| Prolyl aminopeptidase(PAP) is a type of exopeptidase which catalyzes the cleavage of N-terminal proline residue from peptides and proteins specifically. Coupled with endoprotease, PAP can improve the hydrolysis of proteins. Additionally, PAP is widely used to remove the bitterness of hydrolysate, particularly hydrolysate from proteins with high-content of proline. The heterogenous expression of pap gene in safe strain B. subtilis can greatly reduce the cost of industrial production of PAP.In this study, the pap gene was amplified by a pair of primers using pMD19-pap as templates. Then it was inserted into plasmid pMA5 to construct a recombinant expression vector pMA5-pap. The resultant pMA5-pap plasmid was transformed into B. subtilis WB600 by electroporation and the positive clones were screened in LB solid medium containing 50 μg/m L kanamycin.The intracellular and extracellular PAP activity reached 39.6 U/m L and 7.5 U/m L respectively after the recombinant strain was cultured in TB medium(containing 50 μg/m L kanamycin) at 37 oC and 220 rpm for 24 h. When the recombinant B. subtilis was fermented in an optimized secretion medium which was consisted of extra 5% sorbitol and 2 mM CaCl2 on the basis of TB medium, the PAP activity in the fermented supernatant increased from 7.5 U/mL to 36.0 U/m L.The optimized conditions for shaking flask fermentation were optimized and set as follows: culture temperature 37 oC, shaking speed 250 rpm, medium volume 40 m L/250 m L, initial pH 5.5, inoculum size 8% and the fermentation time 24 h. Extracellular enzyme activity increased to 52.5 U/m L. It was 7 times of the PAP activities before optimization. When cultured in a 7 L fermentor, the cell density and intracellular PAP activity were improved.The purified recombinant PAP was obtained after stepwise salting-out at 40-50% ammonium sulfate, overnight dialysis, Hitrap Q anion exchange chromatography, ultrafiltration, and Superdex 75 gel filtration chromatography. And the specific activity of the purified PAP was 247.3 U/mg. The recovery was 1.7% and the purification factor was 8.8. The purified PAP reached electrophoretically pure which was verified by SDS-PAGE.The enzymatic properties of the purified recombinant PAP were investigated. The optimum temperature and pH were 50 oC and 7.5, respectively. It presented good stability within pH 6-11 and had good thermostability below 50 oC. The activity of PAP was suppressed by CuSO4 and ZnSO4. However, CoCl2, MgCl2, MnCl2, CaCl2 and NiSO4 had neglected effect on the activity. The PAP activity was guadually inhibited at increased concentration of PMSF. β-mercaptoethanol can repress the inhibition effect of Zn2+. In addition, PAP was activated in the presence of high concentration of several salts. The relative enzyme activity reached 127.02% in 4.3 M NaCl. For substrates specificity, the recombinant PAP only exhibited hydrolysis towards Pro-pNA. A proline residue from the N-terminus of dipeptides and oligopeptides was cleaved specifically. Whereas prolines from the C-terminus cannot be hydrolyzed.Combined with recombinant PAP with alkaline protease and leucine aminopeptidase, hydrolyzation of casein was carried out. The free amino acids of hydrolysate sharply increased and the total free amino acids content was 63 times of the sample without enzymolysis. And the content of oligopeptides and small peptides increased significantly. The compounds in hydrolysate with molecular weight lower than 180 Da were accounted for 36.66%, while the macromolecules with molecular weight larger than 2000 Da were almost been degraded. |
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