Immobilization of lipase from Candida rugosa on synthetic polymer beads for use in the synthesis of fatty esters

Lipase from Candida rugosa was immobilized on three different supports, i.e. Amberlite XAD7, poly(methylmethacrylate) (PMMA) and celite. With the conditions tested, maximum adsorption can be achieved after 30 min. The activities of the immobilized lipases were determined by the esterification reaction of oleic acid and butanol. The immobilized lipases were found to be very effective in the esterification reaction. The immobilized activities generally were high in apolar organic solvents with log P values from 2·0 to 4·0. The preference for fatty acids as acyl donors differed in all cases of immobilized lipases. Lipase immobilized on XAD7 and PMMA exhibited high preference of acyl donors (fatty acids) with chain lengths 12–18 and 8–18, respectively. Lipase immobilized on celite, however, showed high activity in all cases of fatty acids. The nucleophile (alcohol) selectivity studies showed that lipase immobilized on XAD7 and celite was more accessible to alcohols of chain lengths 3–12. However, lipase immobilized on PMMA showed a significant preference towards alcohols of chain lengths from 3 to 10.     

固定化磷酸盐选择性催化合成2-O-己二酸单乙烯基蔗糖酯

因为蔗糖含有8个反应活性相近的羟基,蔗糖与脂肪酸酯的转酯化反应很难得到单一的产物,本研究以固定化磷酸盐作催化剂可以选择性地催化蔗糖和己二酸二乙烯酯(Divinyl adipate,DVA)的转酯化反应.方法是在含有磷酸盐的丙酮中加入酸洗过的硅藻土,振荡1 h,可制成固定化磷酸盐;取0.01 mol蔗糖和0.02 mol DVA,加入固定化磷酸盐10 g,以100 mL DMF为溶剂,在50℃、220 r·min~(-1)的恒温摇床中连续反应1 d,可以选择性地获得蔗糖单酯产品.转酯化反应的转化率和产率分别达到60.0%、54.8%.经过MS和~(13)C-NMR鉴定,转酯化位置为蔗糖基的C-2位,合成产物为2-O-己二酸单乙烯基蔗糖酯.     

酸性脲酶的固定化研究

以明胶为包埋材料,戊二醛为交联剂固定化粪产碱菌所产的酸性脲酶。对酸性脲酶的固定化条件(包括明胶含量、戊二醛浓度、吸附时间、交联时间和粗酶液用量)及酶学性质(温度和pH值)进行了研究。结果表明,固定化酶的最适宜条件为：明胶的质量分数15％,戊二醛质量分数0.3％,吸附时间4 h,交联时间20 min,粗酶液用量4 mL。...     

明胶膜固定化脲酶的制备及性质

以明胶为载体,戊二醛为交联剂,采用包埋-交联联用法制备了明胶膜固定化脲酶,其酶活力为6 07U/g载体,酶活力收率为66 1%。最优固定化条件是包酶量为10mg酶/g明胶,ρ(明胶)=100g/L,φ(戊二醛)=0 5%。研究了固定化酶的性质,并与游离酶作了比较,游离酶的最适pH=7 0,固定化酶的最适pH=6 5;游离酶的最适温度为60℃,固定化酶的最适温度升至70℃;固定化酶与游离酶的米氏常数Km分别为11 7mM和12 4mM;固定化酶在80℃下180min仍保留初始活力的10%,而游离酶几乎完全失活。固定化酶重复使用20次其活力仅下降15%,4℃下贮存35d后仍保持初始活力的55%。     

Integration of flocculation and adsorptive immobilization of Pseudomonas delafieldii R‐8 for diesel oil biodesulfurization

BACKGROUND: Deep desulfurization of hydrocarbon fuels is receiving increasing worldwide attention because of the increasingly stringent regulations to meet the requirement of environmental protection. Biodesulfurization (BDS) is being explored as either an alternative or complementary process to the conventional oil refining technologies. The whole cell immobilization technique is of great importance for accelerating the industrialization of BDS. An effective technique for a BDS process employing flocculation and integration with immobilization was developed. RESULTS: Pseudomonas delafieldii R‐8 cells were successfully flocculated and immobilized by directly adding chitosan and celite into the culture broth. The one‐step immobilized R‐8 cells exhibited good catalytic activity and retained at least 85% activity after six cycles of repeated‐batch desulfurization. Extensive biodesulfurization of diesel oil resulted in 82% reduction of total sulfur from 123 to 22 µg g^?1 in 24 h. CONCLUSIONS: A novel and simple technique was developed using chitosan flocculation and integration with cell immobilization onto celite for dibenzothiophene BDS. The present report indicates that integration of flocculation and immobilization may provide a continuous and efficient method of BDS. Copyright © 2010 Society of Chemical Industry     

