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。     

海藻糖对固定化酶的保护作用

研究了海藻糖对固定化纤维素酶在干燥和存放过程中的保护作用 ,发现海藻糖能有效地减少固定化酶干燥过程中酶的热失活 ,而且能提高固定化纤维素酶存放过程中的热稳定性 .但温度越高海藻糖的保护效果越差 .借助红外分析和差示扫描量热方法 ,初步推测了海藻糖对固定化纤维素酶保护的机理为 :一是糖的羟基同酶分子以氢键的形式结合 ,提高了酶的热变性温度 ;二是糖分子包裹在酶分子周围 ,或填充在酶分子的空间结构内 ,特别是酶的活性部位附近 ,并形成玻璃态 ,将酶蛋白的空间结构固定住而避免酶的失活 .     

磁性微球的制备及其固定化脂肪酶的研究

将Lipozyme CALB脂肪酶成功地固定于磁性纳米颗粒Fe3O4@SiO_2-p(NIPAM-co-GMA)的表面,研究了固定化过程中给酶量、温度、时间对固载量的影响。此外,稳定性试验表明:固定化脂肪酶的操作稳定性以及热稳定性都有提高。     

毕赤酵母表面展示脂肪酶催化合成肉豆蔻酸糖酯

研究了毕赤酵母表面展示脂肪酶(CALB)全细胞催化剂的较佳活性条件,结果显示,适宜的反应温度为50~60℃。以CALB为催化剂,比较了不同底物对糖酯合成的影响。以1,2-O-异亚丙基-D-呋喃葡萄糖(Ip Glc)为酰基受体,肉豆蔻酸为酰基供体,考察了有机溶剂种类、CALB的添加量、底物摩尔比、分子筛的添加量、初始水活度对合成6-O-肉豆蔻基-1,2-O-异亚丙基-α-D-呋喃型葡萄糖酯(Ip Glc-C14)的影响,得到较佳的合成条件为:丙酮5 m L、CALB(干粉)0.3 g、n(Ip Glc)∶n(肉豆蔻酸)=1∶3(其中Ip Glc 0.5 mmol)、4A分子筛0 g、初始水活度a_w=0.11、反应温度50℃、200 r/min反应72 h。在此条件下,Ip Glc-C14的收率为91.25%。比较了CALB和固定化脂肪酶Novozym 435对Ip Glc-C14合成的反应进程的影响,结果显示使用Novozym 435的反应速率快,而CALB的最终收率较高。     

溶胶-凝胶法固定化木瓜蛋白酶的活性保护

选择糖和脂质体作为溶胶-凝胶法固定木瓜蛋白酶(Papain)过程中的保护剂. 研究了蔗糖、葡萄糖、海藻糖、木糖、麦芽糖及构成脂质体的正癸烷溶液中胆固醇含量、卵/胆比等因素对固定化酶活性的影响. 结果表明,80 mL 20 mg/mL Papain溶液与15 mg木糖、500 mL正癸烷溶液[溶有1.5%(w)胆固醇,卵磷脂/胆固醇质量比为4.5:1]充分混合后制备的固定化酶活性最高. 在优化条件下制备的固定化酶包封率为42.0%,活力回收率为61.2%. SEM分析表明,固定化酶形态呈球状且大小均匀,内孔分布呈蜂窝状. 破膜剂对固定化酶活性发挥的影响研究表明,TritonX-100的效果最好.     

烷基侧链对Sol-gel固定化脂肪酶酯化活性的影响机理

以甲基三甲氧基硅烷(MTMS)、乙烯基三甲氧基硅烷(VIMOS)、乙烯基三乙氧基(VTEOS)、辛基三甲氧基硅烷(OTMOS)和四甲氧基硅烷(TMOS)为前趋体制备4种不同的固定化脂肪酶,并系统考察了烷基侧链对sol-gel固定化酶胶体结构和酶活性的影响.结果表明,随着烷基侧链的增长和数目的增加,固定化酶活力均逐渐增加,固定化脂肪酶颗粒平均孔径逐渐增大,孔体积逐渐增加,对底物的传质阻力逐渐降低;同时颗粒逐渐由球形变成不定形或团块状结构.脂肪酶活性的增加不仅来源于疏水性烷基侧链引起的脂肪酶的界面激活效应,同时固定化颗粒结构的改变了增加了底物和酶分子的结合,提高了固定化酶的表观活性.     

