固定化扩展青霉脂肪酶的制备及其在玉米油转酯反应中的应用

采用吸附法对来源于扩展青霉Penicillium expansum的脂肪酶进行了固定化.从20种不同来源的树脂中筛选出固定化效率高且价格低廉的D4020树脂作为载体,系统研究了固定化条件对固定化效率及固定化酶转酯活力的影响.结果表明,最适加酶量、缓冲液pH和吸附时间分别为0.7 g/g、9.4和4 h.冻干时添加0.5%的半乳糖有助于提高固定化酶的转酯活力.在上述优化条件下,固定化酶的转酯活力为404.0 U/g,而所用的游离酶不能催化该转酯反应.利用该固定化酶催化玉米油转酯反应生产生物柴油时,叔戊醇为适宜的反应介质,其最适添加量为0.5 ml/g;适宜的酶量、加水量和反应温度分别为60.6 U/g、油重的1.2%和35℃.按醇/油摩尔比为1的比例分别在反应0、2和6 h时加入甲醇,在优化反应条件下,反应24 h后甲酯产率达85.0%;固定化脂肪酶具有较好的操作稳定性,反应10批次时,相对酶活力为62.8%.     

黑曲霉脂肪酶的耦合固定化及特性

研究了吸附-絮凝耦合的方法固定脂肪酶的工艺条件.结果表明:在33℃下,用0.03mol/L的磷酸二氢钾?氢氧化钠缓冲液控制体系pH为7.0,酶与树脂(质量比1∶8)作用吸附1h后,用0.2mL絮凝剂聚丙烯酰胺(w(PAM)=0.5%)处理,得到活力较高的固定化脂肪酶.固定化酶最适pH9.0,最适温度为45℃,活力为405U/g,酶活回收率可以达到40%.固定化脂肪酶制备简便,可重复使用,稳定性较高.     

海藻酸钠固定化根霉脂肪酶的制备及其性质

 研究了以海藻酸钠为载体,用包埋法制备固定化德氏根霉(Rhizopus delemar)脂肪酶的条件. 将酶粉和海藻酸钠溶于pH 5.0的HAc-NaAc缓冲溶液,用注射器将此混合液滴入到0.05 mol/L无菌CaCl2溶液中,静置固化45 min, 经过滤、洗涤和干燥后得到球状固定化酶. 固定化酶的活力回收约为34.1%. 酶学性质研究表明,此固定化酶的热稳定性较好. 游离酶在 60 ℃下保温1 h已完全丧失活力,而固定化酶在100 ℃下保温1 h仅损失36.2%的活力,在100 ℃下保温6 h仍可保持46.8%的酶活力. 酶经固定化后,其橄榄油水解反应的最适温度由40 ℃上升至90 ℃, Km值由13.8 mg/ml下降为8.1 mg/ml. 常见有机溶剂对固定化酶的活力影响较小. 将该固定化脂肪酶用于非水溶剂中正戊酸异戊酯的合成,重复使用6次后,固定化酶仍保持95%的酶活力.     

脂肪酸分子印迹对sol-gel固定化脂肪酶酯化活性和胶体结构的影响

基于sol-gel工艺的生物印迹对提高脂肪酶在非水相体系中的活性,扩大其应用范围具有重要作用.本研究以乙烯基三甲氧基硅烷(VTMOS)和四甲氧基硅烷(TMOS)为前趋体,系统考察了脂肪酸印迹分子对sol-gel脂肪酶固定化率和活性的影响,获得了最优的工艺条件.当VTMOS/TMOS为7∶1时,印迹后脂肪酶的比酶活和总酶活分别达到3311.0μmol/h/mg和2506.9μmol/h/gGel,比未印迹同摩尔比的固定化脂肪酶分别提高了2.06和1.79倍.系统考察了脂肪酸生物印迹效应和胶体结构对固定化酶表观活性的影响.氮气吸附—解吸附分析和扫描电镜观察表明,随着脂肪酸分子的参入,固定化脂肪酶颗粒平均孔径增大,对底物的传质阻力逐渐降低,但比表面积和孔体积的变化并不显著;印迹酶平均颗粒直径明显减小,各有机硅烷单体之间的成键能力减弱.表明脂肪酶活性的增加主要来源于疏水性脂肪酸侧链引起的脂肪酶的界面激活效应(生物印迹效应),同时固定化颗粒孔径的改变增加了底物和酶分子的结合,提高了固定化酶的表观活性.     

