Immobilization and Properties of Lipase from Candida rugosa on Electrospun Nanofibrous Membranes with Biomimetic Phospholipid Moities

Reported here is a protocol to fabricate a biocatalyst with high enzyme loading and activity retention, from the conjugation of electrospun nanofibrous membrane having biomimetic phospholipid moiety and lipase. To improve the catalytic efficiency and activity of the immobilized enzyme, poly（acrylonitrile-co-2-methacryloyloxyethyl phosphorylcholine）s（PANCMPCs） were, respectively, electrospun into nanofibrous membranes with a mean diameter of 90 nm, as a support for enzyme immobilization. Lipase from Candida rugosa was immobilized on these nanofibrous membranes by adsorption. Properties of immobilized lipase on PANCMPC nanofibrous membranes were compared with those of the lipase immobilized on the polyacrylonitrile（PAN） nanofibrous and sheet membranes, respectively. Effective enzyme loading on the nanofibrous membranes was achieved up to 22.0 mg/g, which was over 10 times that on the sheet membrane. The activity retention of immobilized lipase increased from 56.4% to 76.8% with an increase in phospholipid moiety from 0 to 9.6%（molar fraction） in the nanofibrous membrane. Kinetic parameter Km was also determined for free and immobilized lipase. The Km value of the immobilized lipase on the nanofibrous membrane was obviously lower than that on the sheet membrane. The optimum pH was 7.7 for free lipase, but shifted to 8.3-8.5 for immobilized lipases. The optimum temperature was determined to be 35 ℃ for the free enzyme, but 42-44℃ for the immobilized ones, respectively. In addition, the thermal stability, reusability, and storage stability of the immobilized lipase were obviously improved compared to the free one.     

壳聚糖固定化碱性脂肪酶的研究

以蟹壳为原料提取壳聚糖,用戊二醛作交联剂,将碱性脂肪酶固定于壳聚糖上。同时探讨了一定量干壳聚糖载体与交联剂浓度、给酶量等关系的最适固定化酶条件,并对固定化酶的热稳定性、操作稳定性、米氏常数、最适温度、离子强度的影响以及使用半衰期等理化性质进行探讨。     

Physical and Covalent Immobilization of <Emphasis Type="Italic">Lipase</Emphasis> onto Amine Groups Bearing Thiol-Ene Photocured Coatings

In this study, amine groups containing thiol-ene photocurable coating material for lipase immobilization were prepared. Lipase (EC 3.1.1.3) from Candida rugosa was immobilized onto the photocured coatings by physical adsorption and glutaraldehyde-activated covalent bonding methods, respectively. The catalytic efficiency of the immobilized and free enzymes was determined for the hydrolysis of p-nitrophenyl palmitate and also for the synthesis of p-nitrophenyl linoleate. The storage stability and the reusability of the immobilized enzyme and the effect of temperature and pH on the catalytic activities were also investigated. The optimum pH for free lipase and physically immobilized lipase was determined as 7.0, while it was found as 7.5 for the covalent immobilization. After immobilization, the optimum temperature increased from 37 °C (free lipase) to 50–55 °C. In the end of 15 repeated cycles, covalently bounded enzyme retained 60 and 70 % of its initial activities for hydrolytic and synthetic assays, respectively. While the physically bounded enzyme retained only 56 % of its hydrolytic activity and 67 % of its synthetic activity in the same cycle period. In the case of hydrolysis V _max values slightly decreased after immobilization. For synthetic assay, the V _max value for the covalently immobilized lipase was found as same as free lipase while it decreased dramatically for the physically immobilized lipase. Physically immobilized enzyme was found to be superior over covalent bonding in terms of enzyme loading capacity and optimum temperature and exhibited comparable re-use values and storage stability. Thus, a fast, easy, and less laborious method for lipase immobilization was developed.     

