脂肪酶固定化的新方法研究及其应用
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摘要
脂肪酶是一种水解酶,其突出特点是能够在油-水界面实施催化功能,被广泛应用于食品、医药及手性化合物合成等领域。由于固定化酶具有稳定性高、易回收和重复利用等优点,一直以来是酶工程的热点研究领域之一。本文以有机相中酶法拆分消旋烯丙醇酮为对象,以制备高活性、高稳定性和高对映体选择性的固定化脂肪酶催化剂为目标,从固定化载体材料和固定化方法两个方面研究固定化过程对酶催化性能的影响规律及作用机制,研究新型的脂肪酶固定化方法。主要内容如下:
(1)以廉价的天然无机材料硅藻土为载体,通过表面改性使之表面连接上不同的疏水性有机官能团,具体包括甲基丙烯酰氧基丙基、辛基、十二烷基、乙烯基等。采用吸附法对Arthrobacter sp.脂肪酶(ASL)进行固定化,研究载体表面性质对固定化酶催化性能的影响。结果表明,经疏水表面改性的载体能明显影响固定化酶的蛋白结合量、活力和稳定性,但影响规律不同。其中,甲基丙烯酰氧基丙基载体所得固定化酶的活力最高,但稳定性相对较低,而十二烷基固定化酶的活力和稳定性均相对较高。以十二烷基修饰的硅藻土为载体,研究载体疏水性强弱、酶与载体比例以及缓冲液pH等因素对吸附固定化酶性能的影响规律,并对固定化工艺进行优化,最终确定较佳的条件为:载体硅烷化程度0.08g/g载体、酶与载体的比例1/30-1/40、缓冲液为pH9.0,0.03M的Tris-HCl。在吸附的基础上用戊二醛进一步处理,得到酶聚集体包被法固定化酶。该固定化酶的蛋白结合量、活力及稳定性等均较吸附法有明显提高,总活力回收达到游离酶的8.5倍;E≥200;重复利用20天后,残余酶活力为原来的76%左右;
(2)以氨丙基三甲氧硅烷和正硅酸乙酯(TEOS)为前体,采用溶胶凝胶法合成氨丙基凝胶,并用戊二醛对其进行活化使其末端带有游离的醛基。以该凝胶材料为载体分别采用共价结合和酶聚集体包被两种方法对脂肪酶ASL进行固定化。结果表明,这两种方法所得固定化酶的稳定性明显升高。其中,基于共价结合的酶聚集体包被法固定化酶在蛋白结合量和酶活力等方面均比单纯的共价结合法要高,其总酶活回收为游离酶的84%;E≥200;重复利用9次后,残余酶活在95%以上;
(3)以非烷烃类的硅烷化试剂甲基丙烯酰氧基丙基三甲氧基硅烷(MAPTMS)和正硅酸乙酯(TEOS)为混合前体采用溶胶凝胶包埋法对脂肪酶ASL进行固定化,并将之与已报道的烷烃类硅烷前体所制备的包埋固定化酶进行比较。结果表明,该固定化酶在p-NPP水解以及烯丙醇酮转酯化拆分两个反应体系中均表现出比其它前体更高的酶活力,并且这些固定化酶活力的高低与凝胶材料的疏水性强弱无直接对应关系;此外,在烯丙醇酮的转酯化反应体系中,固定化酶的对映体选择性明显升高。研究包埋条件对固定化酶催化性能的影响规律,并对包埋工艺进行优化,包括MAPTMS与TEOS的摩尔比,前体水解过程中水的添加量以及酶的添加量等,最终在较佳的条件下固定化酶的总活力回收为游离酶的13.6倍,蛋白负载量为1.4mg/g固定化酶;最佳固定化条件下所得的包埋酶表现出比游离酶更高的稳定性,在烯丙醇酮转酯化反应体系中,固定化酶重复利用60天后残余酶活力为原来的54%左右;而游离酶重复利用20天后,残余酶活力为52%;
(4)将溶胶凝胶包埋固定化酶用于烯丙醇酮转酯化拆分反应体系中,考察了反应温度、外扩散和内扩散效应、底物浓度和产物浓度等条件对酶活力、选择性以及反应过程的影响,结果表明上述反应条件的改变对酶的对映体选择性基本无影响;当摇床转速大于200r/min时可基本消除外扩散的影响;当固定化酶的粒径小于1.0mm时可基本消除内扩散限制;当底物浓度和产物浓度小于1.0mol/l时,酶催化过程无抑制现象存在。在无内外扩散及底物和产物抑制的前提下研究了溶胶凝胶包埋固定化酶催化转酯化反应拆分烯丙醇酮的动力学。根据乒乓反应机制建立了动力学模型,反应速率方程为(?),其中,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。此外,尝试了一种合成S-烯丙醇酮的新工艺,即以消旋烯丙基呋喃甲醇为起始原料,先经过生物拆分得到其S-对映体,然后采用化学重排的方法将S-烯丙基呋喃甲醇转化为S-烯丙醇酮。采用脂肪酶催化的转酯化反应对烯丙基呋喃甲醇进行动力学拆分,通过筛选催化剂和有机溶剂,优化反应条件,最终建立了较佳的酶法转酯化拆分烯丙基呋喃甲醇的反应体系,即脂肪酶Rhizopus arrhizus为催化剂,烯丙基呋喃甲醇与乙酸乙烯酯(酰基供体)的摩尔比为1/5,正己烷为溶剂,反应温度30℃。在该条件下,S-烯丙基呋喃甲醇的e.e.>98%,E为56;将前面开发的多种形式的固定化酶用于烯丙基呋喃甲醇的转酯化拆分中,酶的对映体选择性大幅度提高(E=99)。按照消旋烯丙醇酮的制备工艺,对S-烯丙基呋喃甲醇进行化学转化,最终得到消旋的烯丙醇酮。
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.
