Study On The Lipase-gold Nanoparticles Hybridase-catalyzed Resolution Reaction

Lipase is an important green and safe industrial enzyme preparation.Because of its wide substrate specificity,it can catalyze a variety of reactions and is widely used in many industrial fields such as oil processing,pharmaceutical and perfumery.Agricultural products and microorganism are an important source of lipase.Due to the problems of poor stability,poor selectivity and difficulty in reuse,the application range of nature lipase is limited.In this thesis,Candida rugosa lipase?CRL?was used as model enzyme,and two novel hybrid strategy of enzymes were proposed by utilizing the excellent biocompatibility and unique physical and chemical properties of gold nanoparticles?AuNPs?.the preparation of CRL-AuNPs hybridase were used to resolute?R,S?-Naproxen methyl esters,which significantly improved the optical purity of the product?S?–Naproxen and the operational stability of CRL lipase.The method of improving the properties of lipase in this article can also be applied to lipases derived from agricultural products.It provide theoretical support for expanding the purposeful modification of lipase.The main research contents and results are as follows:The lipase-catalyzed?R,S?-Naproxen resolution reaction system was constructed.Chiral resolution was catalyzed by 100 mg CRL enzymes in micro-aqueous phase,and the reaction was conducted at 35?for 96 h.The experimental results showed that conversion?C?of CRL-catalyzed stero-selective resolution of naproxen methyl ester was 23.2%,the enantiomeric excess of the product?ee_p?was 94.8%,and the enantiomeric ratio?E?was 50.3.The residual substrate and hydrolyzed products were qualitatively analyzed by HPLC.Under the condition of irreversible kinetic resolution,the parametric equations were established and the theoretical curves plot of enantiomeric excess of substrate?ee_s?and product?ee_p?as a function of the conversion?C?were drawn.AuNPs with different particle sizes were synthesized by the reduction of HAuCl₄ with Na₃C₆H₅O₇?2H₂O.CRL-AuNPs hybridase?1?was obtained by the combined action of ionic bonding and physical adsorption.The experimental results showed that when 10 mL of CRL enzyme?10 mg/mL?,the average particle size of AuNPs was 14 nm,the addition amount of AuNPs was 60 mL,the hybridization reaction temperature was 30?,and the hybridization reaction time was 24 h,the CRL-AuNPs hybridase?1?prepared has the best resolution effect.Under these conditions,the conversion of substrate?C?was 20.0%,the ee_p was 97.3%and the E was 94.1.The UV-vis spectroscopy,Fluorescence spectroscopy,FTIR and TEM were used to characterize and explore the secondary structure changes of CRL in prepared CRL-AuNPs hybridase?1?.The reductive functional groups of amino acids in the CRL molecules were used to one-step synthesize CRL-AuNPs hybridase?2?by the reduction of HAuCl₄.The CRL-AuNPs hybridase?2?was characterized by UV-vis spectroscopy,Fluorescence spectroscopy,FTIR,TEM and XPS.TEM results showed that AuNPs were spherical and uniformly dispersed.XPS results showed that the binding energies of Au4f_7/2 and Au4f_5/2 were 83.5 and 87.2 eV,respectively,and it has been proved that Au???was reduced to Au?0?.The results showed that the optimal synthesis condition of CRL-AuNPs hybridase?2?was to take 10 mL of CRL enzyme?10 mg/mL?and 1 mL of HAuCl₄?1 mg/L?and place them into a shaker with a rotating speed of 100 rpm at 30?for 15 h.Under these conditions,hybridase-catalyzed resolution conversion?C?was 21.4%,the ee_p was 98.6%and the E was 179.3.The catalytic characteristics of CRL-AuNPs hybridase?1?and?2?obtained under the optimal preparation conditions were analyzed.CRL-AuNPs hybridase?1?and CRL-AuNPs hybridase?2?retained 86.2%and 92.2%of the activity of pure CRL enzymes,respectively.The enantiomeric ratios?E?compared with the free enzyme,which increased by 87.1%and256.5%.After 6 batchwise cycles,the enzyme activity remained at 57.3%and 69.4%of the initial value,and stereoselectivity has hardly changed.The study found that the secondary structure of the lipase modified by AuNPs hybridization changed slightly,which improved its stereoselectivity and stability,and made it easy to reuse.