有机硅介质中脂肪酶印迹体系的优化及印迹机制的初步研究

采用溶胶-凝胶固定化工艺,以C6~C14之间的5种饱和脂肪酸和月桂醇为印迹分子,考察了碳链长度、印迹分子浓度对洋葱伯克霍尔德菌脂肪酶PS酯化活性的影响。研究表明,印迹分子的疏水性与底物分子的相似性对印迹效果影响较大,疏水性越强,与底物分子相似性越大,印迹效果越好;异辛烷洗脱印迹分子,对酶活的保持最好;随着碳链长度的增加,印迹酶对甲醇的耐受性逐渐降低;反应体系中加入微量水(10μL),对酯化酶活具有激活作用;经印迹的固定化酶使用5个批次后,相对酶活为0.65。对印迹机理的初步探讨表明,印迹分子对脂肪酶酯化活力的提高主要通过影响聚合物的结构来改变固定化酶的酶活,即分子印迹作用。     

以壳聚糖为载体固定化海藻糖合成酶

以壳聚糖为栽体,采用戊二醛为交联剂的方法来固定海藻糖合成酶。研究结果表明：在戊二醛质量分数为0．5％、液态酶与壳聚糖凝胶的配比为1：1、交联pH值为8．0、交联温度为15℃、交联时间为12h条件下,固定化海藻糖合成酶的活性最高,生成的海藻糖量最多,海藻糖的最高含量能达到40％左右。另外,固定化酶转化麦芽糖为海藻糖的最佳反应时间为18h,这时可以获得最高含量的海藻糖。     

功能化磁性纳米颗粒固定化脂肪酶研究

用葡萄糖酸对Fe3O4磁性纳米颗粒表面进行修饰,然后用水溶性碳化二亚胺（EDC）作偶联剂,对脂肪酶进行固定化。考察了偶联剂浓度、给酶量和反应时间对脂肪酶固定化过程的影响。结果表明,制备功能化磁性颗粒固定化酶的最佳条件为：偶联剂浓度为12．5mg／mL磷酸缓冲液（PBS）,给酶量为2．5mg／mLPBS,反应时间为24h。固定化脂肪酶表现出优异的热稳定性,60℃时酶活为游离酶的6倍。重复使用10次后,酶促活力依然保持80％以上。     

吸附-聚合物修饰组合固定化Candida antarctica脂肪酶研究

通过吸附法联合PEG非共价修饰,研发了一种固定化南极假丝酵母脂肪酶(Candida antarctica lipase)的新方法,可以有效提高固定化酶在非水介质中的催化活性。最佳固定化条件为硅藻土:酶粉(W/W)=8,PEG4000:酶粉(W/W)=0.6,缓冲液pH7.5。采用三油酸甘油酯与甲醇的转酯化反应,测定了固定化酶的转酯活性。结果表明,固定化酶同时加入PEG进行非共价修饰,可显著提高固定化酶的转酯活力。PEG修饰的固定化酶转酯比活是未经PEG修饰的固定化酶的4.1倍,转酯酶活回收率为604.8%,说明PEG两性分子的特性对制备用于非水介质的固定化酶有重要作用。该固定化方法可显著提高Candida antarctica脂肪酶在非水介质中的催化效率,且固定化方法简单、成本低,具有工业应用价值。     

Thermal Stability of Immobilized Lipase from Candida antarctica in Glycerols with Various Water Contents at Elevated Temperatures

The immobilized lipase from Candida antarctica (fraction B, CALB) was incubated in glycerols with various water contents at 80–100 °C to measure the residual activity as a function of time. The glycerol-containing water stabilized the immobilized CALB, especially at 30–60 wt% water contents. The thermal inactivation behaviors of the immobilized CALB were expressed by a model in which the free energy of activation for the inactivation of the immobilized lipase molecules obeyed a Gaussian distribution.     

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.     

固定化酶催化制备乙酸正丁酯及动力学

酯交换反应可用南极假丝酵母脂肪酶B（CALB）作催化剂,采用溶胶-凝胶法固定脂肪酶CALB得到的催化剂颗粒可用于乙酸乙酯和正丁醇的酯交换反应。首先探究了固定化酶的稳定性和重复使用性,然后在间歇反应釜内进行反应动力学实验,考察了转速、催化剂用量、酯醇比、温度等因素对反应的影响,确定了适宜的操作条件。在328.15～343.15 K下,将实验数据拟合得到反应的动力学方程,通过实验值与计算值的比较,验证此宏观动力学方程合理,可用于模拟计算。     

Lipase-Catalyzed Production of Biodiesel by Hydrolysis of Waste Cooking Oil Followed by Esterification of Free Fatty Acids

