Enhancing Lysostaphin Thermostability and Utilizing Lysostaphin-Producing Probiotics to Combat Staphylococcus aureus in Milk

IF 3.5 2区 农林科学 Q2 BIOCHEMISTRY & MOLECULAR BIOLOGY
Shuhang Zhang, Kangshu Wen, Wenxia Song, Qingbin Li, Qingsheng Qi, Xuemei Lu
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Abstract

The emergence and spread of methicillin-resistant Staphylococcus aureus (MRSA) is a major cause of bacterial infections in healthcare. Lysostaphin, a Zn2+-dependent endopeptidase, hydrolyzes the glycine (G)-linked pentapeptide bridge in the pathogen Staphylococcus aureus. In this study, the stability of lysostaphin was improved by engineering the addition of disulfide bonds around the protein’s Zn2+ binding site through molecular dynamics simulation. Further mutagenesis to add polyG at the C-terminus yielded combination variant 319/252-G3, which was 2.6-fold more stable than lysostaphin after 30 min at 60°C. Optimization of the C-terminal glycine number produced variant 319/252-G6, which retained 67.3% of its antimicrobial activity after 30 min at 70°C, and could be resistant to milk pasteurization conditions. Based on this, a probiotic strain of Escherichia coli Nissle 1917 to constitutively express and secrete lysostaphin from a mutated lac operator was engineered, resulting in effective control and prevention of S. aureus contamination in milk. One possible biotechnological application to utilize lysostaphin and probiotic strains can be used in animal feed and food additives for the prevention and treatment of S. aureus infections.

Abstract Image

提高溶血萘素的耐热性并利用产生溶血萘素的益生菌来对抗牛奶中的金黄色葡萄球菌
耐甲氧西林金黄色葡萄球菌(MRSA)的出现和传播是医疗保健领域细菌感染的主要原因。溶葡萄蛋白是一种 Zn2+ 依赖性内肽酶,能水解病原体金黄色葡萄球菌中的甘氨酸(G)连接五肽桥。在这项研究中,通过分子动力学模拟,在蛋白质的 Zn2+ 结合位点周围增加了二硫键,从而提高了溶血肽的稳定性。通过进一步诱变在 C 端添加 polyG,得到了组合变体 319/252-G3,该变体在 60°C 温度下 30 分钟后的稳定性是溶血萘蛋白的 2.6 倍。通过优化 C 端甘氨酸的数量,产生了变体 319/252-G6,该变体在 70°C 温度下 30 分钟后仍能保持 67.3% 的抗菌活性,并能耐受牛奶巴氏杀菌条件。在此基础上,我们设计了一株大肠杆菌 Nissle 1917 的益生菌株,该菌株可通过突变的 lac 操作子连续表达和分泌溶血磷脂,从而有效控制和预防牛奶中的金黄色葡萄球菌污染。溶血磷脂和益生菌株在生物技术方面的一个可能应用是用于动物饲料和食品添加剂,以预防和治疗金黄色葡萄球菌感染。
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来源期刊
Journal of Food Biochemistry
Journal of Food Biochemistry 生物-生化与分子生物学
CiteScore
7.80
自引率
5.00%
发文量
488
审稿时长
3.6 months
期刊介绍: The Journal of Food Biochemistry publishes fully peer-reviewed original research and review papers on the effects of handling, storage, and processing on the biochemical aspects of food tissues, systems, and bioactive compounds in the diet. Researchers in food science, food technology, biochemistry, and nutrition, particularly based in academia and industry, will find much of great use and interest in the journal. Coverage includes: -Biochemistry of postharvest/postmortem and processing problems -Enzyme chemistry and technology -Membrane biology and chemistry -Cell biology -Biophysics -Genetic expression -Pharmacological properties of food ingredients with an emphasis on the content of bioactive ingredients in foods Examples of topics covered in recently-published papers on two topics of current wide interest, nutraceuticals/functional foods and postharvest/postmortem, include the following: -Bioactive compounds found in foods, such as chocolate and herbs, as they affect serum cholesterol, diabetes, hypertension, and heart disease -The mechanism of the ripening process in fruit -The biogenesis of flavor precursors in meat -How biochemical changes in farm-raised fish are affecting processing and edible quality
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