Enhanced The Stability Of Cross-linked Lipase Aggregates By Combined Immobilization

Enzyme immobilization is the basis of the industrial application of enzymes and is a hotspot in the field of bio-catalysis.Cross-linked enzyme aggregate(CLEAs)is a new carrier-free enzyme immobilization techniques.The preparation process has the advantages of simple operation,low requirement on the purity of the enzyme,high enzyme activity recovery rate,and reusability.However,because there is no solid-phase carrier as a scaffold,the structure of CLEAs is relatively loose and lead to leakage of enzymes easily.CLEAs are prone to caking during separation and recovery,which is not conducive to redispersion during secondary reactions.The use of CLEAs for large-scale enzyme reactors is poor.To solve this problem,three new types of immobilized lipase were prepared respectively,which used aqueous in-situ polymerization techniques,layer-by-layer self-assembly techniques,and enzymatic cross-linking techniques,based on cross-linked enzyme aggregates.Its preparation process,morphology and catalytic properties were studied.The main research content was as follows:(1)Cross-linked lipase aggregate gels(LCLEAs-gel)were prepared in combination with aqueous in-situ polymerization.The optimum preparation conditions were determined as follows: the best precipitant was acetone,the concentration was 60%(v/v),the precipitation time was 1.5 h,and the cross-linking time was 2 h.The ratio of lipase to cross-linker was 2 m L of 25% glutaraldehyde solution per gram of lipase.The ratio of enzyme to gel monomer was 1.0 g of acrylamide monomer per gram of lipase,and the gel polymerization time was 4 h.Structural characterization results showed that the gel shell not only forms on the surface of the cross-linked lipase aggregates,but also forms a gel on its internal pore surface,which could protect the internal enzyme proteins more effectively.The internal pore size was 3 nm and the average particle size was 63.839 ?m.The enzymatic properties of LCLEAs-gel showed Km value was 10.92 mmol·L-1 and Vmax value was 101.19 ?mol·min-1·mg-1.Compared with LCLEAs,LCLEAs-gel had stronger acid-base tolerance,temperature tolerance and organic solvent resistance.Moreover,the stability of the operation was also improved,and 75% of catalytic activity was still retained after 6 cycles of methyl oleate synthesis.(2)Layer-by-layer self-assembled silicified cross-linked lipase aggregates(LCLEAs-Si O2)were prepared.The characterization results of surface morphology,internal pore structure,and internal chemical bond changes show that after surface layer self-assembly,the surface morphology of LCLEAs-Si O2 underwent great changes.The surface of the LCLEAs-Si O2 was rough and covered a layer of 20 nm to 30 nm thick particles.XRD and FT-IR indicated that LCLEAs-Si O2 contains polyelectrolyte and nano-silica particles inside.LCLEAs-Si O2 had smaller internal pore size.The Km value of LCLEAs-Si O2 was 11.31 mmol·L-1 and the Vmax value was 149.80 ?mol·min-1·mg-1.LCLEAs-Si O2 had stronger stability,especially acid tolerance,and still retained more than 80% of the enzyme activity after treatment for 24 h under extreme conditions of p H 1.LCLEAs-Si O2 still retained more than 95% of catalytic activity after 6 cycles of methyl oleate synthesis.(3)The bovine serum albumin-encapsulated cross-linked lipase aggregates(LCLEAs-BSA)were prepared by transamidation of Tgase.The surface of LCLEAs-BSA had a number of relatively smooth irregularities,covered with a layer of dense gel-like material,with no obvious pores excepted the accumulation of mesopores.Enzymatic studies showed that LCLEAs-BSA had good temperature tolerance and organic solvent tolerance.Compared with LCLEAs,LCLEAs-BSA had a certain increase in alkali tolerance and its tolerance to organic solvents had been enhanced.And there still retained 80% of catalytic activity after 6 cycles of methyl oleate synthesis.The Km value of LCLEAs-BSA was 7.84 mmol·L-1,and there was a slight decrease compared with LCLEAs.The three new immobilized enzymes had increased the stability of CLEAs to a certain extent,provided a certain reference for the combined immobilization of CLEAs.