触角状环氧链结构对固定化脂肪酶(YCJ01)酶活的影响

为了改善酶的刚性固定化对酶蛋白构象的负面影响,以原子转移自由基聚合法(ATRP)合成了亲水性、柔性、触角状环氧化酶固定化载体PS-acyl-P(AM-co-GMA),通过改变单体总量与引发剂量比例得到不同链长触角状环氧化酶固定化载体,并将其用于耐有机溶剂脂肪酶(YCJ01)的共价柔性固定化,重点考察了链长对固定化酶酶活的影响。结果表明,链长(增重率不超过3200%)越长,酶活越高。     

壳聚糖载体柔性固定化木瓜蛋白酶

用酶柔性固定化模型,以壳聚糖为载体,双醛淀粉为柔性链,对木瓜蛋白酶进行柔性固定化. 通过对固定化条件的优化,得出选用壳聚糖、双醛淀粉制得的柔性载体(Chitosan-DAS50)在酶用量为14.4 mg/g(酶/干球)、pH 8的条件下,固定木瓜蛋白酶18 h,所得的固定化酶活力回收率达72%,相当于采用壳聚糖-戊二醛(Chitosan-GA)手臂载体的3倍. 结果表明,酶的柔性固定化模型可以改善传统共价结合法固定化及手臂固定化酶活力回收率不高的缺陷.     

胺化聚苯乙烯载体柔性固定化木瓜蛋白酶

提出酶的“柔性固定化”模型,并以Mannich反应得到担载量为0.4～6.0 mmol NH₂•g^-1的胺化聚苯乙烯树脂为载体,以双醛淀粉为柔性链,对木瓜蛋白酶进行柔性固定化,酶活回收率可达40%～50％,相当于手臂固定化酶活力回收率的1.8～2.4倍,且柔性固定化酶稳定性较好.该结果说明,酶的“柔性固定化”模型可以改善传统共价结合固定化酶及手臂固定化酶活力回收率不高的缺陷.     

金属有机框架固定化酶及其在环境中的应用

固定化酶克服了游离酶易失活、稳定性差、难以回收利用的缺点,扩大了酶的实际应用范围。近年来,由于金属有机框架（metal-organic frameworks,MOFs）独特的性质,如比表面积大、孔隙率高、孔径可调节、开放的金属位点和多样的结构组成等,其作为固定化酶的新型载体成为研究热点。本文综述了近年来MOFs固定化酶的研究进展,其中重点讨论了酶在MOFs上的固定化方法（从头合成和后合成）和机理（载体包埋、表面吸附、共价连接和孔道扩散）,并且分析了不同合成方法的优势和局限性,如从头合成可以选择孔径小于目标酶尺寸的MOFs,但要求MOFs的合成条件温和;后合成可以选择合成条件苛刻的MOFs,但固定化过程相对复杂。此外,还对MOFs固定化酶在环境污染物检测和去除方面的应用进展进行了总结。最后对MOFs固定化酶在环境领域的应用研究和面临的挑战进行了展望,提出应关注MOFs固定化酶中MOFs和酶对污染物的协同作用以及MOFs固定化酶的可控制备。     

聚乙烯亚胺/多巴胺改性氧化硅固定碳酸酐酶

利用聚乙烯亚胺（PEI）/多巴胺（DA）共沉积法改性氧化硅，并以此为载体固定化碳酸酐酶（CA）。考察了PEI/DA质量比、沉积时间对沉积率的影响，用傅里叶红外光谱（FTIR）和扫描电子显微镜（SEM）对改性前后的微球进行了表征；研究了沉积率、载体用量、酶浓度及戊二醛（GA）浓度对固定化酶活回收率的影响；考察了固定化酶的储存稳定性和重复利用性。结果表明，随PEI/DA质量比增加，沉积率先增加后降低，质量比为1∶1时最大；随沉积时间增加，沉积率线性增长，10 h后PEI/DA体系沉积率为单独DA沉积改性的2.66倍，但沉积时间对N元素含量和酶活回收率影响不大；酶固定化时载体用量存在饱和值，CA和GA浓度的最优值分别为0.8 mg/ml和0.1%(质量)，此时酶活回收率可达78.8%。在25℃下储存30 d后，固定化酶的保留活性为77.2%，而游离酶只有12%；重复使用10次后，固定化酶仍能保持88.3%的相对活性。     

环氧树脂固定化拟南芥腈水解酶NIT的研究

游离酶不易回收,很难重复利用,而固定化酶重复利用度高。利用LX-1000EP(C)环氧树脂作为载体对腈水解酶进行固定化,研究了其最优固定化条件及其稳定性。最佳固定化条件为:固定化温度为20℃,固定化过程中缓冲液为磷酸钾缓冲液,pH8.0,浓度为0.1mol/L,加量为每克载体加10mL的粗酶液。固定化腈水解酶的最高酶活回收率达到98.1%,固定化酶在重复使用6次后,酶活仍能保持在初始酶活的30%以上。     

