Screening、Expression And Molecular Modification Of New Food-grade Prolyl Endopeptidase

The prolyl endopeptidase（EC3.4.21.26, PEP） is an enzyme that belongs to a distinct class of serine peptidases, and has been used in pharmaceutical, brewing and food industry. However, its application might be hindered by its less variety, low catalytic activity, poor operational stability, and so on. Previous studies mainly focused on the mammal PEP or bacterial PEP, while the fungal PEP has been not described. These factors became the barrier to the utilization of PEP in food industry. In our study, fungal food-grade PEP is selected firstly, and then a series of relevant works are carried out as follows.（1） A reusable method, which integrated in the media screening, enzyme activity, identification of molecular biology and morphological identification of microorganisms for screening the strains producing PEP was first established in the present study. By using the method, A. niger 2（PEP2） and A. oryzae 101（PEP101） were obtained with PEP activity 95 U·L-1 and 81 U·L-1, respectively. The molecular weight of PEP2 and PEP101 were both about 60 k Da and they were more stabled in slightly acidic to neutral environments, which was different from other known PEPs. The optimum temperature of PEP2 and PEP101 were in the range between 30°C and 40 °C. Some metal ions(Cu²⁺, Al³⁺, Zn²⁺ and Mn²⁺) had great negative impacts on the PEP2 and PEP101. Kinetic analysis showed PEP2 and PEP101 with no hydrolysis capacity toward a peptide and dipeptide, while with higher substrate affinity（Km: 0.18 m M） and catalytic efficiency（kcat/Km: 996.2 m M-1S-1, 924.3 m M-1S-1） toward Ala-Ala-Pro-p NA. The PEP2 and PEP101 had an effect of the removal of bitterness with casein as substrate.（2） Therefore, cloning and heterologous expression of A. niger gene was carried out using recombinant DNA technology due to the catalytic potential of this strain for brewing industry. The encoding gene（1,578 bp） was cloned from A.niger 2,then transformed into Pichia pastoris GS115 and recombinant strain GS115/p PIC9K-endoprotease EPR was successfully constructed. The recombinant strain displayed good PEP activity（500 U·L-1） with Z-Gly-Pro-p NA as the substrate. SDS-PAGE showed the molecular weight of endoprotease EPR were 60 k Da. The optimum p H and temperature of endoprotease EPR were p H 4.0-5.0 and 35°C, and the stabilities were p H 3.0-7.0 and 15-55°C, respectively. Cu2+, Al 3+, Zn2+ and Mn2+ could inhibit the activity of recombinant enzyme intensively, and Fe2+ and Mg2+ could inhibit activity moderatly, while Ca2+ could active endoprotease EPR. According to the kinetic assay, the endoprotease EPR was with highest substrate affinity（Km:0.24 m M） and catalytic efficiency（kcat/Km:731.7 m M-1S-1） toward Ala-Ala-Pro-p NA, while Z-Pro-p NA and Z-Ala-Ala–Ala-Pro-p NA were better substrates. Furthermore, the recombinant enzyme had the ability to digest peptides with the C-terminal of proline as well as alanine, and was also capable of hydrolyzing larger peptides. The properties made it a highly promising candidate for application in the field of brewing and food process industry.（3） In our previous researches, we found that PEP from A. oryzae had better characteration（such as stability in acidic condition and broader temperature stability） than other PEPs. There was no report about the gene encoding PEP of A. oryzae. Partial c DNA sequence was amplified from the total RNA of wild A. oryzae through RT-PCR and a novel gene encoding prolyl endopeptidase from A. oryzae（AO-PEP） was amplified with degenerate primers. AO-PEP was consisted of 1,743-bp ORF which encoded 581 amino acid residues. Amino acid sequence analysis of AO-PEP showed that this enzyme belongs to a class serine peptide S28 family, which having α/β hydrolase typical structure. The active site of AO-PEP was located in Ser-206, Asp-479, His-528 with conserved region G204SSYSG209, based on the structure of human prolylcarboxypeptidase（PRCP, PDBID: 3n2 z B） with sequence identity of 18.2 %.The AO-PEP gene was inserted into p PIC9 K, then the recombinant p PIC9K-AO-PEP was introduced into host GS115. After induced by methanol, the recombinant GS115/p PIC9K-AO-PEP was successfully expressed with PEP activity 310 U·L-1. Expression, purification and characterization of AO-PEP were analyzed. SDS-PAGE showed the molecular weight of AO-PEP was 64 k Da. The optimum p H and temperature of AO-PEP were p H 5.0 and 40°C, and the stabilities were p H 3.5-7.5 and 15-60°C, respectively. Cu2+, Al 3+, Zn2+ and Mn2+could inhibit the PEP activity intensively, and Fe2+ and Mg2+ could inhibit activity moderatly, K+ and Na+ could not almost inhibit activity, while Ca2+could activate AO-PEP. The Km and kcat values of AO-PEP for different substrates were evaluated. The results implied that the AO-PEP toward the Ala-Pro-p NA showed no activity, while Ala–Ala-Pro-p NA and Z-Ala-Ala–Ala-Pro-p NA were better substrates for the AO-PEP.（4） In order to improve the catalytic activity of AO-PEP, mutagenesis for the active site and substrate pockets was developed. Site mutagenesis of Thr-391, Thr-537 in the loops in the substrate pocket and Cys-529, Gln-533 located the arrangement of the catalytic histidine amino acids near the active site were conducted to improve the specific activity and stability. Ten mutant libraries（T391A, T391 C, T537 A, T537 C, C529 H, C529H/Q533 R, T537A/T391 A, T537A/T391 C, T537A/C529 H and T537A/C529H/Q533R） were constructed and their positive mutants were obtained. The results showed that the PEP activity of these mutants were increased, as well as the ranges of p H stability and temperature stability, except the mutants T391 A and T391 C. Kinetic datas analysis showed that mutants C529H/Q533 R, T391 C, T537A/T391 A, T537A/T391 C and T537A/C529H/Q533 R toward the substrate Z-Gly-Pro-p NA had better affinity than AO-PEP, and the catalytic efficiency of all the mutants had been improved. The AO-PEP m RNA levels in the mutants（except T391 A and T391C） were also enhanced than that in the AO-PEP. The results demonstrated that the tandem His-His arrangement and the loops facing the active site of AO-PEP are crucial to enhance enzyme activity and substrate specificity.（5） To improve the storage stability of recombinnat prolyl endopeptidase and identify its potential improvement in non-biological stability of beer, a series of small molecule compounds were added in the storage mixed, and the prescription was made up which contained glycerol as the main key factor. Addition of recombinant enzymes during the fermentation phase of beer brewing, demonstrated the potential application of our prolyl endopeptidases in the non-biological stability of beer, which will favor further industrial development of these new enzymes in beer stabilization.