非水相脂肪酶在维生素酯类衍生物合成中的应用

脂肪酶在非水介质中催化是目前生物技术中一个颇具基础性和应用性的研究领域。非水介质中的酶催化反应已经被成功应用到了多肽合成、聚合物合成、药物合成、立体异构体拆分及维生素改性等。介绍了脂肪酶在非水介质中的催化机理及其影响催化活性的重要因素,概述了近年来非水介质中脂肪酶催化合成几种维生素酯的研究进展。     

咖啡酸苯乙酯的合成研究进展

咖啡酸苯乙酯是近年来新药研发的热点化合物,其抗肿瘤活性备受关注.介绍了不同底物合成咖啡酸苯乙酯的工艺路线及其催化机理,其中重点分析了绿原酸水解酶和脂肪酶对定向合成咖啡酸苯乙酯的生物催化特性和反应特征,并展望了咖啡酸苯乙酯合成方法的发展趋势及应用前景.     

Candida sp. 99-125脂肪酶及其在化学品合成中的应用

传统的酯化或转酯化产品的合成通常需要高温、强酸、强碱等相对苛刻的条件,脂肪酶由于其生物催化过程具有高效、高选择性、条件温和和环境友好等特点,在化学品的合成中越来越受到人们的关注。本课题组开发了一种可以用于酯类合成的新脂肪酶,并且实现了该酶的工业化生产。来源于Candida sp. 99-125的脂肪酶在非水相中对酯化和转酯化反应具有高效的催化活性和稳定性。本文介绍了该脂肪酶的发酵生产及其在中长链脂肪酸酯、二元酸酯、维生素A棕榈酸酯、手性化合物以及生物柴油等多种化学品的合成中的应用。     

脂肪酶催化合成生物表面活性剂

脂肪酸单甘油酯、脂肪酸糖酯、聚甘油脂肪酸酯和长链脂肪酸蜡酯是重要的生物表面活性剂。传统化学法以碱为催化剂在高温下进行,不仅能耗高且产品纯度低。脂肪酶作为一种天然生物催化剂,可以温和条件下催化合成上述生物表面活性剂,能耗低且产品纯度高。综述对脂肪酶催化合成生物表面活笥剂工艺路线、反应体系及操作参数。     

丙酮中麦芽糖月桂酸单酯的酶法选择性合成

为选择性地合成麦芽糖月桂酸单酯,该文研究了脂肪酶催化合成条件对麦芽糖月桂酸酯浓度和产率的影响,确定了麦芽糖月桂酸单酯的最佳合成参数。当c(麦芽糖)=25 mmol/L、c(月桂酸)=75 mmol/L、ρ(脂肪酶)=20 g/L、ρ(3A分子筛)=60 g/L时,在丙酮中50℃反应72 h,麦芽糖月桂酸单酯的产率和浓度分别可达93%和23.35 mmol/L。     

单体碳数对脂肪酶N435催化合成直链聚酯的影响

通过多级催化法用脂肪酶N435催化不同碳数脂肪族二醇与二酯反应,合成直链聚酯。结果表明:短链二醇与短链二酯难以被脂肪酶催化发生反应,长链二醇与二酯易被催化发生反应。采用脂肪酶N435催化己二酸二乙酯与1,4-丁二醇反应可以获得重均分子量为24 816,相对分子质量分布为1.09的聚酯。脂肪酶催化二醇、二酯与甘油共聚合可以获得重均分子量较高、相对分子质量分布窄的共聚物。     

酵母脂肪酶酶促合成虾青素琥珀酸酯及优化

研究了酵母脂肪酶在有机溶剂中以琥珀酸酐为酰化试剂酯化合成虾青素琥珀酸酯。液相色谱质谱联用检测表明,酯化产物中既有虾青素单酯也有虾青素二酯。对酶促反应脂肪酶、反应介质、反应底物摩尔比、反应温度等酯化影响因素进行了详细探讨和优化。在所选的几种脂肪酶中,假丝酵母脂肪酶(Candida sp.)的催化活性最好,二甲基亚砜为适宜的反应溶剂,最佳反应温度为45℃。底物摩尔比是决定反应能否进行的关键因素,底物摩尔比为40以上,反应才能进行,摩尔比达到800时,虾青素转化率达到90%以上。底物摩尔比还显著影响产物中单酯和二酯分布,高摩尔比下二酯才能产生。对体系初始含水量和酶量的影响进行了优化。实验结果表明酶法合成虾青素琥珀酸酯具有很好的可行性。     

