表面活性剂改性磁性氧化石墨烯共价固定脂肪酶CRL

采用化学共沉淀法制备磁性氧化石墨烯（MGO）,直接在反应体系中加入Span系列表面活性剂,一锅法制备得到表面活性剂改性磁性氧化石墨烯（SMGO）。X射线衍射仪（XRD）、扫描电镜（SEM）和傅里叶变换红外光谱（FTIR）表征结果表明SMGO制备成功,且具有良好的磁分离性能。以1,5-戊二醛（GA）为交联剂,褶皱假丝酵母脂肪酶（CRL）为模型酶,共价固定CRL于SMGO载体上。Span40 MGO固定化酶酶活回收率为116.5%±1.7%,为MGO固定化酶的6倍;比活可达32.5U/mg,为游离酶的1.6倍;k_cat/K_m也有较大的提升,高于游离酶50%。储存稳定性及热稳定性得到提高,用于水解反应6批次后仍然保留73.6%的相对酶活力。初步分析认为MGO经改性后表面从亲水性转为强疏水性,使得共价固定化过程中同时发生疏水性界面活化,这是酶活性提高的原因之一。文章所报道的改性策略可为类似载体改性提供新思路。     

Immobilization and stabilization of <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> SRT9 lipase on tri(4-formyl phenoxy) cyanurate

Lipase was extracted and purified from Pseudomonas aeruginosa SRT9. Culture conditions were optimized and highest lipase production amounting to 147.36 U/ml was obtained after 20 h incubation. The extracellular lipase was purified on Mono QHR5/5 column, resulting in a purification factor of 98-fold with specific activity of 12307.81 U/mg. Lipase was immobilized on tri (4-formyl phenoxy) cyanurate to form Schiff’s base. An immobilization yield of 85% was obtained. The native and immobilized lipases were used for catalyzing the hydrolysis of olive oil in aqueous medium. Comparative study revealed that immobilized lipase exhibited a shift in optimal pH from 6.9 (free lipase) to 7.5 and shift in optimal temperature from 55 °C to 70 °C. The immobilized lipase showed 20–25% increase in thermal stability and retained 75% of its initial activity after 7 cycles. It showed good stability in organic solvents especially in 30% acetone and methanol. Enzyme activity was decreased by ∼60% when incubated with 30% butanol. The kinetic studies revealed increase in K _M value from 0.043 mM (native) to 0.10 mM for immobilized lipase. It showed decrease in the V _max of immobilized enzyme (142.8 μmol min⁻¹ mg⁻¹), suggesting enzyme activity decrease in the course of covalent binding. The immobilized lipase retained its initial activity for more than 30 days when stored at 4 °C in Tris-HCl buffer pH 7.0 without any significant loss in enzyme activity.     

核壳结构磁性树枝状纤维形有机硅固定化脂肪酶制备及其应用

为了提升脂肪酶的稳定性并构建新型固定化酶催化体系,利用改进的Winsor Ⅲ微乳液双连续相体系合成了超顺磁性Fe₃O₄内核和树枝状纤维形氧化硅外壳的核壳结构磁性有机硅纳米粒子（MMOSNs）,用于固定化南极假丝酵母脂肪酶B（CALB）。优化条件后CALB负载量为177.49 mg/g,比水解活性为27390 U/g。磁性有机硅通过与CLAB分子之间疏水相互作用及表面孔道结构,可有效激活CALB的界面活性并保护活性构象免受破坏,比游离酶和磁性无机硅固定化酶表现出更好的活性和稳定性。除此之外,将CALB@MMOSNs用于催化乙酰丙酸与十二醇的酯化反应最高转化率为85.05%,重复使用9次后仍保留68.94%转化率,而商业化N435只保留29.83%。证明疏水性磁性核壳结构有机硅是固定化CALB的良好载体,可有效扩展脂肪酶的工业应用。     

Microwave‐assisted immobilization of lipase from Candida rugosa on Eupergit® supports

