吸附法固定化脂肪酶研究进展简

吸附法固定化酶优点众多,特别是操作简单,载体种类丰富使其在固定化技术上占有一席之地.目前吸附法固定化脂肪酶已经得到广泛应用.介绍了吸附法固定化脂肪酶的研究现状,并对该技术的发展瓶颈及趋势进行了总结.     

膜材料的亲疏水性对固定化脂肪酶的影响

用吸附法固定脂肪酶时,膜材料的亲疏水性对固定化酶的量、比活力和活力稳定性等有很大影响.今以柱状假丝酵母脂肪酶和猪胰脂肪酶为研究对象,选取了8种亲疏水性不同的膜材料(醋酸纤维素、聚丙烯腈、聚酰胺、聚砜、聚醚砜、聚偏二氟乙烯、聚丙烯和聚四氟乙烯)作为固定化载体,用吸附法制备了固定化脂肪酶膜.研究结果表明,强疏水性聚四氟乙烯和聚丙烯膜对两种酶的吸附量都比较大,且固定化酶的比活力和活力回收率比较高,聚四氟乙烯固定化柱状假丝酵母酶比游离态酶的半衰期提高了6倍以上.强亲水性醋酸纤维素膜对猪胰脂肪酶的吸附量比聚四氟乙烯高,但是固定化酶的比活力、活力回收率比强疏水性膜低,而接触角在40°～50°的聚酰胺膜和聚砜膜的吸附量最小.因此吸附法制备固定化脂肪酶膜,选择聚丙烯膜和聚四氟乙烯膜是合适的,制备的优化条件为吸附温度25℃,酶溶液的pH为7.5,吸附时间10 h.     

丙酮中麦芽糖月桂酸单酯的酶法选择性合成

为选择性地合成麦芽糖月桂酸单酯,该文研究了脂肪酶催化合成条件对麦芽糖月桂酸酯浓度和产率的影响,确定了麦芽糖月桂酸单酯的最佳合成参数。当c(麦芽糖)=25 mmol/L、c(月桂酸)=75 mmol/L、ρ(脂肪酶)=20 g/L、ρ(3A分子筛)=60 g/L时,在丙酮中50℃反应72 h,麦芽糖月桂酸单酯的产率和浓度分别可达93%和23.35 mmol/L。     

树脂吸附法固定Candida rugosa脂肪酶

Candida rugosa脂肪酶具有优良的催化性能,对其进行固定化可以很方便地实现酶的回收和再利用。采用南开大学化工厂生产的4种阴离子交换树脂和4种大孔吸附树脂为载体,对来源于Candida rugosa的脂肪酶进行了吸附固定化,结果表明,以大孔吸附树脂AB-8为载体的固定酶比活性最高。固定化酶制备过程中缓冲液的最适宜pH值为7.2,最佳固定化时间为1 h,载体和酶的最佳质量配比为10∶1。与游离酶相比,固定化后酶活损失大约30%,但稳定性平均约提高60%。     

表面活性剂包衣的固定化脂肪酶在有机介质中催化酯化反应

以大孔吸附树脂为载体,将来源于Candidarugosa的脂肪酶用吸附法固定化后用谷氨酸双十二烷基酯核糖醇进行包衣,用于在有机溶剂中催化月桂酸和月桂醇的酯化反应。结果表明,AB 8型大孔吸附树脂为最佳载体,最适有机溶剂为异辛烷,酶固定化后再包衣,酶的比活较未包衣前提高了60%—90%。     

脂肪酶固定化及催化酯化反应

本研究报道了利用吸附法和化学键合法将白地霉脂肪酶固定化于五种载体上，用吸附法固定化的脂肪酶保持了高的活性。在有机溶剂中，固定化酶催化了长链脂肪酸与醇的酯化反应，酯化率可达９４％。固定化过程并未改变脂肪酶的选择性。固定化脂肪酶可重复使用。     

脂肪酶催化合成D-异抗坏血酸月桂酸酯

用固定化脂肪酶Lipozyme 435作催化剂,由D-异抗坏血酸和月桂酸合成D-异抗坏血酸月桂酸酯。考察了反应温度、反应溶剂、反应物配比、酶用量、反应时间、月桂酸浓度、分子筛用量、摇床转速对产物收率的影响。结果表明,以20mL叔戊醇为反应溶剂、0.10g脂肪酶为催化剂、2.0g 4A分子筛为脱水剂,在55℃、170r/min转速的恒温摇床中,0.88g D-异抗坏血酸和2.50g月桂酸反应36h,产物收率68%。产物结构经IR、1H NMR、13C NMR和MS表征。     