柱子型直接固定氯化血红素及其应用

用惰性载体Sepharose 4B、琼脂糖、微晶纤维素、人造沸石等分别直接固定氯化血红素,分别装柱后应用于痕量过氧化氢及血清葡萄糖浓度的检测,并比较它们的检测灵敏度。结果表明,以上几种载体经活化后都能很好地固定血红素．固定化的血红素装柱后保持稳定的催化过氧化物的活性,检测灵敏度高,血红素在几种载体上检测灵敏度的大小为：Sepharose 4B〉琼脂糖〉微晶纤维素〉人造沸石。本法操作简单且成本低,柱子型直接固定氯化血红素可反复使用,能快速检测样品中的痕量过氧化氢及用于血清中葡萄糖含量的检测。     

Production of low methoxyl pectin using immobilized pectinmethylesterase silica from acerola (Malpighia glabra L.)

The aim of this work was to develop an efficient reactor for the production of low methoxyl pectin, using pectinmethylesterase (PME, EC 3.1.1.11) from acerola immobilized on silica. The immobilized enzyme was used in up to 50 successive bioconversion runs at 50 °C with an efficiency loss of less than 20%. The fixed‐bed reactor (6.0 × 1.5 cm) was prepared using PME immobilized in glutaraldehyde‐activated silica operated at 50 °C with an optimum flow rate of 10 mL h^?1. The bioconversion yield was shown to strongly depend on the nature of the enzymatic preparation. An efficiency of 44% was achieved when concentrated PME was used, compared with only 30% with purified PME, both after an 8‐h run. The process described could provide the basis for the development of a commercial‐scale process. Copyright © 2006 Society of Chemical Industry     

海藻酸钠-明胶协同固定化S-腺苷甲硫氨酸合成酶的研究

以海藻酸钠和明胶为载体,对S-腺苷甲硫氨酸合成酶进行固定化。再用戊二醛对其进一步交联,增强固定化酶的稳定性。考察了海藻酸钠和明胶质量分数、CaCl2质量分数、酶和载体比例以及交联剂戊二醛体积分数等因素对固定化酶的影响。结果表明,最佳固定化条件为:海藻酸钠质量分数2.0%、明胶质量分数1.0%、CaCl2质量分数4.0%、固定化酶量为2.5 g/L凝胶、戊二醛体积分数0.6%。交联固定化酶热稳定性得到大幅度提高,在50℃下保温5 h仍保留72%的活力,而游离酶则完全失活。交联固定化酶在碱性溶液中的稳定性较高,在pH=8.0～9.0的缓冲液中4℃保温10 h酶活性仍保留87%以上。将交联固定化酶用于S-腺苷甲硫氨酸的合成,连续反应8批次后酶活性仍保留65%。     

Entrapment of porous and stable concanavalin A–peroxidase complex into hybrid calcium alginate–pectin gel

Ammonium sulfate‐fractionated proteins of turnip (Brassica rapa) were used for the simultaneous purification and immobilization of peroxidase by using crude jack bean extract. The concanavalin A–turnip peroxidase complex retained nearly 70% of the original activity. Calcium alginate–pectin‐entrapped soluble turnip peroxidase and the concanavalin A complex of peroxidase retained 63% and 52% of the original activity, respectively. The concanavalin A–peroxidase complex, the alginate–pectin‐entrapped soluble peroxidase and the alginate–pectin‐entrapped concanavalin A–peroxidase complex showed very high stability against denaturation mediated by heat, pH, urea, organic solvents and detergents. The exposure of soluble and immobilized turnip peroxidase to trypsin resulted in an enhancement of the peroxidase activity. The concanavalin A–peroxidase complex entrapped preparation was markedly more stable as compared with the directly entrapped soluble enzyme preparation. The results suggested that such preparations have great potential in the construction of bioreactors to be used for the remediation of aromatic compounds present in polluted wastewater/industrial effluents. Copyright © 2006 Society of Chemical Industry     

Batch and Continuous Hydrolysis of Lactose Using P-Galactosidase Immobilized on Gelatin-Cmc

β-Galactosidase enzyme (E.C. 3.2.1.23) was immobilized onto a gelatin-carboxymethylcellulose carrier system by using cross-linker chromium acetate. A relative activity increase of 33% was achieved by adding carboxymethylcellulose to gelatin. The effect of operation temperature and pH on relative activity and reusability of the immobilized enzyme was investigated. A fluidized-bed reactor with a down flow mechanism was used for continuous process hydrolysis, and a constant glucose production rate was obtained.     

