Cloning And Expression Of The Proline Aminopeptidase From Aspergillus Oryzae

PAPs are the aminopeptidases of narrow specificity and can only cleave a proline residuespecifically from the N-terminus of polypeptides and protein. Just like other aminopeptidases,PAPs also have the ability to reduce bitter peptides in food processing industry. Moreover, thenarrow specificity of PAPs towards proline residue makes PAPs are essential for thedegradation of collagen. Therefore, researches on PAPs have scientific significances andapplication values.In this study, double-stranded cDNA was synthesized by the total RNA template whichisolated from A. oryzae JN-412CGMCC8487, and the cDNA sequence which encoded PAPwas amplified by specific primers using PCR method. Then, the recombinant E. coliBL21/pET-28a-pap was successfully constructed and the cDNA sequence coding PAPefficiently expressed in E. coli BL21. The recombinant enzyme was purified3.3-fold tohomogeneity with a recovery of36.7%from cell free extract using Ni2+-NAT affinity columnchromatography.Study on the characterization of the recombinant enzyme showed that the purifiedrecombinant enzyme exhibited the highest activity at60oC and pH7.5. After incubation at30oC for1h in different pH range, the recombinant enzyme showed perfect pH stability andthe residual activity was still above80%. However, this recombinant enzyme showed poorthermal stability, and after incubation at50oC for1h, the residual activity was only10%. Zn2+,Cu2+and PMSF showed strongly inhibition on enzyme activity. DTT, β-mercaptoethanol andEDTA showed no influence on enzyme activity. When Zn2+and β-mercaptoethanol werecoexisted, the inhibition influence of Zn2+was greatly weakened.By using collagen as substrate, the purified recombinant enzyme greatly contributed tothe hydrolysis of collagen when used in combination with neutral protease, free amino acid incollagen hydrolysates was significantly increased compared with three other control groups.The optimal fermentation conditions at shake flask stage for enzyme production wasfixed as follows: glucose5g·L-1, peptone20g·L-1, yeast extract30g·L-1, Mg2+7.5mmol·L-1,K2HPO412.54g·L-1, KH2PO42.31g·L-1, initial pH7.2, logarithmic phase seed2%, volume50mL/250mL. The optimal induction opportunity was for9h after inoculation when OD600wasabout11.44. Induction time and the final concentration of lactose was12h and3g·L-1,respectively. Using the above conditions for fermentation, the final specific activity reached78.42U·mg-1, DCW reached4.55g·L-1and OD600was about13.67.At the fermentor phase, batch fermentation and fed-batch fermentation strategy wereperformed at7L fermentor for investigating the high cell-density culture (HCDC) for therecombinant E. coli which could produce proline aminopeptidase. Exponential feedingstrategy was used for fed-batch fermentation. From the experiment results, exponentialfeeding strategy was good for controlling the acetic acid concentration in the cell growthphase. The OD600reached142(DCW=44.96g·L-1),9.16-fold vs batch fermentation. The finalenzyme activity reached211.20U·mg-1,2.3-fold vs batch fermentation.The most flexible region (amino acid numbers,238-246) out of the protein active center was determined by the molecular dynamics simulation (MDS), and one glycine (241) wasexisted in this region by sequence analysis. Mutant (G241P) was construsted by the BuildMutants module of Discovery studio2.5. The mutant was tested by MDS, and the stability ofthe region (238-241) was enhanced. Furthermore, the thermostability of mutant was measured,the T50of mutant was4.8oC higher than the original enzyme.