Phospholiase D-catalyzed Transphosphatidylation For Production Of Naturally Rare And Non-natural Phospholipids

The aim of present investigation is to develope the efficient technology for the enzymatic synthesis of naturally rare and non-natural phospholipids（PLs）.The widely used strategy for production of PLs is phospholipase D（PLD）-mediated transphosphatidylation.The serious drawbacks of these reported methods were unsatisfactory yields,serious hydrolysis,complicated post-treatment stages and low safety of products.The research field of this work is the enzymic catalytic reaction engineering.The immobilization technology and construction of the aqueous-solid system were employed to systematically investigate the transphosphatidylation to solve the above technical barriers.Thus,this work is devoted to provide the theoretical and technical support for the industrial production of PLs,guarantee the supply in marketing these phospholipids like"brain vitamins（phosphatidylserine,PS）",and make up for the huge gap between supply and demand in the current market of naturally rare and non-natural PLs.The catalytic performace of PLD was imporoved by a novel immobilization technology,called adsorption-precipitation-cross-linking.PLD was immobilized by adsorption and precipitation of the enzyme,followed by chemical cross-linking to form an“enzyme net”covering the surface of on the nonporous silicon dioxide nanoparticles.The core（carrier）-shell（enzyme aggregates）structured cross-linking enzyme aggregates（CS-CLEAs）were prepared.The immobilization method is simple and feasible.The obtained immobilized enzymes can be easily collected and reused.Enzymes can be effectively immobilized.The immobilization yield of PLD reached 80%.The specific activity of immobilized PLD reached15872 U/g_protein during the production of phosphatidylethanolamine（PE）,which was approximately 1.15 times higher than that of free PLD(13813 U/g_protein).The kinetic investigation indicated that the catalytic activity and enzyme-substrate affinity of PLD were improved after immobilization.In addition,the pH tolerance,thermostability,storage and operational stabilities were significantly enhanced compared with free PLD.To improve the immobilization technology of adsorption-precipitation-cross-linking,the bio-imprinting technology was combined to prepare the core-shell structured bio-imprinting cross-linking enzyme aggregates（CS-BI-CLEAs）.For production of phosphatidylglycerol in transphosphatidylation,the bio-imprinting biotechnology was employed to hyperactivate PLD by glycerol through the ligand-induced effect and,then,the obtained hyperactivated PLD molecules were immobilized by adsorption and precipitation,followed by cross-linking covering the surface of on the nonporous silicon dioxide nanoparticles.Finally,the ligand glycerol was washed and the bio-imprinting-immobilized PLD was obtained.The high degree of conformational rigidification provided by CS-BI-CLEAs can make PLD“remember”imprint-induced characteristics even in aqueous solutions.The obtained immobilized PLD showed the excellent catalytic performance.The maximum activity of immobilized PLD reached 166953 U/g_protein,which was about 14 times higher than that of free form(11922U/g_protein).After bio-imprinting-immobilization,the activity and selectivity of PLD were improved.The yield of PG reached 94.0%and the yield of the byproduct phosphatidic acid（PA）was only 5.96%.Generally,transphosphatidylation was carried out in the water-organic biphasic system.Serious drawbacks of this system are the serious pollution,low safety of product,low efficiency of enzymatic catalysis,and poor operational stability of PLD.In this work,PC was attached on the surface of porous carriers,which was dispersed in the aquous phase and react with enzymes and the second subsrate.An aqueous-solid system for transphosphatidylation was constructed by the adsorption-precipitation method.The substrate PC was pre-dissolved in ethyl acetate.The carrier silica was added and,then,the precipitator acetone was used to make PC attach on the surface of carriers by the adsorption and precipitation.The PC loading reached 90.7%.The obtained silica-attached PC was successfully used for PLD-catalyzed transphosphatidylation to produce phosphatidylserine（PS）.In the aqueous-solid system,the surface of silica was employed as an“artificial interphase”between subtrates and enzymes.The nature of the surface of the silica was changed with PC attachment,allowing the creation of a hydrophobic microenvironment for minimal hydrolysis and maximal reaction efficiencies.Special attention has been paid to the effect of the pore