The Study On Expression And Characterization Of Phospholipase B And Its Application

Phospholipase B(PLB),having the ability of hydrolyzing both the Sn-1 and Sn-2 acyl ester bonds of phospholipids,can produce L-alpha-glycerylphosphorylcholine(GPC)using phosphatidylcholine(PC)as substrate and transform the non-hydratable phospholipids into their hydratable forms.GPC,is well known as the precursor for producing PC and acetylcholine in the body,and for having important medical applications in psychiatric and neurological disorders of the human brain.However,GPC is scarce in natural sources and difficult to obtain by extraction,Degumming,the first step in the refining process of vegetable oils,can remove phospholipids and mucilaginous gums from crude vegetable oils,which is facilitate the following refining steps including neutralization,bleaching,and deodorization.There are three genes plb1,plb2,plb3,with PLB activity in Saccharomyces cerevisiae.The gene plb2(plb),encoding PLB from S.cerevisiae,was obtained by PCR and inserted into the plasmid pPIC9K to generate the recombinant plasmid pPIC9K-plb.Then,pPIC9K-plb was transformed into Pichia pastoris GS115,resulting in the construction of recombinant GS115/pPIC9K-plb for the expression of recombinant PLB(rPLB).The fermentation conditions of recombinant GS115/pPIC9K-plb were optimized in shake falsk at single factor levels.It was found that the optimum fermentation conditions for GS115/pPIC9K-plb were:methanol 1.0%(v/v),volume 20 mL,pH 6.0,temperature 32?,and speed 280 r/min.The rPLB activity was up to 1723 U/mL with an increase of 33%compared to that with the original conditions.The PLB displayed maximum enzymatic activity at 40 ? and pH 5.5 and was stable with the range of 30 ?-40 ? and pH 5.0-6.0.GPC was synthesized by the rPLB with PC as substrates in single water phase.The optimized catalysis condition is summarized as follows:160 mg PC,3000 U rPLB,pH 5.5,temperature 40 ?,reaction for 28 h.Under the above conditions,the maximum yield of GPC reached 17%.The rPLB was applied in the process of peanut oil degumming.The optimized catalysis condition is summarized as follows:temperature 40 ?,pH 6.0,5500 U rPLB.The phosphorus content of peanut oil was reduced from 91.8 mg/kg to 3.7 mg/kg by enzymatic hydrolysis.