甘油单脂肪酸酯合成中催化剂的研究进展

综述了非离子表面活性剂甘油单脂肪酸酯合成过程中所用催化剂的研究进展以及几种可以用于甘油单脂肪酸酯合成的新型催化剂类型,即非离子碱、离子交换树脂、介孔材料、金属氧化物和各种脂肪酶催化剂,并对各自的催化效果及优缺点作了比较。     

介孔材料SBA-15固定化脂肪酶的研究进展

介孔材料由于其孔道分布有序且孔径均匀等优点而在固定化酶催化领域引起人们的广泛关注.本文综述了新型介孔材料SBA-15对脂肪酶固定化的研究进展.总结了SBA-15的孔径大小、形貌及等电点等因素对脂肪酶固定化的影响.归纳了SBA-15上三种不同固定化方法的优缺点,并介绍了SBA-15固定化脂肪酶在手性拆分、酯水解、酯合成及转醇化反应等领域的应用.最后提出SBA-15固定化脂肪酶在发展过程中存在的问题以及今后的发展趋势.     

技术市场

脂肪酶合成维生素A棕榈酸酯维生素A、维生素A醋酸酯、维生素A棕榈酯是我国可允许添加的维生素A系列添加剂,但是维生素A、维生素A醋酸酯对热、光极不稳定。维生素A棕榈酯相对维生素A、维生素A醋酸酯具有化学性质稳定,不易分解等优点,已广泛应用于化妆品、药物、饲料等。该项目中的发酵法生产脂肪酶部分属于国家“九五”攻关课题,现已经完成了中试,积累了成熟的技术资料,通过了国家鉴证,并于1997年申请了国家专利:固定化脂肪酶催化合成脂肪酸低碳醇酯,专利授理号为02117514·0。该技术的特点:酶法合成工艺简便,具有反应条件温和、催化反应…     

Immobilization of <Emphasis Type="Italic">Candida</Emphasis> sp.99-125 lipase onto silanized SBA-15 mesoporous materials by physical adsorption

SBA-15-NH₂ and SBA-15-Cl Mesoporous Materials were prepared by modifying SBA-15 with silane coupling agent 3-aminopropyltriethoxysilane and 3-chloropropyltriethoxysilane using the post-synthesis method. The mesoporous samples were characterized by Fourier transform infrared spectra and nitrogen adsorption. Compared with SBA-15-NH₂, SBA-15-Cl has suitable pore opening for further utilization in the immobilization of Candida sp.99-125 lipase by physical adsorption. The influences of lipase concentration and immobilizing time on the immobilization efficiency were investigated. Meanwhile, the lipase immobilized on SBA-15-Cl showed higher thermal, pH and storage stability than that of free lipase. Further study demonstrated that lipase immobilized on SBA-15-Cl could be used eight times without significant decrease of enzyme activity. The phenomenon was associated with the shrinkage of the pore opening of SBA-15-Cl keeping lipase from leaping out.     

融合蛋白CR2-GDH固定化及不对称还原制备(S)-4-氯-3-羟基丁酸乙酯

比较介孔分子筛材料SBA-15、MCM-41、海藻酸钙、改性二氧化硅4种载体固定化融合蛋白CR2-GDH其酶固载量和酶活回收率,选择SBA-15为固定化载体。研究固定化条件对固定化融合酶量的影响以及固定化酶的稳定性,固定化酶在双相体系催化不对称还原反应。结果表明,在pH值为5.5、酶浓度为1.4mg/mL、反应1h条件下,固定化酶量为27.7mg/g。加入25mmol/L的Ca²⁺,固定化酶的酶活回收率由58.6%提高到78.1%。与游离酶相比,固定化酶的热稳定性显著提高,40℃条件下酶活回收率提高19.1%。固定化酶水相中反复使用7批次后,剩余活性仍超过30%,具有较好的操作稳定性。与游离酶相比,固定化酶更耐受烷烃类有机溶剂。在水/有机溶剂双相反应体系中,Ca²⁺/SBA-15固定化酶和游离酶催化相比,产物得率提高23.8%。     

One-dimensional crosslinked enzyme aggregates in SBA-15: Superior catalytic behavior to conventional enzyme immobilization

