Enzymatic Properties Of D2 Protease And Establishment ELISA For Detecting D2

Objective:1. To isolate and purify the serine alkaline protease from the D2 fermentation.2. To investigate the effect of metal ions and laundry detergent on the enzymatic properties and stability of D2 protease.3. To establish the ELISA for quantitative analysis of D2 protease by preparing the polyclonal antibody.4. To investigate the amino acid sequence of D2 protease.Methods:1. The protease was purified by a stepwise procedure including centrifugation, gelsiccation and size-exclusion chromatography, and identified by SDS-PAGE.2. The protease activity and stability was investigated by adding the mental ions Ca2+,Cu2+,Mg2+,K+and commercial detergents.3. The double antibody sandwich ELISA and indirect ELISA for quantitative analysis of D2 protease were established by the antisera against D2 protease obtained from the rabbits and guinea pigs.4. By means of electrospray ionization-quadrupole-time of fight-mass spectrometry (ESI-Q-TOF-MS), amino acid sequence of purified D2 protease was deduced.Results:1. The three-step purification protocol resulted in a 3.0-fold purification with 39.1% activity recovery.2. Enzymatic activity is enhanced by the addition of mental ions such as Ca2+,Cu2+,K+, Mg2+. The stability and activity of D2 protease have a significant activation with 5mM CaCl2. The protease can be stable in the commercial detergents.3. In double antibody sandwich ELISA, the optimal working concentrations of coating and detection antibodies were 1:4000 and 1:5000 respectively. The linear range and detection limit for determination of D2 were 28～1180μg/L,4.6μg/L respectively. The recovery rates of D2 ranged from 92.44%～105.26%. The developed method showed good relationship to that by Bradford method. In indirect ELISA, the linear detection range was from 18 to 1160μg/L. The coincidence rate was 95.5% between this method and agar diffusion reaction.5. By means of ESI-Q-TOF-MS, the amino acid sequence of D2 was determined as QVAWATGETAAPK,FAVQSTFK,LGYVSPPALVLADDNAAAR,QLNLNVTQLG CGVFSGK,AHGFLPLTK,APSATGGSALYPLEFVVGK。 Conclusion:The ELISA technique was established successfully, it has laied the foundation for the study of enzymology and biology. The confirmation of D2 N terminal sequence provide theoretical evidence for the follow-up study of D2 gene structure and primary amino acid structure.