对选定的革兰氏阳性细菌细胞的崩解作用，革兰氏阴性菌,和酵母。AFM结果显示,与革兰氏阴性细菌相比，需要较低浓度的溶菌酶（Lyz1）来分解革兰氏阳性细菌的细胞。