多酚氧化酶的改性蛭石固定化及其催化特性研究

以马铃薯为原料,提取其中的多酚氧化酶,采用改性蛭石为载体,对其进行固定化,研究游离与固定化酶的最适催化温度、pH值,固定化酶的储存稳定性、重复使用稳定性及对苯酚的清除性能。结果表明,改性后蛭石出现了较多沟壑状孔隙,颗粒质感疏松,有利于固定化酶分子;最佳固定化条件为:改性蛭石含量0.4 g,戊二醛浓度2.0%,搅拌时间2 h,在此条件下,酶活力回收率可达到60.67%;游离与固定化酶的最适催化温度均为50℃,最适催化pH值均为7.0;固定化酶4℃下储存28 d后酶活力可保留52.1%,具有较好的储存稳定性;重复使用5次后,仍能保持61.0%的初始活性,说明固定化酶构象较稳定,固定化马铃薯多酚氧化酶对苯酚具有较好的清除性能。     

多酚氧化酶的改性蛭石固定化及其催化特性研究

以马铃薯为原料,提取其中的多酚氧化酶,采用改性蛭石为载体,对其进行固定化,研究游离与固定化酶的最适催化温度、pH值,固定化酶的储存稳定性、重复使用稳定性及对苯酚的清除性能。结果表明,改性后蛭石出现了较多沟壑状孔隙,颗粒质感疏松,有利于固定化酶分子;最佳固定化条件为:改性蛭石含量0.4 g,戊二醛浓度2.0%,搅拌时间2 h,在此条件下,酶活力回收率可达到60.67%;游离与固定化酶的最适催化温度均为50℃,最适催化pH值均为7.0;固定化酶4℃下储存28 d后酶活力可保留52.1%,具有较好的储存稳定性;重复使用5次后,仍能保持61.0%的初始活性,说明固定化酶构象较稳定,固定化马铃薯多酚氧化酶对苯酚具有较好的清除性能。     

DEAE-D/H树脂固定化氨基酰化酶

选用6种弱碱性大孔树脂固定化氨基酰化酶,结果发现DEAE-D/H（二乙基氨基乙基–二乙烯基苯聚合物）树脂固定化效果较好,固定化酶活1 356.41 U/g。考察了不同酶液浓度下的DEAE–D/H固定化酶活收率。研究发现：酶液浓度115 U/mL时,酶活收率最高,为59.49%。具体分析了此固定化酶拆分乙酰–DL–蛋氨酸的最佳操作条件。结果表明,最佳pH值、温度和Co2+浓度分别为6.5、65℃和5×10－4　mol/L。同时考察了固定化酶的稳定性、再生性。固定化酶连续拆分乙酰-DL-蛋氨酸30天,酶活降为最初的72.6%。失活的固定化酶经洗脱、再生后,酶活达1380.64 U/g,达到新树脂固定化效果,证明载体可重复使用。     

大孔载体固定化脂肪酶

用自制大孔载体固定化脂肪酶,对固定化条件进行了优化,比较了固定化酶与游离酶的酶学参数. 结果表明,酶粉与载体质量比为1:1、固定化温度在20~25℃之间、固定化时间1.5 h的条件下,所得固定化酶的酶活最高. 固定化酶的最适pH为8.5,最适温度为40℃,其热稳定性、操作稳定性都比游离酶高,4℃下保存7 d后,酶活仍剩余94%.     

Immobilization of lipase on a polymeric microsphere with an epoxy group prepared by radiation‐induced polymerization

A polymeric microsphere (PM) with an epoxy group was prepared by the radiation‐induced polymerization of glycidyl methacrylate and diethylene glycol dimethacrylate in reaction conditions with variations in solvents, irradiation dose, and monomer composition. The epoxy group of the PM was analyzed by solid‐state ¹³C‐NMR, Fourier transform infrared spectroscopy (FTIR), Fourier transform Raman spectroscopy, and elemental analysis (EA) after amination. In EA after amination, the epoxy group content was in the range 0.20–0.50 mmol/g. The lipase was immobilized to the epoxy group of the PM in experimental conditions with variations in the pH and the epoxy group content. The activity of the lipase‐immobilized PM was in the range 148–342 unit/mg min. The activity of lipase‐immobilized PM increased in accordance with the epoxy group content. The lipase‐immobilized PM was also characterized by FTIR and EA. © 2003 Wiley Periodicals, Inc. J Appl Polym Sci 88: 1153–1161, 2003     

Immobilization of Pseudomonas cepacia lipase by sol-gel entrapment and its application in the hydrolysis of soybean oil

