Study On Preparation Of Lipase@MOFs And Kinetic Resolution Of Enantiomers

The enzymatic kinetic resolution has the advantages of high stereoselectivity,high resolution efficiency,and large-scale production,making it ideal for the resolution of racemates.However,there are some shortcomings of free enzyme in enzymatic kinetic resolution.It is difficult to separate free enzyme from the product,to recycle,and the enzyme activity is low.To solve the above problems,while improving the stability of the enzyme and expanding the scope of operation.In this paper,the immobilized enzyme was synthesized by physical adsorption or covalent bonding with MOFs as the carrier,and its structure was characterized,which was applied to the kinetic resolution of enantiomers.With Pseudomonas cepacia lipase（PCL）as the biocatalyst,the free PCL was successfully immobilized into the water-stable material ZIF-8 by physical adsorption.The effects of temperature and time during the immobilization process on the relative enzyme activity and enzyme loading of PCL@ZIF-8 were investigated.The performance of PCL@ZIF-8 catalyzed by ester hydrolysis resolution（R,S）-2-phenylpropionic acid enantiomer and transesterification resolution（R,S）-1-phenylethanol enantiomer were studied.The results confirmed that the majority of PCL was adsorbed inside the pores of ZIF-8,and an amount of PCL was adsorbed on the surface of ZIF-8.The amount of enzyme per gram of support was 200.5 mg PCL/g ZIF-8.In the resolution reaction of ester hydrolysis,the reaction rate of PCL@ZIF-8 was three times that of the free PFL in the first 2 h,and the time required for the reaction to reach equilibrium was much shorter than that of free PCL.In addition,the PCL@ZIF-8 showed good reusability in both hydrolysis（30.57%of initial activity,4 cycle）and transesterification reaction systems（69.34%of initial activity,6 cycle）.The immobilized lipase（PFL@NH₂-MIL-88B/Fe₃O₄）was successfully prepared by using NH₂-MIL-88B/Fe₃O₄ as the carrier to covalently immobilize pseudomonas fluorescens lipase（PFL）.The effects of p H,temperature,amount of enzyme and reaction time during the immobilization process on the relative enzyme activity and enzyme load of the immobilized enzyme were investigated.30 mg free PFL and 60mg NH₂-MIL-88B/Fe₃O₄,p H=7.5,T=15℃,t=24 h,the amount of enzyme per gram of support was 241.79 mg PCL/g NH₂-MIL-88B/Fe₃O₄.Characterization results confirmed that the majority of PFL was fixed on the outer surface of NH₂-MIL-88B/Fe₃O₄.PFL@NH₂-MIL-88B/Fe₃O₄ as biocatalyst was used for the lipase-catalyzed resolution of（R,S）-3-phenyllactic acid enantiomers.Meanwhile,the effects of temperature,amount of immobilized enzyme and reaction time on conversion（c）and enantiomeric excess of the product（ee_p）were investigated for the lipase-catalyzed transesterification reaction.The optimal reaction conditions were obtained by optimizing the reaction conditions,including temperature of 50℃,50 mg of PFL@NH₂-MIL-88B/Fe₃O₄ and 20 h of the reaction time.Under these reaction conditions,the total conversion and enantiomeric excess of the product were 38.15%and 96.94%,respectively.PFL@NH₂-MIL-88B/Fe₃O₄ exhibited good reusability,and it still remained 46.75%of the initial activity at the sixth cycle.The immobilized lipase（PFL@NH₂-Ni-MOF）was firstly prepared by using NH₂-Ni-MOF as carrier to covalently immobilize pseudomonas fluorescens lipase,and NH₂-Ni-MOF was modified with polyvinyl pyrrolidone（PVP）to improve the biocompatibility of the carrier.The effects of p H,temperature,amount of enzyme and reaction time during the immobilization process on the relative enzyme activity and enzyme load of the immobilized enzyme were investigated.The amount of enzyme per gram of support was 116.78 mg PFL/g NH₂-Ni-MOF.PFL@NH₂-Ni-MOF as biocatalyst was used for the lipase-catalyzed resolution of（R,S）-mandelic acid enantiomers.Meanwhile,the effects of temperature,amount of immobilized enzyme and reaction time on conversion and enantiomeric excess of the product were investigated for the lipase-catalyzed transesterification reaction.The optimal reaction conditions were obtained by optimizing the reaction conditions,including temperature of 55℃,50 mg of PFL@NH₂-Ni-MOF and 5 h of the reaction time.Under these reaction conditions,the total conversion and enantiomeric excess of the product were49.18%and 97.90%,respectively.While only 26.81%of c and 98.51%of ee_pwere obtained with free PFL.In addition,PFL@NH₂-Ni-MOF exhibits good reusability,and it still remained 50.18%of the initial activity at the tenth cycle.