Preparation,Characterization And MPEG Modification Of Recombinant Phospholipase C

Phospholipase C（EC3.1.4.3,PLC）is a kind of hydrolase which catalyzes the hydrolysis of C3 phosphodiester bonds in glyceryl phospholipids to generate a water-soluble phosphate monoester and diacylglycerol（DAG）.Phospholipase C exists in prokaryotic and eukaryotic organisms widely and is broadly used in medicine,food and enzymatic degumming,etc.Phospholipase C originated from microorganisms are easy to be produced largely and become the main sources of industrial enzymes.In this study,a high phospholipase C producing strain was isolated,and the enzyme production condition was optimized.Furthermore,the characterization and mPEG modification of the recombinant Phospholipase C were discussed.The main results are as follows:1.Using egg-yolk solid medium,11 phospholipase C producing microorganisms were isolated from Xiamen mangrove sediments.One isolate named HSL3 showed high phospholipase C activity and hemolytic activity.It was identified as Bacillus cereus based on morphological,physiological and biochemical characteristics,and 16 S rRNA sequence phylogenetic analysis.By single-factor experiments,the optimal conditions for B.cereus fermenting were obtained to be 25℃,liquid volume 75 mL/250 mL flask,pH 7.0,0.25% inoculation amount,0.75%（v/v）egg yolk and 24 h.Under the optimized conditions,the phospholipase C activity was up to 22.8 U/m L which had been enhanced by 30.46%.2.The phospholipase C gene was successfully cloned by using PCR from HSL3 and the fusion expression strain Er 2566/pSmart I-PLC was constructed.Induced by IPTG（isopropyl β-D-1-thiogalactopyranoside）at 25℃ for 5h,a fusion protein（SUMO-PLC）with significant phospholipase C activity was detected in recombinants.After removal of the SUMO tag,the SDS-PAGE showed a heterologous expression of phospholipase C with a molecular weight of approximate 28 KDa,which was identified by LC-MS/MS.3.The analysis of enzymatic properties showed that the optimum temperature was 80℃.It remained 85% phospholipase C activity at 60℃ and 63% phospholipase C activity at 70℃ for 50 min respectively,which suggested that it was a thermal stability phospholipase C;The optimum pH was 8.0 and it was stable at pH 7.2⁸.0;Phospholipase C activity was stimulated by Zn²⁺,Mn²⁺ and inhibited by Cu²⁺,EDTA and EGTA;The analysis of substrate specificity showed that the enzyme could hydrolyze phosphatidylcholin,phosphatidylserine,phosphatidylinositol and phosphatidylglycerol;Kenetic analysis indicated an apparent Km value of 0.27 mM,an apparent Vmax value of 131.57 mM/min,and Kcat is 89.5 s^-1.4.The recombinant phospholipase C was modified with mPEG-Succinimidyl Succinate（SS-mPEG）.When the molar ratios（modifier to the phospholipase C）were 10,20 and 30,the modification rates were 10.68%,15.85% and 19.98% respectively.The purified SS-PEG-PLC had a molecular weight of 33.649 KDa analyzing by mass spectrum,which was 5 KDa larger than the that of the unmodified enzyme.Compared with unmodified PLC,the optimum temperature and pH of SS-PEG-PLC have been not changed,but the thermal stability and pH stability of SS-PEG-PLC have been improved.The optimum modification degree and recovery of the enzyme activity were 10.68% and 92.87%,respectively.The phospholipase C activity at 70℃ for 50 min increased from 63.20% to 70.24% and increased from 7.40% to 10.80% at 80℃ for 50 min.Kenetic analysis of the 10.68% modificed SS-mPEG-PLC indicated an apparent Km value of 0.62 mM,an apparent Vmax value of 222 mM/min,and Kcat is 622.89 s^-1.And the SS-mPEG-PLC with 10.68% modification degree had good storage stability at 4℃.The second structure of the PLC and SS-mPEG-PLC didn’t changed analysised by circular dichroism,and the stability of the SS-mPEG-PLC has improved because of the increased of antiparallel.