混合溶剂系统中固定化脂肪酶对酮基布洛芬的催化酯化反应

以硅藻土吸附的脂肪酶为催化剂,对外消旋酮基布洛芬[2-(3-苯甲酰苯基)丙酸]进行对映选择性酯化反应;考察了不同的脂肪酶制剂,固定化时所加缓冲液的体积与pH值,酰基受体(醇)的种类以及混合溶剂系统的组成等因素对酶活性的影响.结果表明,在所考察的7种脂肪酶中,以LipaseOF的酪化活性最高;用硅藻土吸附固定化酶时,缓冲溶液的最适pH为7.0左右,每克酶粉加1.0mL缓冲溶液为最佳;固定化酶催化酯化的活性比游离的脂肪酶高.在酮基布洛芬与不同酰基受体(醇)的酶促酯化反应中,以丙醇的反应速度为最快.在由一种主溶剂与一种助溶剂组成的混合溶剂系统中,酶促酯化的速度要比在单一的主溶剂或助溶剂系统中快.当以1gP值较大的环己烷或异辛烷等为主溶剂,甲苯为助溶剂时,脂肪酶催化酮基布洛芬酯化反应的活性最高.     

STUDY ON IMMOBILIZED PORCINE PANCREATIC LIPASE CATALYZING TRANSESTERIFICATION BETWEEN METHYL－BUTYRATE AND 1－BUTANOL IN NONAQUEOUS SYSTEMS

ＳＴＵＤＹＯＮＩＭＭＯＢＩＬＩＺＥＤＰＯＲＣＩＮＥＰＡＮＣＲＥＡＴＩＣＬＩＰＡＳＥＣＡＴＡＬＹＺＩＮＧＴＲＡＮＳＥＳＴＥＲＩＦＩＣＡＴＩＯＮＢＥＴＷＥＥＮＭＥＴＨＹＬ－ＢＵＴＹＲＡＴＥＡＮＤ１－ＢＵＴＡＮＯＬＩＮＮＯＮＡＱＵＥＯＵＳＳＹＳＴＥＭＳＸｉ...     

STUDY ON IMMOBILIZED PORCINE PANCREATIC LIPASE CATALYZING TRANSESTERIFICATION BETWEEN METHYL—BUTYRATE AND 1—BUTANOL IN NONAQUEOUS SYSTEMS

Transesterification between methyl-butyrate and 1-butanol in nonaqueous systems was catalyzed by porcine pancreatic lipase which was immobilized on cross-linked polystyrene.Organic solvents,substrate concentration,contents of water and other parameters which affect the immobilized enzyme activity were studied.Lipase immobilized on hydrophobic crosslinked polystyrene can reduce its diffusion limit in the reaction.It was found that the activity of immobilized lipase in organic systems was two times as high as that of free lipase.     

PDA/SiO2大孔复合材料固定化荧光假单胞菌脂肪酶

以具有三维骨架结构的大孔聚合物为模板制备SiO_2大孔材料,通过多巴胺在SiO_2大孔材料孔道表面的原位聚合制得聚多巴胺表面功能化修饰的二氧化硅大孔材料(PDA/SiO_2)。应用SEM、EDX、MIP、BET、TG-DTA和FTIR等技术对修饰前后的材料进行表征。以PDA/SiO_2为载体固定荧光假单胞菌脂肪酶(PFL),优化固定化条件并对比游离脂肪酶和固定化脂肪酶的性质。结果表明SiO_2大孔材料具有三维连续贯通的孔道结构,孔径分布在300~500 nm,聚多巴胺修饰后形成聚多巴胺/二氧化硅复合纳米薄膜构筑的大孔材料。在固定化时间为14 h、p H值为8、初始脂肪酶浓度为0.4 mg·m L-1时,固定化效果最佳,酶活回收率达246%。与游离脂肪酶相比,固定化脂肪酶有更宽的温度和p H适用范围、热稳定性显著提高,并展现出良好的储存稳定性和操作稳定性,固定化脂肪酶的Km低于游离脂肪酶的,酶与底物的亲和性较好。     

