| Leucine aminopeptidase(LAP; EC 3.4.11.1) is an important flavourzyme especially in protein hydrolysate debittering by removing hydrophobic amino acid residue at the N-terminal end. Besides, it is also applied to preparation of active peptides and analysis of protein sequence. In this study, leucine aminopeptidase from Bacillus Subtilis was cloned and expressed in Pichia Pastoris, a widely used heterologous protein expression host. The research included the following: the optimization of fermentation conditions, separation and purification of aminopeptidase, enzymatic properties, analysis of N-glycosylation and research on preliminary applications of recombinant aminopeptidase.Flask-shaking optimization of the recombinant strain P. pastoris was completed. The optimized conditions were as follows: initial induction time was 20 h, initial methanol concentration was 10 g·L-1, the medium volume was 25 m L/250 m L, initial induction p H was 7.0, induction temperature was 23 oC, induction time was 96 h, and the amount of sorbitol was 6 g·L-1. Finally, the yield reached 51.58 U·m L-1 which was about 13 times higher than that of wild-type strain.The purification of recombinant aminopeptidase from the culture supernatant was achieved through several steps including ammonium sulfate salting(50%-70% saturation), Hitrap Q HP anion exchange chromatography, and Sephadex 75 10/300 GL gel filtration chromatography. The specific activity of the purified recombinant enzyme was 240.48 U·mg-1. The final recovery of the enzyme was 9.4% and the purification factor was 8.4.The properties of the pure recombinant enzyme were characterized and the results showed that the optimal temperature and p H of the purified recombinant enzyme were 60 oC and 8.5, respectively. The purified aminopeptidase was stable within 30-60 oC and p H8.0-9.0. It was intensively inhibited by Ni2+, Ca2+, DL-dithiothreitol(DTT) and ethylene diamine tetraacetic acid(EDTA), but activated by Co2+. Mn2+, Cu2+, Zn2+, Mg2+ and PMSF had slight inhibition on the recombinant enzyme. Using the Leu-p NA as the substrate, the Km and Vmax were estimated to be 0.97 mmol·L-1 and 10.95 mmol·L-1·min-1, respectively.The sequence analysis of aminopeptidase indicated three potential N-glycosylation sites. In the process of expression in P. pastoris, leucine aminopeptidase from B. subtilis Zj016 occurred different levels of N-glycosylation modification and it was further verified via MALDI-TOF-MS analysis and deglycosylated with Endo Hf restriction enzyme. Research on characterization between the wild and recombinant animopeptidase indicated that glycosylation modification had little effect on the secondary structure and substrate specificity of this enzyme, but significantly improved its thermostability(70oC for 1 h, remain 63.5% enzyme activity), and substrate affinity(of 1.9-fold).Conditions(especially initial cell density) for the fed-batch cultivation of the recombinant P. pastoris in a 7 L fermentor was optimized. When the initial cell density(OD600) reached 400, began to flow in methanol immediately. After the induction was maintained for 96 h, the activity of aminopeptidase reached 224 U?m L-1 and was about 4.4-fold higher than that of flask.Using the soybean, pea protein and corn gluten meal as the substrate, they were hydrolyzed with aminopeptidase and alkaline protease enzyme alone or together. Compared with the control group, in the three enzymatic substrate solution, the free amino acids increased significantly, especially the content of Leu in the hydrolyzates of complex enzymes was improved by 223.5 times, 58.8 times and 35.7 times, respectively. The content of small peptides, oligopeptides and polypeptides were significantly increased, of which small peptide(with molecular weight under 180 Da) content accounted for 72.89%, 70.93% and 59.42%, respectively. While the content of macromolecules have been reduced and macromolecular substance(with molecular weight with 3000-5000 Da) content is only 0.98%, 0.32% and 0.07%. Thoes all showed that the recombinant aminopeptidase had a good compound application characteristics as the original enzyme. |
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