微波辐射高效共价固定青霉素酰化酶

为提高青霉素酰化酶的共价固定化效率, 在微波辐射条件下将酶蛋白共价固定于介孔泡沫硅(MCFs)的孔道中. 通过正硅酸四乙酯水解缩合制备介孔泡沫硅, 再于微波辅助下将青霉素酰化酶共价固定在其孔道中. 以固定化酶相对活力和活力回收为指标, 考察了加酶量、固定化温度、微波辐射时间等条件对酶固定化效率的影响. 实验结果表明: 当加酶量为60 mg/g, 固定化温度为20 ℃, 微波辐射140 s, 固定化酶相对活力达到178.1%, 表观活力为1191.3 U/g(以湿重计). 与常规方法相比, 微波辅助固定化酶时, 固定化酶相对活力提高34.5%, 固定化时间亦大幅缩短至数分钟, 这为青霉素酰化酶的高效共价固定化提供了一条新的途径.     

复合纤维素膜固定化胰蛋白酶反应器及其应用于蛋白质酶解

合成了甲基丙烯酸缩水甘油酯-纤维素复合膜,并以此膜为基质共价键合固定化胰蛋白酶,以N-苯甲酰-L-精氨酰乙酯(BAEE)为底物,应用高效液相色谱系统测定了酶固定化膜柱的催化反应特性。研究结果表明:温度、pH值、离子强度、有机溶剂及蛋白变性剂等都对固定化酶的活力有一定的影响。在最适条件下,固定化胰蛋白酶的活力为17800U/g干膜,蛋白载量为3.6mg/g(≈0.15μmol/g)干膜,活性回收率达到52%.固定化酶表现出较高的使用和储藏稳定性,在40℃下,水解BAEE底物24h活力无显着变化。固定化酶膜柱在4℃冷藏保存100d仍保存90%以上的水解活力。固定化酶反应器被应用于蛋白质酶解的肽谱实验。     

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。     

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。     

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。     

重组大肠杆菌固定床间歇生产β-葡聚糖酶

以多孔陶瓷为载体,吸附法固定化重组大肠杆菌并进行固定床间歇培养.在循环流速44.19mL/min,曝气量为0.6mL/min时,培养48h后发酵液的酶活力达100.3U/mL.固定化细胞具有良好的重复使用能力,在连续5批次实验中,培养48h后的酶活力均在100U/mL左右.     

Selection of stabilizing additive for lipase immobilization on controlled pore silica by factorial design

Candida rugosa lipase was covalently immobilized on silanized controlled poresilica (CPS) previously activated with glutaraldehyde in the presence of several additives to improve the performance of the immobilized from in long-term operation. Proteins (albumin and lecithin) and organic molecules (β-cyclodextrin and polyethylene glycol [PEG]-1500) were added during the immobilization procedure, and their effects are reported and compared to the behavior of the immobilized biocatalyst in the absence (lacking) of additive. The selection of the most efficient additive at different lipase loadings (150–450 U/g of dry support) was performed by experimental design. Two 2²full factorial designs with two repetitions at the center point were employed to evaluate the immobilization yield. A better, stabilizing effect was found when small amounts of albumin or PEG-1500, were added simul-taneou sly to the lipase on to the support. The catalytic activity had a maximum (193 U/mg) for lipase loading of 150 U/g of dry support using PEG-1500 as the stabilizing additive. This immobilized system was used to perform esterification reactions under repeated batch cycles (for the synthesis of butyl butyrate as a model). The half-life of the lipase immobilized on CPS in the presence of PEG-150 was found to increase fivefold compared with the control (immobilized lipase on CPS without additive).     

CSTR和批式反应器中酶法拆分萘普生的比较

比较了批式反应器和连续流动搅拌反应器中酶动力学拆分萘普生的不同之处。从宏观反应器平衡角度,推导出了在ＣＳＴＲ中不同于在批式反应器中的酶立体选择性,产物对映体过量值和反应转化率的定量关系式,并通过脂肪酶催化的萘一甲酯的不对称水解反应得到了证实。     

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。     

Covalent immobilization of Candida antarctica lipase B on nanopolystyrene and its application to microwave-assisted esterification