Enhancing enzymatic properties by the immobilization method

Effects of some immobilized carriers on enzymatic properties have been studied. The following results were obtained: (1) When cholinesterase was immobilized on the hydrophobic carrier with either α-naphthylamine, benzylamine, orp-methylbenzylamine groups, the affinities of immobilized cholinesterase for toxic organophosphors, GB (Isopnopy 1-methylphophonofluoridate) and Vx [o-ethyl-S-(2-diisopnoylomino-thyl) methyl phosphonothiolate], were enhanced 60–90 times and 700–1200 times, respectively, whereas the thermal stability of the immobilized cholinesterase increased to 110%. Approximately 82–88% activity of the immobilized cholinesterase remained after continuously operating for 8 h; and (2) Lipase was immobilized on the carrier that was made up of 6% polyethylenimine, 1% alginate gel, and 1% glutaraldehyde. The initial reaction rate of the esterification of lauric acid with lauric alcohol catalyzed by this kind of immobilized lipase was increased 21 times, as compared to lipase powder. About 72% esterification activity of lipase remained after continuous operating for 10 d.     

猪胰脂肪酶的固定化及其在有机相中催化丁酸甲酯和正丁醇的酯交换反应研究

考察了大孔苯乙烯－二乙烯苯交联共聚物的交联度、致孔剂量及胺化试剂对载体固定化猪胰脂肪酶的影响。选择出一种最佳载体对猪胰脂肪酶进行固定化，对比了自由酶和固定化酶在有机相中催化丁酸甲酯和正丁醇的酯交换反应。结果表明，酶经固定化后催化反应活力比自由酶提高近１倍。     

铁氨基黏土纳米结构脂肪酶的构筑 及催化特性

以铁氨基黏土(Fe-aminoclay)为载体, 1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐(EDC)为共价交联剂, 构筑了铁氨基黏土纳米结构脂肪酶催化剂(Feclay-lipase). 利用X射线衍射(XRD)、 透射电子显微镜(TEM)和傅里叶变换红外光谱(FTIR)等技术对Feclay-lipase进行了表征, 并通过酶动力学对比研究了游离脂肪酶和Feclay-lipase的酶学特性. 结果表明, Fe-aminoclay的载酶量为414.4 mg/g, 固定化效率可达82.88%, Feclay-lipase的酶活较游离酶提高了3倍, 最适反应温度提高了10 ℃, 最适反应 pH向碱性偏移, 储存稳定性更好, 在4 ℃下贮存30 d后其酶活无明显减弱.     

聚乙烯亚胺改性聚丙烯腈中空膜固定脂肪酶

聚丙烯腈是富腈基的高分子聚合物,易修饰改性,广泛应用于膜分离应用.我们以聚丙烯腈中空膜为载体,采用化学法交联聚乙烯亚胺并固定脂肪酶,固定过程中引入海藻酸钠,用CaCl_2进行后处理,得到固定化脂肪酶PAN-PEI-SA/E-CaCl_2载酶量为31.70(mg enzyme)/(g support),酶活为50.20 U/(g support),15次重复使用可保留58.77%的酶活,与游离酶相比耐酸性和耐温性有所提高,相同条件下与Nov 435相比,酶活更高,这表明最终得到的固定化脂肪酶有良好的工业应用前景.     

Screening and Immobilization <Emphasis Type="Italic">Burkholderia</Emphasis> sp. GXU56 Lipase for Enantioselective Resolution of (<Emphasis Type="Italic">R</Emphasis>,<Emphasis Type="Italic">S</Emphasis>)-Methyl Mandelate

Microorganisms producing lipase were isolated from soil and sewage samples and screened for enantioselective resolution of (R,S)-methyl mandelate to (R)-mandelic acid. A strain designated as GXU56 was obtained and identified as Burkholderia sp. Preparing immobilized GXU56 lipase by simple adsorption on octyl sepharose CL-4B, the optimum temperature was shifted from 40 °C (free lipase) to 50 °C (immobilized lipase), and the optimum pH was shifted from 8.0 (free lipase) to 7.2 (immobilized lipase). The immobilized enzyme displayed excellent stability in the pH range of 5.0–8.0, at the temperatures below 50 °C and in organic solvents compared with free enzyme. Enantioselectivity ratio for (R)-mandelic acid (E) was dramatically improved from 29.2 to more than 300 by applying immobilized lipase in the resolution of (R,S)-methyl mandelate. After five cycles of use of immobilized lipase, conversion and enantiomeric excess of (R)-mandelic acid were 34.5% and 98.5%, respectively, with enantioselectivity ratio for (R)-mandelic acid (E) of 230. Thus, octyl-sepharose-immobilized GXU56 lipase can be used as a bio-resolution reagent for producing (R)-mandelic acid.     

