Abstract: 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.

Key words: Nucyep, extracellular expression, Bacillus subtilis, optimization of cultivation conditions, activity assay