BACKGROUND: Immobilization of lipase (triacylglycerol acylhydrolase EC 3.1.1.3) from Candida rugosa on Eupergit^® C and Eupergit^® C 250L was performed under microwave irradiation in order to reduce immobilization time. Lipase loading, hydrolytic activity, esterification activity and operational stability in organic solvent of immobilized lipase preparation were determined. RESULTS: The microwave‐assisted procedure resulted in a 29% lower lipase loadings, compared with immobilized lipase obtained without microwaves. In hydrolytic activity assay, lipase immobilized under microwaves exhibited a 23% higher specific activity. Slight activation of lipase by microwave‐assisted immobilization was observed, since specific activity was around 5% higher than for free lipase. Lipase of highest activity was obtained after 2 min immobilization on Eupergit^® C. The same preparation exhibited high esterification activity in organic medium and a half life of 212 h was determined in multiple use assay. CONCLUSION: The application of microwave irradiation leads to reduction of immobilization time from 2 days to only 2 min. The immobilized lipase obtained has prospects for further application due to its high retained activity and stability. Copyright © 2009 Society of Chemical Industry     

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.     

Synthesis of geranyl acetate by esterification with lipase entrapped in hybrid sol-gel formed within nonwoven fabric

Candida cylindracea lipase was entrapped in organic-inorganic hybrid sol-gel polymers made from tetramethoxysilane (TMOS) and alkyltrimethoxysilanes. By forming the gels within the pores of a nonwoven polyester fabric, a novel immobilized biocatalyst in sheet configuration based on sol-gel en-trapment of the enzyme was obtained. Lipases immobilized in sol-gel matrices efficiently catalyzed the direct esterification reaction of geraniol and acetic acid in anhydrous hexane to produce geranyl acetate. The optimal formulation of the sol-gel solution for enzyme immobilization was at a 20∶1 molar ratio of water to total silane; a 4∶1 molar ratio of propyltrimethoxysilane to TMOS; hydrolysis time at 30 min; and enzyme loading of 200 mg lipase/g gel. Under these conditions, protein immobilization efficiency was 91%, and the specific activity of the immobilized enzyme was 2.6 times that of the free enzyme. Excellent thermal stability was found for the immobilized enzyme in dry form or in hexane solution in the presence of acetic acid, in which case severe inactivation of free enzyme was observed. The immobilized enzyme retained its activity after heating at 70°C for 2 h, whereas the free enzyme lost 80% of its activity.     

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.     

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.     

Lipase immobilization on glutaraldehyde-activated nanofibrous membranes for improved enzyme stabilities and activities

Poly(vinyl alcohol-co-ethylene) (PVA-co-PE) nanofibrous membranes were successfully fabricated and activated with glutaraldehyde (GA) to interact with enzyme molecules. A lipase isolated from Candida rugosa was employed as a model biocatalyst and successfully immobilized onto the membrane surfaces via covalent bonds with the aldehyde groups. Scanning electron microscopy images revealed that the membranes retained uniform nanofibrous and open porous structures after the treatments. The results indicated that the increment of the initial glutaraldehyde concentration induced an increase of the enzyme loading on the membrane surfaces but a decrease in the activity of the immobilized enzyme. Under an optimum condition, the glutaraldehyde activated PVA-co-PE nanofibrous membrane reached the highest enzyme activity at 676.19 U/g of the membrane. The pH tolerance, thermal and storage stability of the immobilized lipase were significantly improved. In addition, the immobilized lipase can be easily recovered and retained at 67% of its initial activity after 10 time uses. Therefore, the glutaraldehyde activated PVA-co-PE nanofibrous membrane is a promising solid support media for enzyme immobilization, and the immobilized enzymes could have broad biocatalytic applications.     

Zwitterionic polymer-coated porous poly(vinyl acetate–divinyl benzene)microsphere: A new support for enhanced performance of immobilized lipase

Enzyme immobilization has attracted great attention for improving the performance of enzymes in industrial applications. This work was designed to create a new support for Candida rugosa lipase(CRL) immobilization.A porous poly(vinyl acetate–divinyl benzene) microsphere coated by a zwitterionic polymer, poly(maleic anhydride-alt-1-octadecene) and N,N-dimethylethylenediamine derivative, was developed for CRL immobilization via hydrophobic binding. The catalytic activity, reaction kinetics, stabilities and reusability of the immobilized CRL were investigated. It demonstrated the success of the zwitterionic polymer coating and subsequent CRL immobilization on the porous microsphere. The immobilized lipase(p2-MS-CRL) reached27.6 mg·g~(-1) dry carrier and displayed a specific activity 1.5 times higher than free CRL. The increase of Vmax and decrease of Kmwere also observed, indicating the improvement of catalytic activity and enzyme-substrate affinity of the immobilized lipase. Besides, p2-MS-CRL exhibited significantly enhanced thermal stability and p H tolerance. The improved performance was considered due to the interfacial activation regulated by the hydrophobic interaction and stabilization effect arisen by the zwitterionic polymer coating. This study has thus proved the advantages of the zwitterionic polymer-coated porous carrier for lipase immobilization and its potential for further development in various enzyme immobilizations.     