基于酶催化合成单甘油酯工艺优化及抑菌活性研究

以月桂酸和甘油为原料，叔丁醇为介质，采用脂肪酶催化合成了月桂酸单甘油酯（GML），采用单因素试验验证了GML的较佳合成工艺，并研究了GML对大肠杆菌的抑制效果，结果表明，叔丁醇与甘油的质量比为1.5∶1，脂肪酶用量为12%，反应温度70℃，甘油和月桂酸的摩尔比为3∶1，反应时间为1.5 h为较优条件。抑菌测试表明GML对大肠杆菌有着显著的抑制作用，抑菌效果随GML质量浓度增大而增强，GML质量浓度达到16 mg/mL时抑菌圈直径不再增大，在pH=5.7的弱酸环境下的抑菌效果和抑菌率的半衰期均强于pH=7.2的中性条件，72 h时pH=5.7的环境下GML对大肠杆菌的抑菌率依旧能够保持在3.9%。     

阳离子聚合物/膨润土纳米复合吸附材料的性能及对红色染料的脱色

利用阳离子聚合物-聚环氧氯丙烷二甲胺为插层剂,采用 “有机高聚物溶液直接插层复合法”制备了一系列阳离子聚合物聚环氧氯丙烷二甲胺/膨润土纳米复合吸附材料,经特性分析表明,经聚环氧氯丙烷二甲胺插层复合后的膨润土比表面积显著增加,表面电性由负值变为正值,颗粒的聚集程度增加,有较好的吸附和沉降性能.在此基础上,研究了还原大红R、分散大红S-R、活性艳红K-2BP等红色染料在复合膨润土上的吸附行为.结果表明,聚环氧氯丙烷二甲胺/膨润土对三种染料的吸附脱色能力明显大于钠基膨润土,平衡吸附量q_e与平衡浓度C_e之间的关系均符合Langmuir和Freundlich等温吸附模型,吸附动力学行为遵循Langmuir方程所述规律.     

新型合成聚合物重金属离子吸附剂及其吸附性能

阐述了新型聚合物重金属离子吸附剂的合成与改性及其对水溶液中Cd(Ⅱ)、Pb(Ⅱ)、Cu(Ⅱ)、Hg(Ⅱ)等重金属离子的吸附性能。这些聚合物重金属离子吸附剂对低浓度的重金属离子表现出了较好的吸附能力，其中聚合物吸附剂PGHyFeO—COOH对mg／L级的Pb(Ⅱ)、Cd(Ⅱ)、Hg(Ⅱ)的最大吸附量分别为211．4mg／g、155．0mg／g、147．2mg／g。这些吸附剂的吸附速度也较快，平衡吸附时间一般在60min左右，有的甚至只有几分钟。新型聚合物重金属离子吸附剂在水处理工业中具有广阔的应用前景。     

Transesterification of soy lecithin by lipase and phospholipase

Soy lecithin was modified by enzymatic transesterification in a solvent-free system. 1,3-SpecificRhizomucor miehei lipase was found to be efficient in the transesterification with lauric acid and oleic acid, where oleic acid was more incorporated into soy lecithin. Phospholipase A₂ incorporated lauric acid hardly at all, but it hydrolyzed lecithin efficiently. The mixture of lipase and phospholipase A₂ (1:1, w/w) incorporated lauric acid to the same extent as did 1,3-specific lipase alone at the same total enzyme concentration. The main fatty acids replaced were palmitic and linoleic acids by 1,3-specific lipase and its mixture with phospholipase A₂, and linoleic and linolenic acids by phospholipase A₂ alone, suggesting an improved oxidative stability of the resulting product. Hydrolysis could not be prevented, but it could be regulated by incubation time and by enzyme dosage. The minimal water content for significant incorporation of lauric acid into lecithin was below 0.5% of the weight of the reaction mixture.     