Biodiesel is conventionally produced by alkaline‐catalyzed transesterification, which requires high‐purity oils. However, low‐quality oils can be used as feedstocks for the production of biodiesel by enzyme‐catalyzed reactions. The use of enzymes has several advantages, such as the absence of saponification side reactions, production of high‐purity glycerol co‐product, and low‐cost downstream processing. In this work, biodiesel was produced from lipase‐catalyzed hydrolysis of waste cooking oil (WCO) followed by esterification of the hydrolyzed WCO (HWCO). The hydrolysis of acylglycerols was carried out at 30 °C in salt‐free water (WCO/water ratio of 1:4, v/v) and the esterification of HWCO was carried out at 40 °C with ethanol in a solvent‐free medium (HWCO/ethanol molar ratio of 1:7). The hydrolysis and esterification steps were carried out using immobilized Thermomyces lanuginosus lipase (TLL/WCO ratio of 1:5.6, w/w) and immobilized Candida antarctica lipase B (10 wt%, CALB/HWCO) as biocatalysts, respectively. The hydrolysis of acylglycerols was almost complete after 12 h (ca. 94 %), and in the esterification step, the conversion was around 90 % after 6 h. The purified biodiesel had 91.8 wt% of fatty acid ethyl esters, 0.53 wt% of acylglycerols, 0.003 wt% of free glycerol, viscosity of 4.59 cP, and acid value of 10.88 mg KOH/g. Reuse hydrolysis and esterification assays showed that the immobilized enzymes could be recycled five times in 10‐h batches, under the conditions described above. TLL was greatly inactivated under the assay conditions, whereas CALB remained fully active. The results showed that WCO is a promising feedstock for use in the production of biodiesel.     

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.     

Selective Ethanolysis of Fish Oil Catalyzed by Immobilized Lipases

Selective ethanolysis of fish oil was catalyzed by immobilized lipases and their derivatives in organic media. Lipases from Candida antarctica B (CALB), Thermomyces lanuginosa (TLL) and Rhizomucor miehei (RML) were studied. The three lipases were immobilized by anion exchange and hydrophobic adsorption. The discrimination between the ethyl ester of eicosapentaenoic acid (EE-EPA) and the ethyl ester of docosahexaenoic acid (EE-DHA) depends on the lipase, the immobilization support, the physico-chemical modifications of the immobilized lipase derivatives and on the solvents used. TLL and RML were much more selective than CALB. EE-EPA is released 20-fold faster than EE-DHA when ethanolysis was catalyzed, in cyclohexane, by TLL hydrophobically adsorbed on Sepabeads C18. The selectivity and stability of the different derivatives in these polar organic solvents were further improved after physico-chemical modification. The best results for activity-selectivity-stability were obtained in cyclohexane for TLL adsorbed on Sepabeads C18 and further modified via solid-phase physical modification with a polyethylenimine polymer. In this case, the initial selectivity was higher than 20, and a 80 % of EPA was released as ethyl ester after 3 h at 25 °C. At this conversion, mixtures of ethyl esters highly enriched in the ethyl ester of EPA with less than 5 % of the EE-DHA were obtained. TLL derivatives remained fully active after incubation for 24 h in anhydrous solvents.     

Use of polyelectrolyte complex for immobilization of microorganisms

Cells from Escherichia coli (IAM 12119) were immobilized with the polyion complex of trimethylammonium glycol chitosan iodide (TGCI) and potassium poly(vinyl alcohol) sulfate (KPVS). The immobilization was carried out at pH 8 by mixing TGCI with the cell suspension, followed by addition of KPVS. The immobilized cells were characterized by investigating the glucose oxidizing activity. The results obtained indicated that the glucose consumption with immobilized cells is due not only to the cells released from the complex support but also to the entrapped cells which are grown in the complex; therefore, the cells entrapped in the complex have the glucose oxidizing activity. The physicochemical studies on the immobilization mechanism showed that cells are immobilized via two stages: the aggregation of cells with TGCI and the entrapment of the aggregates in the TGCI–KPVS complex. In the aggregation process, a part of TGCI which is added to cell suspension adsorbs on the cells and the other remains in the suspension. In the entrapment process, the remainder forms the polyion complex with KPVS added and the aggregated cells are coprecipitated with the complex.     

Lipase catalyzed synthesis of polycaprolactone and clay-based nanohybrids

This study presents the use of original systems based on Candida antarctica lipase B (CALB) immobilized on montmorillonite and sepiolite nanoclays as efficient catalysts for the enzymatic polymerization of ε-caprolactone (ε-CL) and the in situ elaboration of nanohybrids.