Key Laboratory of Feed Biotechnology, Ministry of Agriculture, Beijing, 100081, China.
Appl Microbiol Biotechnol. 2014 Jan;98(2):681-94. doi: 10.1007/s00253-013-4881-2. Epub 2013 Apr 30.
NZ2114, a new variant of plectasin, was overexpressed in Pichia pastoris X-33 via pPICZαA for the first time. The total secreted protein of fermentation supernatant reached 2,390 mg/l (29 °C) and 2,310 mg/l (25 °C), and the recombinant NZ2114 (rNZ2114) reached 860 mg/l (29 °C) and 1,309 mg/l (25 °C) at 96 h induction in a 5-l fermentor, respectively.The rNZ2114 was purified by cation exchange chromatography, and its yield was 583 mg/l with 94.8 % purity. The minimal inhibitory concentration (MIC) of rNZ2114 to four ATCC strains of Staphyloccocus aureus was evaluated from 0.028 to 0.90 μM. Meanwhile, it showed potent activity (0.11-0.90 μM) to 20 clinical isolates of MRSA. The rNZ2114 killed over 99.9 % of tested S. aureus (ATCC 25923 and ATCC 43300) in Mueller-Hinton medium within 6 h when treated with 4 × MIC. The postantibiotic effect of rNZ2114 to S. aureus ATCC 25923 and ATCC 43300 was 18.6-45.6 and 1.7-3.5 h under 1×, 2×, and 4× MIC, respectively. The fractional inhibitory concentration index (FICI) indicated a synergistic effect between rNZ2114 and kanamycin, streptomycin, and vancomycin against S. aureus ATCC 25923 (FICI = 0.125), and additivity between rNZ2114 and ampicillin, spectinomycin (FICI = 0.625), respectively. To S. aureus ATCC 43300 [methicillin-resistant S. aureus (MRSA)], rNZ2114 showed a synergistic effect (FICI = 0.125-0.3125) with kanamycin, ampicillin, streptomycin, and vancomycin, and antagonism with spectinomycin (FICI = 8.0625). The rNZ2114 caused only less than 0.1 % hemolytic activity in the concentration of 128 μg/ml, and showed a good thermostability from 20 to 80 °C. In addition, it exhibited the highest activity at pH 8.0. These results suggested that large-scale production of NZ2114 is feasible using the P. pastoris expression system, and it could be a new potential antimicrobial agent for the prevention and treatment of S. aureus especially for MRSA infections.
NZ2114 是一种新型的 plectasin 变体，首次通过 pPICZαA 在毕赤酵母 X-33 中过表达。发酵上清液中的总分泌蛋白达到 2390mg/L（29°C）和 2310mg/L（25°C），在 5L 发酵罐中诱导 96 小时后，重组 NZ2114（rNZ2114）达到 860mg/L（29°C）和 1309mg/L（25°C）。rNZ2114 通过阳离子交换色谱法纯化，其产量为 583mg/L，纯度为 94.8%。rNZ2114 对 4 株 ATCC 金黄色葡萄球菌的最小抑菌浓度（MIC）评估范围为 0.028-0.90μM。同时，它对 20 株耐甲氧西林金黄色葡萄球菌（MRSA）的临床分离株表现出很强的活性（0.11-0.90μM）。rNZ2114 在 6 小时内将测试的金黄色葡萄球菌（ATCC 25923 和 ATCC 43300）的数量减少了 99.9%以上，当用 4×MIC 处理时，在 Mueller-Hinton 培养基中的 MIC 为 4×MIC。rNZ2114 对金黄色葡萄球菌 ATCC 25923 和 ATCC 43300 的后抗生素效应分别为 1×、2×和 4×MIC 下的 18.6-45.6 和 1.7-3.5 h。rNZ2114 与庆大霉素、链霉素和万古霉素对金黄色葡萄球菌 ATCC 25923 的部分抑菌浓度指数（FICI）表明存在协同作用（FICI=0.125），与氨苄西林和大观霉素（FICI=0.625）存在相加作用。对金黄色葡萄球菌 ATCC 43300（耐甲氧西林金黄色葡萄球菌），rNZ2114 与庆大霉素、氨苄西林、链霉素和万古霉素表现出协同作用（FICI=0.125-0.3125），与大观霉素表现出拮抗作用（FICI=8.0625）。rNZ2114 在 128μg/ml 的浓度下仅引起小于 0.1%的溶血活性，并且在 20-80°C 之间表现出良好的热稳定性。此外，它在 pH8.0 时表现出最高的活性。这些结果表明，使用巴斯德毕赤酵母表达系统大规模生产 NZ2114 是可行的，它可能成为预防和治疗金黄色葡萄球菌尤其是耐甲氧西林金黄色葡萄球菌感染的一种新型潜在抗菌药物。