The immobilization of Lipase PS from Pseudomonas cepacia by entrapment within a chemically inert hydrophobic solgel support was studied. The gel-entrapped lipase was prepared by the hydrolysis of tetramethoxysilane (TMOS) with methyltrimethoxysilane (MTMS), isobutyltrimethoxysilane (iso-BTMS), and n-butyltrimethoxysilane. The immobilized lipase was subsequently used in the hydrolysis of soybean oil to determine its activity, recyclability, and thermostability. The biocatalyst so prepared was equal to or better than the free enzyme in its hydrolytic activity. The catalytic activity of the entrapped lipase strongly depended on the type of precursor that was used in its preparation. The lipase entrapped within TMOS/iso-BTMS showed the highest activity. The catalytic activity of the immobilized lipase was more pronounced during the earlier stages of the reaction. Thermostability of the lipase was significantly improved in the immobilized form. The immobilized lipase was stable up to 70°C, whereas for the free enzyme, moderate to severe loss of activity was observed beyond 40°C. The immobilized lipase was consistently more active and stable than the free enzyme. The immobilized lipase also proved to be very stable, as it retained more than 95% of its initial activity after twelve 1-h reactions.     

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.     

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.     

以凹土颗粒稳定的乳液为模板制备复合微球固定化酶

以凹土颗粒稳定的Pickering乳液为模板聚合有机/无机复合微球,并以此为载体固定化脂肪酶,当脂肪酶浓度为0.020wt％,固定化温度为45 ℃及pH=7.4的条件下,固定化效果较好,酶活达到最大.脂肪酶固定化后显示出较好的热稳定性、储存稳定性,重复使用三次后酶活仍与游离酶的初始酶活相近.从而为酶的固定化的提供了一条新的途径.     

Immobilization of <Emphasis Type="Italic">Candida rugosa</Emphasis> lipase by sol-gel entrapment and its application in the hydrolysis of soybean oil

Immobilization of lipase AY from Candida rugosa by entrapment within a chemically inert hydrophobic sol-gel support was studied. The gel-entrapped lipase was prepared by polycondensation of hydrolyzed tetramethoxysilane and isobutyltrimethoxysilane. Certain modifications were incorporated into the conventional immobilization procedure, including the use of glucose as additive and the application of vacuum during the drying and aging stages. The activity and thermostability of immobilized enzyme were subsequently determined in hydrolyzing soybean oil. Hydrolysis results showed more than 95 mol% of the theoretical yield for the formation of FF after 1 h of reaction at 40°C. The level of FFA was 3.3 times greater than that seen when an immobilized enzyme was prepared by the conventional sol-gel process. The immobilized enzyme retained most of its hydrolytic activity compared to the free enzyme and kept more than 95% activity after 120 h of incubation at 40°C, whereas the free enzyme lost 67% of its activity after 24 h of incubation and almost all of its activity after 96 h of incubation at 40°C. The immobilized enzyme also proved to be very stable, as it retained more than 90% of the initial activity after 16 one-hour reactions. Surface characterization studies suggested that the enzyme-containing sol-gel particles have amorphous morphology and are void of micro/meso pores.     

Synthesis of magnetically modified palygorskite composite for immobilization of Candida sp. 99–125 lipase via adsorption

Magnetically modified palygorskite composites were synthesized withγ-Fe2O3 dispersing on the external surface of clay mineral. The magnetic clay was characterized with Fourier transform infrared, X-ray diffrac-tion, transmission electron microscopy, and vibrating sample magnetometer. Candida sp. 99–125 lipase was immobilized on magnetic palygorskite composites by physical adsorption with enzyme loading of 41.5 mg·g-1 support and enzyme activity of 2631.6 U·(g support)-1. The immobilized lipase exhibit better thermal and broader pH stability and excellent reusability compared with free lipase.     

Immobilization of lipase on poly(N-vinyl-2-pyrrolidone-co-2-hydroxyethyl methacrylate) hydrogel for the synthesis of butyl oleate

Lipase from Candida rugosa was immobilized by entrapment on poly(N-vinyl-2-pyrrolidone-co-2-hydroxyethyl methacrylate) [poly(VP-co-HEMA)] hydrogel, cross-linked with ethylene glycol dimethacrylate (EDMA). The immobilized enzyme was used in the esterification of oleic acid with butanol in hexane. The activities of the immobilized enzyme preparations and the leaching of the enzyme from the hydrogel supports with respect to composition were investigated. The thermal, solvent, and storage stability of the immobilized preparations also were determined. Increasing the percentage VP from 0 to 90, which corresponds to the increase in the hydrophilicity of the hydrogels, increased the activity of the immobilized enzyme. Lipase immobilized onto VP(%):HEMA(%), 90:10 hydrogel had the highest activity. Increasing the hydrophobicity of the hydrogel (increasing the percentage HEMA) seemed to decrease leaching of the enzyme from the support. Immobilized lipase on 100% HEMA hydrogel indicated highest entrapment and lowest leaching by hexane washing. The lipase immobilized on VP(%):HEMA(%), 50:50 hydrogel showed highest thermal, solvent, and storage stability compared to lipase immobilized on other hydrogel compositions as well as the native lipase.