Characterization of Sol-gel bioencapsulates for ester hydrolysis and synthesis

Candida rugosa lipase was entrapped in silica sol-gel particles prepared by hydrolysis of methyltrimethoxysilane and assayed by p-nitrophenyl palmitate hydrolysis, as a function of pH and temperature, giving pH optima of 7.8 (free enzyme) and 5.0–8.0 (immobilized enzyme). The optimum temperature for the immobilized enzyme (50–55°C) was 19°C higher than for the free enzyme. Thermal, operational, and storage stability were determined with n-butanol and bytyric acid, giving at 45°C a half-life 2.7 times greater for the immobilized enzyme; storage time was 21 d at room temperature. For ester synthesis, the optimum temperature was 47°C, and high esterification conversions were obtained under repeated batch cycles (half-life of 138 h).     

界面活化的溶胶凝胶包埋Candida rugosa脂肪酶催化合成维生素E琥珀酸酯

采用界面活化的溶胶凝胶包埋Candida rugosa脂肪酶(CRL)催化合成了维生素E琥珀酸酯.考察了影响溶胶凝胶包埋固定化CRL的因素,获得的最佳固定化条件为:丙基三甲氧基硅烷/正硅酸四乙酯摩尔比为1/1,水与硅烷前体摩尔比为15,酶的添加量为0.5mg/ml,PEG400的添加量为12μl/ml溶胶. 溶胶凝胶包埋的CRL在50℃,18h后其活性仍然保持了70.58%,是游离酶的2.6倍,且稳定性得到了明显的改善.基于CRL的界面特性,采用五种表面活性剂对其进行界面活化.结果表明,采用橄榄油活化的溶胶凝胶包埋的CRL合成维生素E琥珀酸酯的酯化活力最高,相比原酶和未界面活化的溶胶凝胶包埋酶分别提高了6.7和1.43倍.     

改性Ultrastable-Y分子筛固定化对非水相中脂肪酶拆分(R,S)-2-辛醇的影响

采用改性Ultrastable-Y分子筛固定Penicillium expansum PED-03 脂肪酶(PEL), 利用固定化PEL在非水相中对(R,S)-2-辛醇进行手性拆分, 考察了改性Ultrastable-Y分子筛固定化处理对PEL催化性能的影响. 结果表明, 与游离PEL及经其它载体固定化的PEL相比, 改性Ultrastable-Y分子筛固定的PEL所催化的拆分反应的转化率(c)和对映体过量值(ee)以及对映体选择性(E)均得到了较大提高. 经固定化处理后, PEL的最适反应温度明显升高, 适宜反应温度范围变宽, 其稳定性也得到了明显改善, 而适宜反应pH值则具有“记忆”性. 在间歇式反应器中利用Ultrastable-Y分子筛固定化PEL对(R,S)-2-辛醇进行手性拆分, 50 ℃反应24 h转化率(c)可达理论值的97.68%, 对映体过量值(ee)可达98.75%. 连续8批拆分反应的结果表明: 改性Ultrastable-Y分子筛固定化脂肪酶催化效率高、立体选择性强(平均E 值＞460), 且催化性能稳定, 显示了该固定化酶在(R,S)-2-辛醇的手性拆分方面具有良好的应用前景.     

