Preparation And The Catalytic Performance Of Lipase Immoblized On Magnetic Nanoparticles

Enzyme is a highly effective biocatalyzer.However,the price of free enzyme is relatively expensive,and there are some defects such as difficulty in recycling and poor stability,which limit its large-scale industrial application.The application of nanomaterials represented by magnetic Fe₃O₄ nanoparticles in immobilized enzyme is a potential effective way to solve the above problems.Natural polyhydroxyl compounds have many biological and pharmacological activities,but their structure has some problems,such as poor stability,short half-life and low bioavailability.Among many acylation methods,the enzymatic method has great application prospect due to its advantages of mild,high efficiency,simple operation and high selectivity.Therefore,lipase was immobilized on the surface modified magnetic nanometer Fe₃O₄ as a carrier,and the acylation system of polyhydroxy compounds based on this nanometer biocatalyst was constructed,and the mechanism of the influence of related factors on the acylation reaction of this enzyme and the substrate recognition rule were explored.The main results were as follows:Magnetic Fe₃O₄ nanoparticles were synthesized by coprecipitation and their surfaces were modified by Polydopamine?PDA?.On this basis,the active groups in the surface layer of Fe₃O₄@PDA were used to react with those in the enzyme protein to produce a new type of nano-biocatalyst with magnetic properties,high activity and high stability.TEM and DLS characterization showed that Fe₃O₄,Fe₃O₄@PDA and Fe₃O₄@PDA@TLL were irregular spheres with an average particle size of 18.70±2.28 nm?23.89±3.12 nm and 29.95±3.41 nm respectively,indicating that the surface thickness of Fe₃O₄ nanoparticles formed after PDA loading was 2.5 nm.After immobilized enzyme,the lipase TLL coated with Fe₃O₄@PDA was about 3.0 nm thick,forming a core-shell structure.The XPS,FTIR,XRD,TGA and VSM characterization of the three kinds of particles further proved that PDA and TLL enzymes were successfully loaded on Fe₃O₄ and Fe₃O₄@PDA,respectively.The prepared Fe₃O₄ and Fe₃O₄@PDA have high purity,the lattice structure has not changed,and they retain good magnetic properties,which can be effectively recovered by using magnetism.The factors of the immobilized enzyme preparation such as enzyme-support ratio,immobilization time,pH and temperature on the enzyme recovery and protein load were studied.The results showed that when the enzyme loading ratio was 1.57:1 mg/g,and the optimal immobilization time,pH and temperature were 4.0 h,8.5 and 25°C,respectively,the immobilization effect was the best,and the recovery of enzyme activity and protein loading capacity were 90.9±0.8%and 156.4±2.1 mg/g,respectively.Studies on the enzymatic properties of Fe₃O₄@PDA@TLL showed that the optimum pH and temperature had a wider range of optimum reaction conditions than that of free TLL enzymes.Further studies showed that the pH stability,thermal stability,organic solvent resistance and storage stability of Fe₃O₄@PDA@TLL were significantly higher than those of the free TLL enzyme.Among them,Fe₃O₄@PDA@TLL maintained 70.2±1.9%catalytic activity after repeated use for 8 times,while the enzyme activity recovery rate was still as high as 64.3±1.4%after 120 days of storage,far higher than 20.1±1.7%of free enzymes.Using hypericin decanoylation as a model,the characteristics of Fe₃O₄@PDA@TLL to catalyze the regioselective acylation of hyperoside were studied.The optimum reaction medium,substrate molar ratio,reaction temperature,adding enzyme quantity and shaking rate for this reaction were MeTHF,11,55°C,1444 U and 200 r/min,respectively.Under these conditions,the reaction rate,maximum substrate conversion rate and 6"-OH selectivity were up to12.6±0.41 mM/h,100±2.1%and 100%,respectively.In the kinetic study of enzymatic reaction,the minimum K_m?52.7mm?,the maximum V_max/K_m(1.13h^-1)and the minimum activation energy E_a?16.3 KJ/mol?all indicated that MeTHF was the best medium for the acylation,and further verified that Fe₃O₄@PDA was an appropriate carrier for the immobilized TLL enzyme.The effects of enzyme sources,organic solvents and acyl donors with different structures on the region-selective acylation of polydatin were studied,so as to clarify the substrate recognition rule of nano-immobilized enzyme Fe₃O₄@PDA@TLL.The results showed that Fe₃O₄@PDA@TLL showed significant acylation activity and good substrate conversion?90.0%-98.7%?in the selected solvents.The structure of acyl donors?such as straight chain,branched chain,unsaturated bond and benzene ring,etc.?has a significant influence on the enzymatic reaction.The aliphatic chains in 11 of the 14 acyl donors could be connected to polydendroids,and the substrate conversion rate was up to 90.1%-99.1%.In terms of enzymatic reaction rate,Fe₃O₄@PDA@TLL is more likely to catalyze the reaction of medium-long chain acyl donor?C10-C14?,with a reaction rate of 12.6 mM/h-13.7 mM/h.When the position of an acyl donor contains a C=C double bond that can form conjugation with carbonyl carbon atoms,the rate of enzymatic reaction was significantly reduced.When the double bond was far away from the carbonyl carbon,it had little effect on the acylation reaction.In addition,it was found that the catalytic reaction was difficult to occur when there were branched chains or benzene rings in the position of acyl donors due to steric hindrance.The effects of structure difference of 14 flavonoid glycosides and their analogues on Fe₃O₄@PDA@TLL catalytic acylation were studied.The results showed that eight flavonoid glycosides and their analogues could be converted into corresponding ester derivatives with excellent conversion rates?94.1%-99.7%?,indicating a good substrate spectrum of the immobilized enzyme.However,in terms of regional selectivity,the acylation sites mainly occurred on the primary hydroxyl group of the substrate glycogen structure,and the secondary hydroxyl group's larger steric hindrance and lower reactivity may be the reasons for its non-acylation.In addition,the influence of substrate on the enzymatic reaction showed that the nano-immobilized carrier Fe₃O₄@PDA changed the catalytic behavior of the enzyme to a certain extent,which may be caused by the complex interaction between active groups and enzyme proteins in the structure of Fe₃O₄@PDA@TLL that changed the microenvironment of the enzyme active center.In terms of regional selectivity,the acylation site of the acylation reaction mainly occurs on the primary hydroxyl group of the glucose group in the substrate,forming monoester derivatives,among which the gastroside was converted into the selective diester compound of82%due to the presence of two more active primary hydroxyl groups.In addition,Fe₃O₄@PDA@TLL did not show selectivity for the secondary hydroxyl group in the substrate structure,which may be attributed to the larger steric hindrability and the lower reactivity of the secondary hydroxyl group.This study not only enriched the basic theoretical knowledge of nano-biocatalysts,but also opened up a new way for nano-immobilized enzymes to prepare derivatives of polyhydroxyl active compounds with high efficiency and high selectivity,which will lay the foundation for the application of nano-immobilized biocatalysts,the study of structure-activity relationship of natural active compounds and the screening of active compounds,and has certain theoretical significance and application value.