Polyaniline‐functionalized magnetic mesoporous nanocomposite: A smart material for the immobilization of lipase

Magnetically separable mesoporous silica nanocomposites with polyoaniline functionalization (Pani‐MS@Fe₃O₄) were synthesized for the immobilization of lipase via electrostatic adsorption. The as‐prepared Pani‐MS@Fe₃O₄ nanocomposites as well as immobilized lipase were characterized by FTIR, XRD, HRTEM, FESEM, BET, and TGA techniques. The BET surface area was calculated to be 779.27 m²/g, 425 m²/g, and 230.45 m²/g for magnetic mesoporous nanoparticle (MS@Fe₃O₄), Pani‐MS@Fe₃O₄ nanocomposite, and lipase immobilized Pani‐MS@Fe₃O₄ nanocomposite respectively. The comparison experiments verified that the immobilized lipase exhibited slightly higher optimal pH and temperature value with a wider pH‐activity and temperature stability in comparison with the free lipase. From Michaelis–Menten kinetic study, the lower K_m value (0.25 mM) and higher V_max value (0.0341 mM/min) for the immobilized lipase revealed the higher affinity of immobilized lipase toward the substrate. Further, reusability studies of the immobilized lipase indicated that up to 70% of the original activity was retained after having been recycled seven times. POLYM. COMPOS. 37:1152–1160, 2016. © 2014 Society of Plastics Engineers     

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%。     

Preparation of a promising whole cell biocatalyst of Geotrichum sp. lipase and its properties

BACKGROUND: As a new protein expression and self‐immobilization system, cell‐surface displayed enzymes have attracted increasing attention. In this study, Geotrichum sp. lipase (GSL), an important enzyme for the enrichment of polyunsaturated fatty acids (PUFAs), was first displayed on the cell surface of Saccharomyces cerevisiae. RESULTS: The activity of displayed GSL was higher (43.7 U g^?1 dry cell) than that of Candida antarctica lipase B (26.26 U g^?1 dry cell) and that of Rhizopus oryzae lipase (4.1 U g^?1 dry cell). It also exhibited higher thermostability than the free lipase, and retained 89% of the original activity after incubation at 40 °C for 3 h, compared with 48% at 35 °C for the free lipase at pH 8.5. Interestingly, the displayed lipase had a wider pH range and better pH stability. It had higher activity at all pH values than the free GSL, and retained 86% of the original activity in the pH range 9.5 to 10.5, whereas the activity of the free GSL could not be detected at pH 10. CONCLUSION: This work presented a method to prepare a whole‐cell biocatalyst with better stability and broader pH tolerance which will provide a useful strategy for other cost‐effective self‐immobilized industrial lipases. Copyright © 2011 Society of Chemical Industry     

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%.     

溶胶-凝胶法固定化Candida sp.99-125脂肪酶

利用正硅酸甲酯(TMOS)和丙基三甲氧基硅烷(PTMS)为复合硅源,以PEG(MW=20000)为稳定剂,以HCl为催化剂,经过溶胶-凝胶过程包埋假丝酵母99-125脂肪酶. 研究得到最适的固定化条件为：PTMS与TMOS的摩尔比4: 1, R值(水与硅源的摩尔比)20, 给酶量(酶占硅源的质量百分数)3.71%, PEG与酶的质量比(1~1.5):1, 硅源水解时间35 min. 在该条件下,固定化脂肪酶的最高酯化活力是游离酶最高酯化活力的2.02倍. 固定化脂肪酶在100℃保温2 h后酶活仍维持为59.1%,固定化酶催化特定酯化反应,经过8批连续反应96 h后酶活维持不变.     