α-Chymotrypsin (CT) and lipase (LP) were immobilized in SBA-15 mesoporous silica by crosslinking adsorbed enzymes. This simple approach resulted in one-dimensional crosslinked enzyme aggregates (CLEAs) in the linear pore channels of SBA-15, which was very effective in preventing the enzyme leaching and consequently improving the enzyme stability. Both CLEAs of CT and LP showed negligible activity decrease under harsh shaking condition for one week while the conventional approaches including adsorption and covalent attachment resulted in more than 50–90% enzyme inactivation under the same condition. This effective stabilization results from the bent pore structure of SBA-15 with a high aspect ratio, which prevents the leaching of one-dimensional CLEAs and thereby achieves the higher enzyme loading capacity. Along with the higher specific activity than that of adsorbed enzymes, this CLEA approach is much simpler than that of covalent attachment by obviating the tedious processes for silica functionalization and enzyme attachment.     

黏土吸附法固定化脂肪酶的初步研究

为研究天然黏土为载体固定化脂肪酶的可行性,采用羟基化、硅烷化处理,对黏土进行改性,并以此为载体吸附固定化脂肪酶,探讨黏土固定化脂肪酶的条件对酶活及蛋白吸附量的影响,优化固定化脂肪酶条件。研究结果表明:黏土经羟基化、硅烷化改性处理后能显著提高固定化酶活和蛋白固定量,其中硅烷化改性最优;载体固定脂肪酶最优条件为:加酶量50 mg/g,载体粒径180—250μm,pH值为4.0,固定化温度25℃,固定化时间2.0 h;与游离酶相比,固定化酶显示出更广的pH值适应性。黏土固定化脂肪酶重复使用10批次后,仍能保留76.85%的初始活力。以天然黏土为载体固定化脂肪酶,具有较好的实际可应用性及操作稳定性,在较低pH值条件下应用具有一定优势。     

Enzyme immobilization: Part 1. Modified bentonite as a new and efficient support for immobilization of Candida rugosa lipase

Immobilization of Candida rugosa lipase onto modified and unmodified bentonites is described. The effect of hydrophilic or hydrophobic nature of the support, the reuse efficiency, and kinetic behavior of immobilized lipase were studied. The modified bentonite with monolayer surfactant (BMS), was the best support, for immobilization. The activity of the immobilized enzyme was examined under varying experimental conditions. The effect of various factors such as concentration of enzyme solution, pH and temperature, stirring and various thermodynamic parameters were also evaluated. The activity of lipase on Na-bentonite, on BMS and on bentonite with bilayer surfactant (BBS) at the optimum pH was 7.2%, 56.6% and 3.6%, respectively. The adsorption isotherm was modelled by the Langmuir equation. The amounts of immobilized lipase on Na-bentonite, BMS and BBS at the highest activity were 42.6%, 61.2% and 28.3%, respectively. The effect of substrate concentration on enzymatic activity of the free and immobilized enzymes showed a good fit to the Michaelis–Menten plots. The immobilized enzyme exhibited an activity comparable to the free enzyme after storage at 30 °C. The thermal stability of free and immobilized lipase were also studied.     

Adsorption of lipase from Candida rugosa on multi walled carbon nanotubes

In this work lipase from Candida rugosa was adsorbed on unmodified surface of multi walled carbon nanotubes (raw-MWCNT). The effects of immobilization time, initial enzyme concentration and buffer ionic strength on enzyme loading and activity of immobilized preparations were tested. High loadings are attained. The immobilized enzyme obtained at lowest initial enzyme concentration and high ionic strength retained 85% of initial enzyme activity. It is assumed that immobilization on hydrophobic surface led to conformational changes that resulted in the adsorption of lipase in active conformation. Immobilized preparations were characterized, with FT-IR spectroscopy, AFM, and cyclic voltammetry.     

