Study On Heterologous Expression And Enzymatic Properties Of Proline Specific Endopeptidase

Proline Specific Endopeptidases （PSEases） are the enzymes that hydrolyze substrates on the carboxyl site of proline residues located internally in a peptide. They are widely distributed in plants, mammals, bacteria and fungi. The enzymes are hignly homologous in structures, but they are very different in their catalytic mechanism. A large number of studies have been reported that the roles of these enzymes among various organisms are diverse, and the functions in different organs of the same organism are also discrepant. Based on the current research progress of this class of enzymes, as well as the development prospects of its value, to meet the domestic market demand to find a biological enzyme, is both an opportunity and a challenge.We have entrusted Sangon Biotech synthetized cDNA from Flavobacterium meningsepticumingsepticum. We connected psease to the vector pGBT-NH, then transformed into the Ecoli BL-21 star, we got the protein GBl-PSEase. The protein molecular mass was analyzed by SDS-PAGE and Western-Blot, the result showed the GBl-PSEase molecular mass was 84.5kDa, it was the same with the theoretical value. We got the purified GBl-PSEase by Nickel ion affinity chromatography technology.The activity of GBl-PSEase was assayed for Z-Gly-Pro-pNA, The result showed that GBl-PSEase hydrolyzed Z-Gly-Pro-pNA, the Km was 0.921 mM/min, the Vmax was 0.666 mM/min, the optimum pH was 6.4 and the optimum temperature was 37℃The pH stability and thermostability of GBl-PSEase was monitored. The result showed GBl-PSEase was more stable in above pH=6.4 enviroments than below pH=5.0. Thermostability of GBl-PSEase was more stable in below 37℃ environments than above 37℃.The effects of different metal ions on GBl-PSEase activity was assayed. The result showed 1 mmol/L Ca²⁺ and Mn2- could increase GBl-PSEase activity up to 18.57% and 23.73%, but 1 mmol/L Mg²⁺, Cu²⁺, Ni²⁺ and Zn²⁺ could inhibite GBl-PSEase activity up to 24.83%,4.55%,18.06%and 43.05%,5 mmol/L Ca²⁺, Mg²⁺and Mn²⁺ could stimulate GBl-PSEase activity by 43.42%,8.44%and 61.96%,5 mmol/L Cu²⁺, Ni²⁺ and Zn²⁺ could inhibite GBl-PSEase activity up to 55.73%,46.25%and 43.05%, which hinted the effects on GBl-PSEase activity was related with the concentration and the character of above ions,Stability of GBl-PSEase that was storaged in 4℃ environment for 3 months was detected. The result showed dry GBl-PSEase activity droped by 9.57%, but liquid GBl-PSEase activity droped by 37.44%, that implied dry GBl-PSEase was more stable than liquid GBl-PSEase, and we might storage dry GBl-PSEase for long time.