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 Candida rugosa lipase on modified natural wool fibers

A method has been developed to immobilize lipase from Candida rugosa on modified natural wool fibers by means of graft copolymerization of poly ethylacrylate in presence of potassium persulphate and Mohr’s salt redox initiator. The activities of free and immobilized lipase have been studied. FTIR spectroscopy, scanning electron microscopy, and the Bradford method were used to characterize lipase immobilization. The efficiency of the immobilization was evaluated by examining the relative enzymatic activity of free enzyme before and after the immobilization of lipase. The results showed that the optimum temperature of immobilized lipase was 40 °C, which was identical to that of the free enzyme, and the immobilized lipase exhibited a higher relative activity than that of free lipase over 40 °C. The optimal pH for immobilized lipase was 8.0, which was higher than that of the free lipase (pH 7.5), and the immobilization resulted in stabilization of enzyme over a broader pH range. The kinetic constant value (km) of immobilized lipase was higher than that of the free lipase. However, the thermal and operational stabilities of immobilized lipase have been improved greatly.     

Characterization of tributyrin hydrolysis by immobilized lipase on woolen cloth using conventional batch and novel spinning cloth disc reactors

Optimal loading and operating conditions for a new, superior immobilization of amano lipase from Pseudomonas fluorescens on woolen cloth were determined. The optimal enzyme loading was 46.8 mg g dry cloth⁻¹ with activity of 200 U. A batch reactor was used to characterize process conditions important to industrial application of the wool immobilized lipase. The optimal pH for immobilized lipase in tributyrin hydrolysis was 7, slightly lower than that of free lipase (pH 8). The optimal temperature for both free and immobilized lipase was 45 °C. The immobilized lipase was more stable to reuse than some other lipase immobilizations, maintaining 85% of its activity after 6 long term runs and 75.8% of the original activity after storage of 40 weeks at 4 °C. The thermal stability of lipase was improved by 2.4 times after immobilization. The thermal deactivation rate of immobilized lipase followed the Arrhenius law with E_d = 199 kJ mol⁻¹. The Michaelis–Menten constant (K_m) of the lipase increased from 1.63 mM to 4.48 mM after immobilization. The immobilized lipase was also successfully applied for tributyrin hydrolysis in a novel enzyme process intensification technology – the spinning cloth disc reactor (SCDR): conversion increased by around 13% under similar conditions compared to a conventional batch stirred tank reactor. The SCDR is therefore key to exploiting the advantages of the wool immobilized lipase developed in this work.     

Spouted Bed Drying as a Method for Enzyme Immobilization

Usually immobilization is a requirement for the use of enzymes as an industrial biocatalyst. In this work, endophytic fungus Cercospora kikuchii lipase was immobilized by covalent binding on agricultural by-products and microcrystalline cellulose. The enzyme support system was submitted to spouted bed drying. Lipase immobilized on microcrystalline cellulose with 1.5% of glutaraldehyde showed the best results, presenting 179.1% of the original activity after drying, followed by rice husk (173.9%), corn stover (169.8%), sugarcane bagasse (157.3%), green coconut fiber (102.3%), and corncob (99.8%). The immobilized derivatives obtained showed a decreased enzyme activity with an average of only 17.31%, whereas the enzyme in its free form lost 85.8% of its initial activity after storage for 6 months. The operational stability showed that the biocatalysts prepared retained an average of 67.2% of the initial activity after five reuse cycles. The results showed that the use of agricultural by-products as low-cost support material associated with the spouted bed drying is promising and can contribute to industrial application of biocatalysts.     

仿生合成氧化锆固定化漆酶处理孔雀石绿

以溶菌酶作为诱导剂,仿生合成了ZrO₂固定化漆酶纳米颗粒,其酶活回收率达59%,采用场发射扫描电子显微镜（FESEM）、能谱仪（EDS）、热重分析仪（TGA）等手段对ZrO₂纳米颗粒及ZrO₂固定化漆酶颗粒进行表征,结果表明漆酶可成功固定到ZrO₂颗粒中,同时还证明了溶菌酶既作为诱导剂催化ZrO₂的形成,又作为生物模板同酶一起包埋在ZrO₂颗粒中。固定化漆酶的最适pH为3,最适温度为70℃,相比于游离酶,其pH、温度稳定性都有明显提高;固定化漆酶纳米颗粒在4℃下储存30d,活性为初始酶活的95%,重复使用5次,固定化酶的残余酶活力仍有60%。此外,固定化漆酶在6h内对孔雀石绿染料的脱色率高达95%以上,通过紫外-可见吸收光谱分析（UV-vis）可知,固定化漆酶对孔雀石绿染料的处理是由吸附和降解联合作用引起的脱色。