Preparation And Refolding Of Phospholipase A₂ Inclusion Body And Its Application

Phospholipase A₂（PLA₂）is an enzyme could hydrolysis glycerol phospholipid by cleaving C-2 ester bond.It plays key role in enzymatic degumming of plant oil and modification of phospholipid.The enzymatic degumming of plant oil by PLA₂ has significant advantages in environment protection and economy compared with traditional oil degumming process.As PLA₂ is too expensive due to the scarcity of PLA₂ even no product domestic,we have constructed an engineering bacterium（Streptomyces LEE2261）with the ability of producing phospholipase A₂（extracellular）,and the enzyme activity could satisfy the requirement of industry applications,but the low expression of the protein is detrimental to subsequent purification and preservation.In this study,we expressed PLA₂ in Escherichia coli expression system,studied the way to produce inclusion body effectively,the process of extraction,purification and refolding of inclusion body.Further,we studied theenzymatic property of refolded enzyme and preliminary application in oil degumming and modification of phospholipid.The main results are listed below:1.For high expression of PLA₂ in Escherichia coli,PLA₂ gene was optimized by related websites and software,the GC content of optimized gene was reduced to 52.5%,codon adaptation index was 0.95 and the optimized gene no longer utilize rare codons that codons ratings below 50.2.The selection of expression vector and host:the optimized gene was inserted into vector p ET-30c,the constructed vector p ET-30c-PLA₂ was transferred into Rosetta,and the protein was successfully expressed as inclusion body.3.The optimum culture medium and induce conditions for PLA₂ inclusion body producing:the optimum medium is as follow:20g/L of yeast extract,10g/L of glycerol,2mmol/L K₂HPO₄,58mmol/L KH₂PO₄,4g/L of Na Cl,and 2g/L of Mg Cl₂.The engineering bacterium were inoculated on the medium according to the proportion of 2%,and cultured for 4h,then induced by 0.6mmol/L of IPTG for 6h,the biomass could reach18g/L（flask fermentation）,and the object protein was about 38%of total protein,biomass could reach 38g/L by 100L scale fermentation and the expreesion of target protein is about 34%。4.The extraction and purification of inclusion body:more than 90%of the bacteria at concentration of 100g/L could be broken by high pressure slurry under the condition of60Mpa for 4times.The purity of inclusion body could reach 85%after crude inclusion body washed for 3 times at the concentration of 1g crude inclusion/20 ml washing buffer.The washing buffer is as follow:20mmol/L of Tris-HCl,1.5mol/L of urea,0.6～0.8%Triton X-100,p H8.0.5.The refolding of inclusion body:the refolding buffer solution is as follow:20mmol/LTris-HCl,0.2mol/Larginine,0.5mol/Lurea,15mg/m LPEG8000,0.2mol/L Na Cl,p H8.5.The inclusion body dissolved by 6mol/L of urea less than 8h then diluted by refolding buffer to the concentration of 0.4mg/m L,and keep the diluted solution for 10h under the condition of 4℃,the enzyme activity of refolding enzyme would reach1900U/m L and the max enzyme activity could last for more than 3d.6.The characteristics of refolding enzyme:the optimum temperature for refolding enzyme is 45～55℃,the max enzyme activity depends on more than 4mmol/L of Ca²⁺,and the refolding enzyme could be stable below 45℃,but enzymatic activity declined rapidly at the temperature of 55℃or higher.7.The application of refolding enzyme:when apply refolding enzyme to oil degumming,the content of degummed oil is 10mg/kg or below;when use refolded enzyme to produce enzyme hydrolyzed phospholipids,the conversion of phospholipid could reach 60%.