Trypsin immobilization on mesoporous silica with or without thiol functionalization

The effects of pore size, structure, and surface functionalization of mesoporous silica on the catalytic activity of the supported enzyme, trypsin, were investigated. For this purpose, SBA-15 with 1-dimensional pore arrangement and cubic Ia3d mesoporous silica with 3-dimensional pores were prepared and tested as a support. Materials with varying pore diameters in the range 5–10 nm were synthesized using a non-ionic block copolymer by controlling the synthesis temperature. Thiol-group was introduced to the porous materials via siloxypropane tethering either by post synthesis grafting or by direct synthesis. These materials were characterized using XRD, SEM, TEM, N₂ adsorption, and elemental analysis. Trypsin-supported on the solids prepared was active and stable for hydrolysis of N-α-benzoyl-DL-arginine-4-nitroanilide (BAPNA). Without applying thiol-functionalization, cubic Ia3d mesoporous silica with ca. 5.4 nm average pore diameter was found to be superior to SBA-15 for trypsin immobilization and showed a better catalytic performance. However, enzyme immobilized on the 5% thiol-functionalized SBA-15 prepared by directly synthesis was found to be the most promising and was also found recyclable.     

Effect of pore diffusional resistance on biocatalytic activity of Burkholderia cepacia lipase immobilized on SBA-15 hosts

The present study was focused on elucidating the effects of nanopore diffusional resistance on the activity of Burkholderia cepacia (BC lipase) lipase immobilized in ordered mesoporous silica hosts. BC lipase was immobilized in ordered SBA-15 hosts possessing 55 and diameter pores by physical adsorption. A colorimetric assay of p-nitrophenyl acetate was employed to determine the lipase catalytic activity. The effect of diffusional resistance on catalytic activity of lipase immobilized in SBA-15 hosts was investigated by determining the effective substrate diffusivity as a function of pore size of the SBA-15 host and enzyme loading. Lipase immobilized in SBA-15- exhibited 20-30% of free lipase activity and the activity was further reduced with enzyme loading due to limited accessibility of substrate to the enzyme active sites. Lipase immobilized in SBA-15- hosts showed catalytic activity similar to free lipase activity suggesting that diffusional limitations were minimal. Large pore SBA-15 hosts provided an improved environment for BC lipase to retain its catalytic activity.     

A novel structured bioreactor: Development of a monolithic stirrer reactor with immobilized lipase

Cordierite monoliths were functionalized with polyethylenimine (PEI) and with different types of carbon, consisting of carbonized sucrose, carbonized ployfurfurryl alcohol, or carbon nanofibers, in order to create adsorption sites for a lipase from Candida antarctica. The prepared supports were compared in terms of immobilization capacity, activity, and stability. The supports with a carbon nanofiber coating displayed the highest enzyme adsorption capacity. The biocatalysts were assayed in the acylation of 1-butanol with vinyl acetate in toluene, yielding butanyl acetate and acetaldehyde. For catalyst performance testing a novel reactor type was employed, the monolithic stirrer reactor, in which monolithic structures are applied as stirrer blades. No profound effect of stirrer rate on the reaction rate was observed, implicating the absence of external mass transfer limitations. For comparison, free enzyme and a commercial (particulate) immobilized lipase were also included in the study. Compared to the free enzyme, the immobilized lipase shows a significantly lower activity. Increased stability, easy catalyst separation and the possibility to reuse the enzyme in immobilized form can overcome this difference. The commercial immobilized lipase initially has a significantly higher activity than the monolithic biocatalysts, but deactivates relatively fast. For the monolithic biocatalysts, no deactivation was observed; the prepared catalysts were stable for several weeks.     

Trypsin immobilization on mesoporous silica with or without thiol functionalization

The effects of pore size, structure, and surface functionalization of mesoporous silica on the catalytic activity of the supported enzyme, trypsin, were investigated. For this purpose, SBA-15 with 1-dimensional pore arrangement and cubic Ia3d mesoporous silica with 3-dimensional pores were prepared and tested as a support. Materials with varying pore diameters in the range 5–10 nm were synthesized using a non-ionic block copolymer by controlling the synthesis temperature. Thiol-group was introduced to the porous materials via siloxypropane tethering either by post synthesis grafting or by direct synthesis. These materials were characterized using XRD, SEM, TEM, N₂ adsorption, and elemental analysis. Trypsin-supported on the solids prepared was active and stable for hydrolysis of N-α-benzoyl-DL-arginine-4-nitroanilide (BAPNA). Without applying thiol-functionalization, cubic Ia3d mesoporous silica with ca. 5.4 nm average pore diameter was found to be superior to SBA-15 for trypsin immobilization and showed a better catalytic performance. However, enzyme immobilized on the 5% thiol-functionalized SBA-15 prepared by directly synthesis was found to be the most promising and was also found recyclable.