小肠结肠炎耶尔森菌非特异性核酸酶培养条件优化

Optimization of cultivation conditions of a non-specific nuclease from Yersinia enterocolitica subsp.Palearctica

为实现小肠结肠炎耶尔森菌非特异性核酸酶(non-specific nuclease from Yersinia enterocolitica subsp.palearctica,Nucyep)的高水平分泌表达,研究从pET24a-nucyep中得到nucyep基因片段,插入穿梭质粒pBE-S得到重组载体pBE-S-nucyep,将其线性化后与枯草芽孢杆菌信号肽混合物连接并转化大肠杆菌感受态细胞,经测序验证共获得43个含正确信号肽序列的克隆。将以上重组载体分别转化枯草芽孢杆菌(Bacillus subtilis,B.subtilis)RIK1285,通过比光密度法筛选得到Nucyep分泌水平最高的工程菌株B.subtilis RIK1285/pBE-S-nucyep-YoaW。对其进行培养条件优化,结果表明,采用工程菌B.subtilis RIK1285/pBE-S-nucyep-YoaW在培养基3×LB中以体积分数5%接种量接种,培养温度30℃,摇床220 r/min振荡培养48 h时,发酵上清液中重组Nucyep酶活达到(8.27±0.41) U/μL,较优化前提高了约20倍。结果表明,通过对信号肽的筛选及培养条件的优化,重组Nucyep在B.subtilis中可获得高效分泌表达,为其进一步研究及应用奠定基础。

To obtain the high-level extracellular expression of a non-specific nuclease from Yersinia enterocolitica subsp.palearctica(Nucyep),the gene fragments of nucyep from pET-24a-nucyep were cloned into the shuttle vector pBE-S to yield pBE-S-nucyep.The linearized pBE-S-nucyep was ligated to the DNA mixtures of different signal peptides through In-Fusion cloning enzyme,then transformed into competent E.coli Stellar cells.43 positive clones were obtained and the corresponding recombinant plasmids pBE-S-nucyep-SP were separately transformed into Bacillus subtilis(B.subtilis)RIK1285 to yield 43 recombinant strains.The Nucyep activities of 43 recombinant strains were determined using the specific optical density method which has been developed in our lab previously.B.subtilis RIK1285/pBE-S-nucyep-YoaW carrying the signal peptide YoaW showed the highest enzymatic activity level among the engineered strains and was employed in the next study.Through optimization of cultivation conditions,the highest Nucyep activity of(8.27±0.41)U/μL was obtained and it exhibited 20-fold higher than that of the cultivation conditions before optimization.The optimized cultivation conditions were as follows:volume fraction 5%of seed culture inoculum,culture medium 3×LB,incubated at 30℃,48 h and 220 r/min.In conclusion,the results indicated that the high activity of Nucyep could be achieved through the employment of suitable signal peptide in combination with applying the optimized cultivation conditions.It will provide some support for further study.