Highly Efficient Expression Of Recombinant Paired-basic Endopeptidase And Its Application In Soybean Proteolysis

Paired-basic endopeptidase（PBE）can specifically cleave the C-terminal peptide bond of double basic amino acids precisely.The PBE domain is complex,including signal peptide,propeptide,catalytic domain,P domain,Thr/Ser rich region,TMD and C-tail.The existence of redundant domain lead to the imbalance between function and expression quantity,resulting in low expression quantity.In this study,PBE from Saccharomyces cerevisiae and Pichia kudriavzevii were selected for research,its codon-optimized gene was truncated to different degrees,and the constructed recombinant vectors were transferred into Escherichia coli BL21 and Pichia pastoris GS115 for high expression and proteolytic applications.The main contents are as follow:（1）Construction and protein expression of recombinant PBE in E.coli BL21.Based on domain analysis,the signal peptide,propeptide,TMD and C-tail of the PBE were excised.And cloned into the expression vector p GEX-6P-1 after codon optimized.The recombinant vector was transferred into E.coli BL21,and thirteen recombinant strains were constructed.SDS-PAGE results showed that the recombinant enzymes except for Pk PBE?1 were expressed in the recombinant E.coli.（2）Purification and enzymatic properties of recombinant PBE expressed in E.coli.The enzyme was purified by GST affinity chromatography and prescission enzyme.The purified wild type Sc PBE and Pk PBE were lower than 1.0 U·g^-1.The enzyme activity of Sc PBE?1,Sc PBE?2,Pk PBE?1 and Pk PBE?2 was lower than 10 U·g^-1.Sc PBE?3,Sc PBE?4,Pk PBE?3,and Pk PBE?4 have the highest enzyme activity 47.5 U·g^-1 and p H 5.0.The optimal temperature of Sc PBE?3,Pk PBE?3 and Pk PBE?4 was 40°C,while that of Sc PBE?4 was 50°C.The k_cat values of Sc PBE?4 and Pk PBE?4presented higher than Sc PBE?3 and Pk PBE?3 by 10.8%and 23.2%,respectively.Sc PBE?4 and Pk PBE?4 increased the k_cat/K_m values by 9.9%and 26.6%than Sc PBE?3 and Pk PBE?3.（3）Construction,high copy screening,optimization of fermentation conditions and protein purification of two kinds of PBE using P.pastoris as expression host.P.pastoris GS115/p PIC9K-Scpbe-678 and P.pastoris GS115/p PIC9K-Pkpbe-724 were successfully constructed.The positive transformants were screened by G418-resistant plates and the fermentation conditions were optimized.The optimal fermentation conditions for Sc PBE-678 and Pk PBE-724 were as follows:p H 5.5-6.0,26°C,the initial inducted OD₆₀₀ was 0.5,1%（v/v）methanol content,after 96 h the highly efficient pretein expression was achieved.The target protein was obtained by dialysis and Ni-NTA affinity chromatography.（4）Enzymatic properties of recombinant PBE expressed in P.pastoris.The results showed that the optimal p H of Sc PBE-678 and Pk PBE-724 was 9.0,and the p H stability of Pk PBE-724 was higher than Sc PBE-678.The optimum temperature of Sc PBE-678 was 45°C,and Pk PBE-724 was 50°C.Thermostability of Pk PBE-724 was higher than Sc PBE-678.Na⁺and ethyl acetate significantly promoted the enzyme activity of Sc PBE-678 and Pk PBE-724.The K_m of Sc PBE-678 was lower than Pk PBE-724,and the k_cat of Sc PBE-678 was smaller than Pk PBE-724.The k_cat/K_m of Pk PBE-724 was higher than Sc PBE-678.（5）Application of the recombinant PBE in hydrolysis.The recombinant PBE can recognize the double basic amino acids on the target short peptide and successfully cut the short peptide.Soybean protein isolate was hydrolyzed by the recombinant PBE and alkaline protease.Compared with alkaline protease hydrolysis alone,the synergistic hydrolysis could degrade soybean protein into smaller peptides.Moreover,the Sc PBE?4 and Pk PBE?4 had higher hydrolysis capacity than Sc PBE?3 and Pk PBE?3 in recombinant E.coli.Among the protein hydrolytic products,the polypeptides with 100-1,000 Da accounted for 45.3%.The recombinant Pk PBE-724 exhibited the better hydrolytic capacity than Sc PBE-678 in P.pastoris.The polypeptides with 100-1,000 Da accounted for 64.2%.