Nanostructure composite ZnFe2O4–FeFe2O4–ZnO immobilized on glass: Photocatalytic activity for degradation of an azo textile dye F3B

An efficient and scalable one-pot synthetic method to prepare nanostructure composite of ZnFe₂O₄–FeFe₂O₄–ZnO (ZFZ) has been investigated. This method is based on thermal decomposition of iron(III) acetate and zinc acetate in monoethanolamine (MEA) as a capping agent. Moreover, thermogravimetric analysis (TG-DTG) was performed to determine the temperature at which the decomposition and oxidation of the chelating agents took place. ZFZ was immobilized on glass using doctor blade method and calcinated at different temperatures. The properties of the ZFZ nanocomposite have been examined by different techniques, such as X-ray diffraction (XRD), field emission scanning electron microscopy (FESEM) and diffuse reflectance (DRS). FESEM shows that nanocomposite is monocrystallines and a narrow dispersion in size of 48 nm. XRD confirms that the prepared nanocomposite is composed of franklinite, ZnFe₂O₄ (54%), magnetite, FeFe₂O₄ (8%) and wurtzite, ZnO (48%). Photocatalytic activity of ZFZ immobilized on glass was carried out by choosing an azo textile dye, Reactive Red 195 (F3B) as a model pollutant under UV irradiation with homemade photocatalytic apparatus and the results indicated that ZFZ exhibited good photocatalytic activity.     

Modulation of the Selectivity of Immobilized Lipases by Chemical and Physical Modifications: Release of Omega-3 Fatty Acids from Fish Oil

Three different lipases (from Candida antarctica fraction B (CALB), Thermomyces lanuginose (TLL), and Rhizomucor miehei (RML)) were immobilized by two different methods, immobilization on CNBr-activated Sepharose via a mild covalent immobilization or adsorption onto hydrophobic supports (Octyl-Sepharose). These immobilized preparations were chemically and physically modified on the protein surface (enzyme carboxylic groups with ethylenediamine, amino groups with succinic anhydride, or coating with polyethyleneimine).The activity and selectivity in the production of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) by enzymatic hydrolysis of sardine oil were evaluated. Activity and selectivity were dependent on the different lipases, the immobilization protocols, the modification methods, and the pH of the reaction media. The selectivity (EPA/DHA ratio) of RML immobilized on CNBr-activated Sepharose was increased after succinylation from 7.5 to 34 at pH 6.0. The selectivity of octyl-RML improved from 1.5 to 8.5 when pH was increased from 6 to 8. The selectivity and activity of octyl-TLL increased twofold after PEI coating at pH 6. The properties of CAL-B derivatives were slightly altered after modification.     

Application of immobilized lipase in production of camptosar (CPT-11)

Lipase fromPseudomonas cepaica (Amano, PS-30) was immobilized on celite and used in organic solvent for the selective acylation of a key alcohol intermediate. The compound was transformed in the synthesis to the anticancer drug Camptosar (CPT-11). Catalyst activity was influenced by the water content and method used to dry the catalyst. This resolution has been conducted on production scale with equal weight of recyclable catalyst.     

Nanodiamonds as Carriers for Address Delivery of Biologically Active Substances

Surface of detonation nanodiamonds was functionalized for the covalent attachment of immunoglobulin, and simultaneously bovine serum albumin and Rabbit Anti-Mouse Antibody. The nanodiamond-IgG^I125 and RAM-nanodiamond-BSA^I125 complexes are stable in blood serum and the immobilized proteins retain their biological activity. It was shown that the RAM-nanodiamond-BSA^I125 complex is able to bind to the target antigen immobilized on the Sepharose 6B matrix through antibody–antigen interaction. The idea can be extended to use nanodiamonds as carriers for delivery of bioactive substances (i.e., drugs) to various targets in vivo.     

