Gene Cloning,heterologous Expression Of Serratia Marcescens Outer Membrane Phospholipase A(A1) And Its Degumming In Rapeseed Oil

The purpose of chemical refining vegetable oils was to remove impurities that adversely affect the quality and shelf life of oil.However,the use of physical refining methods was increasing in view of environmental impacts and economic benefits.Enzymatic degumming was a physical refining method that can refine oil,but the high cost and low efficiency of phospholipase production limits its application.At the same time,phospholipases used for degumming cannot be recovered and reused,which greatly increases the cost of industrial application.In this study,the outer membrane phospholipase A1 gene was cloned,and prokaryotic and eukaryotic expression vectors were constructed to achieve heterologous expression in prokaryotic and eukaryotic expression systems and their enzymatic properties and immobilization processes were studied.The enzymatic degumming of rapeseed oil was also investigated,which provided an effective and feasible method for developing a new phospholipase and improving a degumming technology.The main results were as follows:?1?Cloning and bioinformatics analysis of the outer membrane phospholipase A1gene.The genome of Serratia marcescens was used as a template,and the target gene was amplified by PCR.The pEASY-OM-PLA1 was constructed and transferred into E.coil DH5?.The plasmid was sequenced and verified.The gene has 99.55%homology with Serratia marcescens subsp.marcescens Db11.Phylogenetic tree results showed that the OM-PLA1 produced by this gene showed the highest homology with OM-PLA1 derived from Escherichia coli NC₀00913.3.The protein has the molecular formula of C₁₅₁₃H₂₂₄₉N₄₁₁O₄₄₉S₆ and a relative molecular mass of 33572.34.It was a stable hydrophilic protein containing a signal peptide and was an extracellular secretory protein.The?-helix,?-helix,and irregular curls of secondary structure respectively accounts for 15.75%,36.64%,and 47.60%.The tertiary structure shows that each monomer was a?-barrel structure.?2?Engineered E.coli construction,enzymic properties and enzymatic degumming.The pEASY-OM-PLA1 was transferred into E.coil BL21?DE3?,and the activity of the outer membrane phospholipase A1 was maximized under the conditions of induction of4 h and IPTG concentration of 0.6 mmol/L.After purification of the recombinant protein by Ni²⁺column,SDS-PAGE showed a specific band with a molecular weight of approximately 33 KDa.The results of enzymatic properties showed that the optimum pH and optimum temperature were 7.5 and 50°C,respectively.The stability was good at pH 7.0-8.0 and 45-55°C.The enzyme kinetic parameters were Km=31.954 mM,Vmax=1.5056 mM/min,0.1 mmol/L Ca²⁺,Mg²⁺,Co²⁺,Mn²⁺solution can significantly improve its enzyme activity,and 1 mmol/L Cu²⁺,Zn²⁺,SDS,EDTA,EGTA solution significantly inhibited its enzyme activity.For the degumming of rapeseed oil,when the enzyme addition amount was 15 U and the degumming time was 2 h,the phosphorus content in the degummed oil sample was reduced from 22.6 mg/kg to less than 10mg/kg.?3?Engineered Pichia pastoris construction and induction of expression.After the enzyme cleavage site was inserted upstream and downstream of the gene,pPIC9K-His-OM-PLA1 was constructed by double digestion and T₄ ligase overnight ligation,and introduced into Pichia pastoris GS115.The results showed that the recombinant Pichia pastoris was a His+Muts phenotype,ie methanol slow utilization type,and the genomic DNA was detected by gel electrophoresis to show a single band with a size of about 900 bp.After induction for 72 h,the enzyme activity reached a maximum of 3.21 U/mL.After the target protein was purified by Ni²⁺column and concentrated by ultrafiltration tube,SDS showed a specific protein band with a molecular weight of about 33 KDa and an enzyme activity of up to 21.2.U/mL.The OM-PLA1 activity expressed by recombinant Pichia pastoris was higher when the initial transfer amount was 50 mL,the temperature was 28°C,and the methanol concentration was 1%.?4?Immobilization of outer membrane phospholipase A1 and enzymatic degumming.The carrier Fe₃O₄/graphene oxide was prepared by hydrothermal coprecipitation method.After successfully preparing the carrier material,the outer membrane phospholipase A1expressed by recombinant Pichia pastoris was immobilized on the carrier material by adding glutaraldehyde.The optimum immobilization conditions were pH 6 of the buffer,7%of glutaraldehyde concentration,and 3 h of immobilization time.The prepared immobilized outer membrane phospholipase A1 has an optimum pH of 7.5 and an optimum temperature of 50°C,and retains 58.9%of the original enzyme activity after being stored for 3 months at 4°C.It was used for enzymatic degumming of rapeseed crude oil.The pH was 7.5,the reaction time was 2.5 h,and the reaction temperature was50°C,which was the best degumming condition.After immobilized outer membrane phospholipase A1 was degummed 13 times,it also had 51.7%of the initial enzyme activity.