diameter and surface area of silica covered with PC molecules on the yield of phosphatidylserine（PS）.The highest yield of PS reached 99.5%.Moreover,the free PLD solution could be easily collected and reused.The yield of PS still had 73.6%after being used for six batches.Next,the nonporous silicon dioxide nanoparticle was employed as the carrier to construct the aqueous-solid system by the adsorption-precipitation method,and the non-natural phospholipid,phosphatidylhydroxybutyrate（PB）,was synthesized by PLD-mediated transphosphatidylation.Nanoscale silica dioxide with a very small size can reduce mass transfer resistance and facilitate collisions between the substrate PC and carriers to greatly decrease the boundary layer and improve the mass transfer and adsorption efficiencies.The experimental results indicated that the highest PC loading reached 98.3%to avoided the waste of PC.Special attention has been paid to the effect of the PC coverage on the surface area of silica covered with PC molecules,the PC loading and the yield of PB.The highest PB yield of 97.3%were achieved.The synthesized PB was quantitatively and qualitatively analyzed by High Performance Liquid Chromatography（HPLC）,Fourier Transform Infrared Spectrometer（FT-IR）and Time of Flight Mass Spectrometer（TOF-MS）.The purity of PB prepared by this method was very high and was even directly used for the animal experiment without the purification.It also provided the reliable source of PB during the investigation of its pharmacology.The nonionic surfactant was covalently bound on the surface of silica to prepared functional silica gel which can directly facilitate the adsorption of PC in the aqueous phase.The aqueous-solid system for transphosphatidylation was constructed by the direct adsorption method.t-Octylphenoxypolyethoxyethanol（Triton X-100）was covalently bound to the surface of four kinds of silicas with different sizes and acted as an anchor molecule to facilitate the adsorption of PC in a purely aqueous solution via the lateral hydrophobic interaction.The obtained silica-adsorbed PC was successfully used for PLD-mediated transphosphatidylation in the production of PS.In the batch production,silicas with small sizes showed the better performances in the PC adsorption and transphosphatidylation.The PC loading and PS yield respectively reached 98.9 and 99.0%.Results indicated that the rate-limiting step is the adsorption process of PC,not the dissolution process of phospholipids.One-step and two-step models were employed to investigate the adsorption process of PC and the relevant parameters were calculated.The results showed that the calculated values of the standard free energy for surface aggregation based on two models（-29.8 kJ/mol and-23.0kJ/mol）were always less than that for micellization（19.5kJ/mol）and the hemi-micelle concentration(2.76×10^-6mol/L)was also less than the critical micelle concentration(4.36×10^-4mol/L).Functional silica gel accelerated the adsorption rate of PC molecules on the surface of silica gel in aqueous solutions through the molecular affinity,which promoted the continuous dissolution of PC molecules.Thus,the direct adsorption of PC molecules on the surface of silica gel can be carried out in the aqueous phase.In addition,this aqueous-solid system provides a promising way to continuously biosynthesize PS.A packed-bed reactor in which Triton-X-100-modified silicas was used as the stationary phase,and the PC solution,PLD solution and eluent were respectively employed as mobile phases,to demonstrate the process flow of the continuous production of PS.In the continuous production,silicas with small sizes had higher values of PC loading and PS yield,but the higher space-time yield was obtained from silicas with large sizes.Considering the PC loading,PS yield and space-time yield,the optimal carrier in the continuous production was Triton-X-100-modified silica(5.34×10^-10 mol_Triton/g_silica)with the size of 20-40 mesh.The recyclability and stability of the Triton-X-100-modified silica were excellent,as demonstrated by its use 30 times during continuous operation without any loss of the productivity.After transphosphatidylation,the product（PS）adsorbed on carriers were eluted by coconut oil and used to manufacture microcapsules via the water phase separation method using the PS coconut oil solution as the core materials and chitosan as the wall membrane material.A whole technological process involving the efficient synthesis of PS and the preparation of relevant microcapsules has been proposed in this work to make the aqueous-solid system a promising candidate for the commercial production of PS.