京尼平交联磁性壳聚糖微球的制备及其脂肪酶的固定化

采用反相悬浮法与溶胶凝胶法结合制备磁性壳聚糖微球,并以此为载体,京尼平为交联剂,脂肪酶为模型酶进行固定化,研究了酶固定化的最优条件和固定化酶的性质。结果表明,在京尼平浓度为0.6 g/L、交联温度为55 ℃、交联时间8 h,固定化酶的比活力最大,为4.31 U/g。固定化酶在25~35 ℃,pH值在8.0有最大活性,其米氏常数Km为0.26 mol/L。同时,固定化酶具有良好的热稳定性及pH稳定性,可重复利用,且能进行磁分离。     

Adsorption and activity of lipase from Candida rugosa on the chitosan-modified poly(acrylonitrile-co-maleic acid) membrane surface

Efforts have recently been made to improve the biocompatibility of support surface for enzyme immobilization, which could create a specific microenvironment for the enzymes and thus benefit the enzyme activity. In this work, one natural macromolecule, chitosan, was tethered on the surface of poly(acrylonitrile-co-maleic acid) (PANCMA) membrane to prepare a dual-layer biomimetic support for enzyme immobilization. Lipase from Candida rugosa was immobilized on this dual-layer biomimetic support by adsorption. The properties of the immobilized enzyme were assayed and compared with those of the free one. It was found that the adsorption capacity of lipase on the chitosan-tethered PANCMA membrane increases with the decrease of ionic strength and there is an optimum pH value for the adsorption. The activity retention of the immobilized lipase on the chitosan-tethered membrane by adsorption (54.1%) is higher than that by chemical bonding (44.5%). In comparison with the immobilized lipase by chemical bonding, there is a decrease of the K(m) value and an increase of the V(max) value for the immobilized lipase by adsorption. Additionally, the experimental results of thermal stabilities indicate that the residual activity of the immobilized lipase at 50 degrees C is 38% by adsorption and 65% by chemical bonding.     

单分散磁性纳米粒子固定化猪胰脂肪酶的研究

借助溶热法制备了一种亲水及生物相容良好的Fe3O4磁性纳米粒子,用γ-氨丙基三乙氧基硅烷直接对所得磁性粒子表面改性,然后用戊二醛偶联法制得了固定化猪胰脂肪酶.表征研究显示,所得磁性粒子粒径约200 nm,具有良好的单分散性和磁响应性.考察了戊二醛浓度、给酶量和反应时间对脂肪酶固定化过程的影响,并通过游离酶与固定化酶的比...     

脂肪酶的固定化及其在有机酶促反应中稳定性研究

利用吸附法,将圆柱状假丝酵母脂肪酶固定于４种疏水性不同的载体上,固定化酶的活性及稳定性随载休疏水性的增大而增大。用ＹＧＷ－Ｃ６Ｈ５作为载体,在有机溶剂－水双液相体系中催化萘普生甲酯的不对称水解,反应１２０ｈ转化率为２４．７２％,产品的对映体过量值为９４．８２％。     

Immobilization of Yarrowia lipolytica Lipase—a Comparison of Stability of Physical Adsorption and Covalent Attachment Techniques

Lipase immobilization offers unique advantages in terms of better process control, enhanced stability, predictable decay rates and improved economics. This work evaluated the immobilization of a highly active Yarrowia lipolytica lipase (YLL) by physical adsorption and covalent attachment. The enzyme was adsorbed on octyl–agarose and octadecyl–sepabeads supports by hydrophobic adsorption at low ionic strength and on MANAE–agarose support by ionic adsorption. CNBr–agarose was used as support for the covalent attachment immobilization. Immobilization yields of 71, 90 and 97% were obtained when Y. lipolytica lipase was immobilized into octyl–agarose, octadecyl–sepabeads and MANAE–agarose, respectively. However, the activity retention was lower (34% for octyl–agarose, 50% for octadecyl–sepabeads and 61% for MANAE–agarose), indicating that the immobilized lipase lost activity during immobilization procedures. Furthermore, immobilization by covalent attachment led to complete enzyme inactivation. Thermal deactivation was studied at a temperature range from 25 to 45°C and pH varying from 5.0 to 9.0 and revealed that the hydrophobic adsorption on octadecyl–sepabeads produced an appreciable stabilization of the biocatalyst. The octadecyl–sepabeads biocatalyst was almost tenfold more stable than free lipase, and its thermal deactivation profile was also modified. On the other hand, the Y. lipolytica lipase immobilized on octyl–agarose and MANAE–agarose supports presented low stability, even less than the free enzyme.