Nanopolystyrene was used as a solid support for the covalent immobilization of Candida antarctica lipase B (CalB) using the photoreactive reagent 1-fluoro-2-nitro-4-azido benzene (FNAB) as a coupling reagent. The obtained derivative was then used as a biocatalyst in a microwave assisted esterification experiment. Factors such as contact time, pH, and enzyme concentration were investigated during immobilization. The hydrolytic activity, thermal, and operational stability of immobilized-CalB were determined. The maximum immobilized yield (218 μg/mg support) obtained at pH 6.8 exhibited optimum hydrolytic activity (4.42 × 10³ mU p-nitrophenol/min). The thermal stability of CalB improved significantly when it was immobilized at pH 10, however, the immobilized yield was very low (93.6 μg/mg support). The immobilized-CalB prepared at pH 6.8 and pH 10 retained 50% of its initial activity after incubation periods of 14 and 16 h, respectively, at 60 ℃. The operational stability was investigated for the microwave assisted esterification of oleic acid with methanol. Immobilized-CalB retained 50% of its initial activity after 15 batch cycles in the microwave-assisted esterification. The esterification time was notably reduced under microwave irradiation. The combined use of a biocatalyst and microwave heating is thus an alternative total green synthesis process.     

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.     

AnSBBR Applied to a Personal Care Industry Wastewater Treatment: Effects of Fill Time,Volume Treated Per Cycle,and Organic Load

A study was performed regarding the effect of the relation between fill time, volume treated per cycle, and influent concentration at different applied organic loadings on the stability and efficiency of an anaerobic sequencing batch reactor containing immobilized biomass on polyurethane foam with recirculation of the liquid phase (AnSBBR) applied to the treatment of wastewater from a personal care industry. Total cycle length of the reactor was 8 h (480 min). Fill times were 10 min in the batch operation, 4 h in the fed-batch operation, and a 10-min batch followed by a 4-h fed batch in the mixed operation. Settling time was not necessary since the biomass was immobilized and decant time was 10 min. Volume of liquid medium in the reactor was 2.5 L, whereas volume treated per cycle ranged from 0.88 to 2.5 L in accordance with fill time. Influent concentration varied from 300 to 1,425 mg COD/L, resulting in an applied volumetric organic load of 0.9 and 1.5 g COD/L.d. Recirculation flow rate was 20 L/h, and the reactor was maintained at 30 °C. Values of organic matter removal efficiency of filtered effluent samples were below 71% in the batch operations and above 74% in the operations of fed batch followed by batch. Feeding wastewater during part of the operational cycle was beneficial to the system, as it resulted in indirect control over the conversion of substrate into intermediates that would negatively interfere with the biochemical reactions regarding the degradation of organic matter. As a result, the average substrate consumption increased, leading to higher organic removal efficiencies in the fed-batch operations.     

Strategies in lipase production by immobilizedCandida rugosa cells

Growing cells ofCandida rugosa immobilized in polymethacrylamide-hydrazide and polyurethane foam were employed in fluidized and packed bed reactors, for discontinuous and continuous fermentations to obtain extracellular lipase. In spite of hydrodynamic problems, fermentation cultures using polyurethane foam showed higher lipolytic activity than cultures employing polymethacrylamide-hydrazide beads, which was probably owing to the high immobilized biomass concentration in polyurethane observed by direct microscopy enumeration. Different oleic acid concentrations were assayed. The maximum level of lipase was achieved at 4 g/L of oleic acid. These results reaffirm that lipase production is a direct function of cell-substrate contact and that the organic substrate dispersion is important in this system.     

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...     

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

ＳＴＵＤＹＯＮＩＭＭＯＢＩＬＩＺＥＤＰＯＲＣＩＮＥＰＡＮＣＲＥＡＴＩＣＬＩＰＡＳＥＣＡＴＡＬＹＺＩＮＧＴＲＡＮＳＥＳＴＥＲＩＦＩＣＡＴＩＯＮＢＥＴＷＥＥＮＭＥＴＨＹＬ－ＢＵＴＹＲＡＴＥＡＮＤ１－ＢＵＴＡＮＯＬＩＮＮＯＮＡＱＵＥＯＵＳＳＹＳＴＥＭＳＸｉ...     

Kinetic properties of soluble and immobilizedCandida rugosa lipase

Immobilized lipase (triacylglycerol ester hydrolase, EC 3.1.1.3) fromCandida rugosa has been immobilized on commercially available microporous polypropylene and used for the batch hydrolysis of different animal fats. The effect of the reaction products at concentrations similar to those obtained at 90% hydrolysis, both on soluble and immobilized lipase, was studied. Glycerol showed low inhibitory effect but oleic acid caused 50% inhibition. A mixture of free fatty acids present in the complete hydrolysis of beef tallow inhibited lipase activity more than 70%. The stability of the enzyme (both soluble and immobilized) was highest in the presence of 20% isooctane. The apparent Michaelis constant for each substrate for the soluble enzyme did not change on immobilization.