| Prolyl endopeptidase(PEP) is a special serine protease with a unique ability to cleave peptides bonds on the C-terminal side of internal proline residues. PEP and its hydrolysis products are widely applied in the food manufacturing industry. PEP has also been extensively investigated as a potential pharmaceutical target for various diseases. In our previous studies, we cloned a new PEP gene from Aspergillus oryzae. The present paper describes the efficient expression of active PEP with several fusion partners in P. pastoris GS115 and the properties and application of the recombinant PEP were also studied.(1) When fused with PLA2, CBD, SUMO, MBP, the recombinant expression stains were named as GS115/pPIC9K/pPICZαA-PLMH, GS115/pPIC9K/pPICZαA-CLMH, GS115/pPIC9K/pPICZαASLMH, GS115/pPIC9K/pPICZαA-MLMH, respectively. The parent stain was named GS115/pPIC9K/pPICZαAMOH. The enzyme activities of the recombinant fusion proteins CLMH, SLMH, MLMH and PLMH were increased to 3.8-, 2.7-, 4.9- and 7.4-fold compared with that of the parent MOH. Moreover, western blot analysis of the culture supernatant detected an increase of the fusion secretion level compared with that of MOH. The q-PCR suggested that the PLMH mRNA level in the fusion stain was increased to 2.3-fold compared with that of the parent stain. This phenomenon could be explained by the inactivity of the full-length proenzyme(proPEP) until specific proteolytic processing removes a short N-terminal extension prosequence, with processing that appears to be performed by PEP itself.(2) The optimum temperature and stability of the parent MOH and the fusion protein PLMH were nearly the same. They both showed maximal activity at 40°C and showed high stability(over 80% activity) between 25 and 35°C after incubation for 120 min, but they were not very stable at higher temperatures. When the temperature reached 40°C, the relative activity decreased to 30% of the maximum activity, and the activity was almost lost after incubation for 120 min at 45°C. Additionally, MOH and PLMH had the same pH profile. Within the pH range of 5.5 to 6.5, MOH and PLMH retained more than 80% of the initial activity after incubation at 40°C for 120 min. MOH and PLMH showed the highest activity at pH 5.5, and over 60% of the maximum activity was retained between p H 5.0 and 7.0. Kinetic data for the purified PLMH were evaluated at 40°C and pH 5.0. The Km,kcat,kcat/Km of PLMH were 0.23±0.01 mM, 112.51±0.02 s-1 and 489.17 s-1 mM-1, respectively.(3) The recombinant stain GS115/pPIC9K/pPICZαA-PLMH was studied by high-density fed-batch fermentation in a 7 L bioreactor. When the culture reached an OD600 110, add 100% methanol to a final concentration of 1 g?L-1 to maintain induction. The experimental result indicated that the cell concentration(OD600) achieved the maximum of 660, the total protein concentration achieved the maximum of 0.28 mg?m L-1 and the maximum activity reached at 960 U?L-1.(4) Addition of different concentrations recombinant enzymes during the storage phase of beer brewing, demonstrated the potential application of our fusion PEP in the non-biological stability of beer. Meanwhile, The PLMH also can effectively hydrolyze barley proteins in beer. |
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