海藻酸钠明胶协同固定化黑曲霉脂肪酶

以海藻酸钠明胶为复合载体,采用包埋法制备固定化黑曲霉脂肪酶,考察了海藻酸钠、明胶浓度等因子对固定化效果的影响,比较固定化酶和游离酶对温度、pH等条件的稳定性。结果表明,制备固定化黑曲霉脂肪酶的最优条件为:海藻酸钠、明胶浓度分别为1.25%和0.5%,CaC l2浓度为10%,给酶量为450 IU/g;固定化酶最适温度为35℃,最适pH为9.0,常见有机溶剂和金属离子对固定化酶的活力影响较小。     

米曲霉菌体的固定化及其稳定性

在液相培养基中将米曲霉菌体培养成直径为 1～ 2mm的菌球 ,以甲醛为交联剂、明胶为活性保护剂对其进行固定化研究 .在正交实验L₁₆(4⁵)的基础上 ,利用人工神经网络得到了较优的固定化条件 :菌体与固定液的用量比 (固液比 ) 1∶8,固定液中含明胶 5 g·L^-1、甲醛 5 g·L^-1,固定化时间为 1.5h .在此条件下制备的固定化米曲霉菌体比酶活为 15 0 0U ,比酶活保留率 83% .对固定化米曲霉菌体稳定性进行深入研究表明 ,其最适反应温度和 pH值分别为 6 3℃和 8.0 ,与游离菌体相比最适反应条件范围变宽 .在固定床反应器中用其连续拆分N -乙酰 -DL -丙氨酸 ,半衰期为 77天 ,具有较高的操作稳定性     

壳聚糖微珠亲和层析介质的制备及其初步应用

目的制备壳聚糖(CTS)微珠亲和层析介质,并进行初步应用。方法采用多种化学方法对CTS的表面基团进行改造,使其活化。用此改性CTS进行羊抗人IgG及小牛血清纤维连接蛋白(FN)的纯化。结果此改性的CTS微珠粒径在40￣160μm之间,每克可溶胀至4ml。活化的CTS对人丙种球蛋白的吸附量为15￣25mg/gCTS,对明胶的吸附量为5.8￣6.5mg/gCTS。采用此方法纯化的羊抗人IgG及FN与Sepharose4B法纯化效果一致。结论已制备的壳聚糖微珠亲和层析介质,其性质稳定且价格较低,是一种可以广泛应用的亲和层析介质。     

猪骨基多孔碳固定化酶的研究

本文以猪骨基多孔碳为载体,采用明胶包埋与戊二醛交联结合的方法,制备固定化过氧化氢酶,并对固定化酶制备条件的优化,酶作用最适条件及其稳定性进行研究。结果表明:以猪骨基多孔碳为载体吸附2h、1%明胶包埋、1%戊二醛交联过氧化氢酶制备的固定化酶,在最适的缓冲液浓度、溶液pH和体系温度下,虽然催化效率有所下降,但其贮存稳定性和操作稳定性都有所增加,重复使用8次后,活力仍保持在初始活力的90%以上。这对于过氧化氢酶进一步在食品工业和纺织工业的推广可能有潜在的应用价值。     

Langmuir-Blodgett film based biosensor for estimation of galactose in milk

A mono-enzyme amperometric biosensor has been developed for the estimation of galactose in milk and milk products. Galactose oxidase was immobilized with poly(3-hexyl thiophene)/stearic acid (P3HT/SA) on to indium tin-oxide (ITO) coated glass plates using Langmuir-Blodgett (LB) film deposition technique. The immobilized galactose oxidase in P3HT/SA LB films was characterized using Fourier-transform infrared (FTIR) spectroscopy and scanning electron microscopy. This P3HT/SA/GaO LB film on ITO-coated glass plate was used as working electrode with platinum as reference electrode for development of galactose biosensor. The biosensor shows the linearity 1-4 g/dl galactose. The effect of galactose concentration, pH, temperature, detection limit, response time and stability of the immobilized galactose oxidase in LB films were also studied. The electrode was found stable upto 45 °C and has a shelf life of more than 90 days.     

Efficient Conjugation of Oligosaccharides to Polymer Particles through Furan/Maleimide Diels–Alder Reaction: Application to the Capture of Carbohydrate‐Binding Proteins

Glycan–protein interactions play a crucial role in physiological and pathological events. Hence, improving the isolation of carbohydrate‐binding proteins (i.e., lectins and anti‐glycan antibodies) from complex media might not only lead to a better understanding of their function, but also provide solutions for public health issues, such as water contamination or the need for universal blood plasma. Here we report a rapid and efficient method for producing carbohydrate‐based affinity adsorbents combining enzymatic synthesis and metal‐free click chemistry. Both simple and complex glycans (maltose, blood group antigens A, B, and H) were readily modified by the addition of a furyl group at the reducing end without the need for protecting groups and were then efficiently conjugated to maleimide‐activated Sepharose particles through Diels–Alder cycloaddition. These neoglycoconjugates showed high efficiency for the purification of lectins (concanavalin A and Ulex europaeus agglutinin), as well as for the capture of anti‐A and anti‐B blood group antibodies, opening new prospects for glycoproteomics and for the development of universal blood plasma.