无溶剂体系中共吸附固定化脂肪酶催化合成植物甾醇酯

以大孔树脂NKA吸附固定化脂肪酶为催化剂,以月桂酸和植物甾醇为反应物,在无溶剂体系中合成植物甾醇酯;以NKA为载体共吸附固定葡聚糖与酶,考察了反应温度、底物摩尔比、糖种类及葡聚糖添加量和反应时间对酶催化性能的影响. 结果表明,在酸/醇摩尔比6:1、葡聚糖与酶共同吸附固定时分子量为10 kDa的葡聚糖添加量为酶量7.5%的条件下,60℃下反应12 h,酯化率高达96.2%,酯化反应时间比简单吸附固定化酶缩短4 h.     

Immobilization of hydrophobic lipase derivatives on to organic polymer beads

A simple and effective method of lipase immobilization is described. Lipase from Candida rugosa was first modified with several hydrophobic modifiers before being adsorbed on to organic polymer beads. The soluble hydrophobic lipase derivatives adsorbed more strongly on to the various polymers as compared with the native lipase. The optimal adsorption temperature of the native and modified lipases on all the polymers was 40°C. The optimal pH of adsorption was between 6 and 7. Lipase immobilized in this manner produced high catalytic recoveries which were affected by the type of modifiers, degree of modification and type of supports used. Monomethoxypolyethylene glycol (1900) activated with p-nitrophenyl chloroformate was found to be the best modifier of the enzyme at 95% modification, for adsorption to the polymers. Increasing the degree of modification of the enzyme increased the activity which was immobilized. Generally, both native and hydrophobic lipase derivatives showed higher specific activities when immobilized on polar polymers compared with non-polar polymers.     

Enzymatic esterification of (−)-menthol with lauric acid in isooctane by sorbitan monostearate-coated lipase from Candida rugosa

Esterification of (−)-menthol and (±)-menthol with lauric acid in isooctane was successfully catalyzed by a commercial nonioic surfactant (sorbitan monosterate)-coated lipase from Candida rugosa (Lipase AY “Amano” 30) at the molar ratio of 1∶1 and at 35°C using 1.5 g enzyme/g (−)-menthol and 0.1-g molecular sieves. After 1 h, molar conversion of (−)-menthol reached 81%. Equilibrium was reached after ca. 4 h, giving a (−)-menthol molar conversion of 94%. Under the same conditions, native lipase catalyzed the esterification of (−)-menthol and lauric acid to yield a molar conversion of 93% after 72 h. Coating the lipase with sorbitan monosterate increased the esterification rates of both (−)-menthol and (±)-menthol with lauric acid. After 6 h, the molar conversions of (−)-menthol and (±)-menthol were 94, and 62%, respectively.     

Lymphatic fatty acids from rats fed human milk and formula containing coconut oil

Human milk and infant formula containing coconut/soy oil were infused into the duodenum of rats to determine the incorporation of capric, lauric, myristic and palmitic acids into lymphatic triacylglycerol (TAG). The proportion of capric and lauric acids in the lymphatic TAG reflected the fatty acid composition of the diet. Based on positional analysis, it appears that more than 50% of the capric and lauric acids could have been absorbed from the intestine assn-2 monoacylglycerols. In the rats fed human milk, 50% of palmitic acid in lymphatic TAG was in thesn-2 position. Because of the nonrandom distribution of palmitic acid in the lymphatic TAG, the nonspecific lipase in human milk, i.e., bile salt-stimulated lipase, did not appear to be a factor in milk lipid digestion.     

Modification of butterfat by selective hydrolysis and interesterification by lipase: Process and product characterization

Butterfat was chemically modified via combined hydrolysis and interesterification, catalyzed by a commercial lipase immobilized onto a bundle of hydrophobic hollow fibers. The main goal of this research effort was to engineer butterfat with improved nutritional properties by taking advantage of the sn-1,3 specificity and fatty acid specificity of a lipase in hydrolysis and ester interchange reactions, and concomitantly decrease its level of long-chain saturated fatty acid residues (viz., lauric, myristic, and palmitic acids) and change its melting properties. All reactions were carried out at 40°C in a solvent-free system under controlled water activity, and their extent was monitored via chromatographic assays for free fatty acids, esterified fatty acid moieties, and triacylglycerols; the thermal behavior of the modified butterfat was also assessed via calorimetry. