Department of Biological Sciences, Cork Institute of Technology, Cork, Ireland.
J Appl Microbiol. 2011 Oct;111(4):1025-35. doi: 10.1111/j.1365-2672.2011.05119.x. Epub 2011 Sep 8.
To develop an efficient purification strategy for the bacteriophage lysin CHAP(K) . To evaluate its antibacterial spectrum(,) enzymatic properties, optimal reaction conditions and lytic activity against live Staphlyococcus aureus.
Recombinant CHAP(K) was purified to homogeneity by cation exchange chromatography, with yields of up to 10 mg from 1 l of Escherichia coli culture. The lytic spectrum of CHAP(K) includes all staphylococcal species and also members of the genera Micrcococcus, Streptococcus, Nesterenkonia, Arthrobacter, Leuconostoc and Carnobacterium. The enzyme was active from pH 6 to 11 with an optimum activity at pH 9, from 5 to 40°C, with an optimum activity at 15°C. When cell lysis by CHAP(K) and lysostaphin was compared over a concentration range of 2·5-10 μg ml⁻¹ using live Staph. aureus for 5 min at 37°C, CHAP(K) gave rise to greater turbidity reduction indicating that it works more rapidly than lysostaphin.
This study describes in detail the purification and characteristics of the novel phage-derived enzyme CHAP(K) demonstrating that it has excellent biochemical properties as an anti-staphylococcal agent.
Currently, there is a need for new antimicrobial agents due to the increasing worldwide prevalence of antibiotic resistance. Our findings demonstrate the potential for development of CHAP(K) as an alternative therapeutic against pathogenic staphylococci including MRSA.
开发一种有效的噬菌体溶菌酶 CHAP(K) 的纯化策略。评估其抗菌谱、酶学性质、最佳反应条件以及对活金黄色葡萄球菌的裂解活性。
通过阳离子交换层析，从 1 升大肠杆菌培养物中可将重组 CHAP(K) 纯化至均一性，最高产量可达 10 毫克。CHAP(K) 的裂解谱包括所有葡萄球菌属物种，还包括微球菌属、链球菌属、内斯特龙诺尼亚菌属、节杆菌属、明串珠菌属和肠球菌属的成员。该酶在 pH 6 到 11 之间具有活性，最佳 pH 活性为 9，在 5 到 40°C 之间具有活性，最佳温度为 15°C。当在 37°C 下使用活金黄色葡萄球菌在 2.5 到 10 μg ml⁻¹ 的浓度范围内比较 CHAP(K) 和溶菌酶对细胞的裂解作用 5 分钟时，CHAP(K) 引起的浊度降低更大，表明其作用比溶菌酶更快。
本研究详细描述了新型噬菌体衍生酶 CHAP(K) 的纯化和特性，表明其作为抗葡萄球菌剂具有优异的生化特性。
由于抗生素耐药性在全球范围内的日益流行，目前需要新的抗菌剂。我们的研究结果表明，CHAP(K) 作为治疗包括 MRSA 在内的致病性葡萄球菌的替代治疗方法具有潜在的开发前景。
