Recombinant Expression And Immobilization Of Phospholipase D

Due to improve cognition and anti-depression,relieve stress,assist in activating enzymes,and improve Alzheimer's disease,the application of phosphatidylserine(PS)in the food,cosmetic and pharmaceutical industries has received wide attentions.Although PS is widely distributed in nature,the industrial application of PS is limited due to the low content in plants and the unsafeness of animal brain cells.Therefore,phospholipase D(EC3.1.4.4,PLD)catalyzing phosphatidyl transfer to prepare PS is currently the safest and most efficient method.Although PLD is widely distributed in plants,animals and microorganisms,the cost for separation and purification of PLD from these materials is high,and the obtained PLD activity is very low,which cannot meet the industrial production need of PS.In this work,expression of PLD in Streptomyces and E.coli was attempted with the aim of achieving high level of expression and cheap preparation.The immobilization of PLD was also investigated,which will lay the foundation for the industrial production of PS by biocatalytic method.The main content of this work is divided into three sections:Section I:Expression of PLD in Streptomyces.Adopting the constitutive promoter Perm E*,p SET152 and p CJW93 were separately engineered to construct an integrated plasmid p SET254and an episomal plasmid p CJW254.An episomal plasmid p CJW93 with an inducible promoter Pti PA was also constructed.The PLD genes(sv PLD and sc PLD)derived from Streptoverticillium cinnamoneum and Streptomyces chromofuscus were separately cloned into the above three plasmids,and transferred into Streptomyces lividans TK2,Streptomyces lividans K4-1141 and Streptomyces albus hosts by conjugative transfer.The highest PLD activity only reached 0.085±0.0034 U/m L.Section II:Expression of PLD in E.coli.PLD(sv PLD)from Streptoverticillium cinnamoneum and PLD(sc PLD)from Streptomyces chromofuscus were cloned and expressed in E.coli.Firstly,the effects of p ET vector,culture medium type,inducing conditions on the expression of these two PLDs in E.coli were investigated.The optimal combination was using p ET28a without his-tag to construct p ET28a+sc PLD/BL21(DE3)recombinant strain and inducing in TB medium at 25°C employing 2.5 g/L lactose as the inducer.Then,the components of TB medium were optimized in detail.A medium consisted of 0.7%(w/v)of sorbitol,0.3%(w/v)of starch,1.0%(w/v)of fish peptone,4.0%(w/v)of Angel yeast extract,17 mmol/L of KH₂PO₄ and 72 mmol/L of K₂HPO₄ was determined to improve the sc PLD solubility and enhance the sc PLD yield.The highest sc PLD activity reached 104.28±2.67 U/m L after inducing for 32 h in 250 m L shake flask,which generated a productivity of 3.26±0.083 U m L^-1h^-1,specific activity of 5.11±0.13 U/mg and OD₆₀₀ of 13.15±0.23.Furtherly,the sc PLD production was scaled up to 5.0 L fermenter,and the highest sc PLD activity and OD₆₀₀ achieved 122.94±1.49U/m L and 28.17±1.53 by feeding lactose and inducing at 20°C.Substituting the mixed carbon source(0.7%(w/v)of sorbitol and 0.3%(w/v)of starch)with 1.0%(w/v)of cheap dextrin and adding a feeding medium could still attain a sc PLD activity of 105.81±2.72 U/m L in 5.0 L fermenter.After systematic optimization,the high-level soluble expression of sc PLD was fulfilled in E.coli.The sc PLD activity attained at the shake flask and fermenter levels are both the highest reported in the literature until now.Section III:Immobilization of PLD.The magnetic Fe₃O₄ was used as the carrier,and the magnetic cross-linked sc PLD aggregates was prepared by adsorption-aggregation-crosslinking with sc PLD.The PLD activity recovery reached 72.89%,and the immobilized PLD activity was437.34±6.60 U/g.Compared with free PLD,the tolerance of immobilized PLD to temperature and p H was strengthened to different degree.Due to the reduced affinity of immobilized PLD to the substrate,higher concentrations of PC and Ca²⁺were required.Furthermore,the tolerance of immobilized PLD to different organic solvents was also investigated,which provided reference for choosing appropriate organic solvent to develop two phase system for PS synthesis.After 13cycles of continuous reaction,the activity of immobilized PLD remained above 76%,giving a half-life of 331.67 min,which indicated that the immobilizing method developed in this work could improve the stability of the PLD and its reusability in the catalytic reaction.