(1)以廉价的天然无机材料硅藻土为载体,通过表面改性使之表面连接上不同的疏水性有机官能团,具体包括甲基丙烯酰氧基丙基、辛基、十二烷基、乙烯基等。采用吸附法对Arthrobacter sp.脂肪酶(ASL)进行固定化,研究载体表面性质对固定化酶催化性能的影响。结果表明,经疏水表面改性的载体能明显影响固定化酶的蛋白结合量、活力和稳定性,但影响规律不同。其中,甲基丙烯酰氧基丙基载体所得固定化酶的活力最高,但稳定性相对较低,而十二烷基固定化酶的活力和稳定性均相对较高。以十二烷基修饰的硅藻土为载体,研究载体疏水性强弱、酶与载体比例以及缓冲液pH等因素对吸附固定化酶性能的影响规律,并对固定化工艺进行优化,最终确定较佳的条件为:载体硅烷化程度0.08g/g载体、酶与载体的比例1/30-1/40、缓冲液为pH9.0,0.03M的Tris-HCl。在吸附的基础上用戊二醛进一步处理,得到酶聚集体包被法固定化酶。该固定化酶的蛋白结合量、活力及稳定性等均较吸附法有明显提高,总活力回收达到游离酶的8.5倍;E≥200;重复利用20天后,残余酶活力为原来的76%左右;
(2)以氨丙基三甲氧硅烷和正硅酸乙酯(TEOS)为前体,采用溶胶凝胶法合成氨丙基凝胶,并用戊二醛对其进行活化使其末端带有游离的醛基。以该凝胶材料为载体分别采用共价结合和酶聚集体包被两种方法对脂肪酶ASL进行固定化。结果表明,这两种方法所得固定化酶的稳定性明显升高。其中,基于共价结合的酶聚集体包被法固定化酶在蛋白结合量和酶活力等方面均比单纯的共价结合法要高,其总酶活回收为游离酶的84%;E≥200;重复利用9次后,残余酶活在95%以上;
(3)以非烷烃类的硅烷化试剂甲基丙烯酰氧基丙基三甲氧基硅烷(MAPTMS)和正硅酸乙酯(TEOS)为混合前体采用溶胶凝胶包埋法对脂肪酶ASL进行固定化,并将之与已报道的烷烃类硅烷前体所制备的包埋固定化酶进行比较。结果表明,该固定化酶在p-NPP水解以及烯丙醇酮转酯化拆分两个反应体系中均表现出比其它前体更高的酶活力,并且这些固定化酶活力的高低与凝胶材料的疏水性强弱无直接对应关系;此外,在烯丙醇酮的转酯化反应体系中,固定化酶的对映体选择性明显升高。研究包埋条件对固定化酶催化性能的影响规律,并对包埋工艺进行优化,包括MAPTMS与TEOS的摩尔比,前体水解过程中水的添加量以及酶的添加量等,最终在较佳的条件下固定化酶的总活力回收为游离酶的13.6倍,蛋白负载量为1.4mg/g固定化酶;最佳固定化条件下所得的包埋酶表现出比游离酶更高的稳定性,在烯丙醇酮转酯化反应体系中,固定化酶重复利用60天后残余酶活力为原来的54%左右;而游离酶重复利用20天后,残余酶活力为52%;
(4)将溶胶凝胶包埋固定化酶用于烯丙醇酮转酯化拆分反应体系中,考察了反应温度、外扩散和内扩散效应、底物浓度和产物浓度等条件对酶活力、选择性以及反应过程的影响,结果表明上述反应条件的改变对酶的对映体选择性基本无影响;当摇床转速大于200r/min时可基本消除外扩散的影响;当固定化酶的粒径小于1.0mm时可基本消除内扩散限制;当底物浓度和产物浓度小于1.0mol/l时,酶催化过程无抑制现象存在。在无内外扩散及底物和产物抑制的前提下研究了溶胶凝胶包埋固定化酶催化转酯化反应拆分烯丙醇酮的动力学。根据乒乓反应机制建立了动力学模型,反应速率方程为(?),其中,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。此外,尝试了一种合成S-烯丙醇酮的新工艺,即以消旋烯丙基呋喃甲醇为起始原料,先经过生物拆分得到其S-对映体,然后采用化学重排的方法将S-烯丙基呋喃甲醇转化为S-烯丙醇酮。采用脂肪酶催化的转酯化反应对烯丙基呋喃甲醇进行动力学拆分,通过筛选催化剂和有机溶剂,优化反应条件,最终建立了较佳的酶法转酯化拆分烯丙基呋喃甲醇的反应体系,即脂肪酶Rhizopus arrhizus为催化剂,烯丙基呋喃甲醇与乙酸乙烯酯(酰基供体)的摩尔比为1/5,正己烷为溶剂,反应温度30℃。在该条件下,S-烯丙基呋喃甲醇的e.e.>98%,E为56;将前面开发的多种形式的固定化酶用于烯丙基呋喃甲醇的转酯化拆分中,酶的对映体选择性大幅度提高(E=99)。按照消旋烯丙醇酮的制备工艺,对S-烯丙基呋喃甲醇进行化学转化,最终得到消旋的烯丙醇酮。
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.
引文
[1] Katchalski-Katzir E. Immobilized enzymes-learning from past successes and failures. Trends Biotechnol. 1993. 11:471-478.
[2] Hartmeier W. Immobilisierte Biokatalysatoren. Springer, Berlin Heidelberg New York. 1986,18-20.
[3] Buchholz K. Kasche V. Biokatalysatoren und Enzymtechnologie.VCH,Weinheim. 1997, 7-11.
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