Study On Novel Immobilization Methods Of Lipase And Its Applications

Lipases are a kind of hydrolase. They have been widely used in food industrialand the synthesis of drugs and chiral chemical compounds because of their specificcharacteristic that they can carry out catalytic functions at oil-water interface. In mostcases, enzymes are preferably used in their immobilized states owing to manyadvantages, such as improved stability, ready reutilization of the catalyst andpossibility of continuous operation. So enzyme immobilization techniques havealways been one of the hot research issues. The aim of this dissertation is to prepareimmobilized lipases with high activity, stability and enantioselectivity by exploringnovel immobilization methods. The effects of immobilization including theimmobilized supports and immobilization methods on the catalytic properties of thelipase were studied. All the catalytic characteristics of lipases were investigated in thekinetic resolution of 4-hydroxy-3-methyl-2-(2-propenyl)-2-cyclopenten-1-one(HMPC) in organic solvents. The main contents of this work were as follows:(1) Lipase from Arthrobacter sp. (ASL) was immobilized onto low-costdiatomite materials for the resolution of HMPC by asymmetric acylation. The supportwas modified by the silane treatment and the support surface was grafted variousfunctional organic groups including methacryloxypropyl. octyl, dodecyl and vinyl.The surface modifications had great influences on the bound protein, activity andstability of the immobilized enzyme. Among them, the adsorbed lipase ontododecyl-modified support exhibited both higher activity and stability, while that ontomethacryloxypropyl-modified support showed highest activity but lower stability. Theimmobilization conditions including the activation degree of the support, the massratio of lipase to the support and the pH of the buffer were investigated and theoptimum values were 0.08g/g support, 1/30-1/40 and pH 9.0. 0.03M Tris-HCl buffer,respectively. The enzyme-aggregate coating method was performed based onadsorption, and the characteristics of this immobilized lipase were proved better than that by pure adsorption. It was shown that the enzyme-aggregate coated lipase yieldeda recovered activity of 8.5 folds of the free enzyme, and remained 76% of initialactivity after 20 d. Excellent enantioselectivity (E≥200) was also obtained.(2) The glutaraldehyde activated amino-silica gel which was synthesized bysol-gel technique using (3-aminopropyl)trimethoxysilane and tetraethoxysilane as theprecursors was applied as the support to immobilize ASL. The covalent attachmentand enzyme-aggregate coating methods were performed and the immobilizedenzymes were applied in the asymmetric acylation of HMPC. The results showed thatthe immobilized lipase by enzyme-aggregate coating possessed both higher activityand stability than those by covalent attachment, e.g. it obtained a recovered activity of84% of the free enzyme, and remained 95% of initial activity after 9 recycles.Excellent enantioselectivity (E≥200) was also obtained.(3)γ-(methacryloxypropyl)-trimethoxy silane (MAPTMS) and tetraethoxysilane(TEOS) were choosen as the precursors to immobilize ASL by sol-gel encapsulation.The catalytic properties of the encapsulated lipase were compared with other reportedsilanizing agents. The results turned out that the immobilized enzyme by thecopolymerization of MAPTMS and TEOS exhibited the highest activity in both thehydrolysis of p-nitrophenyl palmitate and the asymmetric acylation of HMPC, andobtained higher enantioselectivity. The effects of various immobilization parameterswere investigated, e.g. MAPTMS/TEOS (mol/mol), water/silane molar ratio (R value)and lipase loading. Under the optimum conditions the total activity of the immobilizedenzyme reached up to 13.6-fold of the free form and the bound protein was 1.4 mg/gimmobilized enzyme. Moreover, the encapsulated lipase exhibited higher stabilitythan the free form and retained 54% of the original activity after uses for 60 d, whilethe free enzyme left 52% after only 20 d.(4) The sol-gel encapsulated lipase was applied in the kinetic resolution ofHMPC in organic solvents. The effects of reaction conditions on the activity,enantioselectivity of the enzyme and the reaction course were investigated includingtemperature, external diffusion, internal diffusion and the concentration of thesubstrate and the product. The results turned out that no changes happened to the enantioselectivity of the enzyme under various reaction conditions; the external andinternal diffusion limitations could be reduced when the rotation speed was above 200r/min and the particle diameter of the immobilized enzyme was less than 1.0 mm.respectively. A kinetic model of this reaction was proposed based on ping-pongmechanism and the reaction rate could be expressed as(?) where V_m=1.00mmol/l/min,K_A=1.80mmol/l. K_B=131.84, K_BA=5.10, K_QA=366.66, K_BQA=0.52 l/mmol.Additionally, a new process to prepare S-HMPC was tried: firstly, synthesis ofS-(1-hydroxy-3-butenyl)-5-methylfuran (S-1) by enzymatic resolution of its racemiccompounds, then conversion of S-1 into S-HMPC by chemical rearrangement. Kineticresolution of racemic 1 via lipase-catalyzed transesterification was successfullyperformed in organic solvents. Lipase Rhizopus arrhizusn and hexane was choosen asthe catalyst and organic solvent, respectively. The e.e. value of S-1 reached up to 98%with an E value of 56 under the optimal reaction conditions, e.g. molar ratio of vinylacetate to rac-1 and reaction temperature were 5/1 and 30℃, respectively. LipaseRhizopus arrhizusn was immobilized by various methods mentioned above, and theenantioselectivity of the lipase was clearly improved with an E value of 99. Thechemical conversion of S-1 into S-HMPC was tried, however, racematic HMPC wasobtained until now and this process needed to be further studied.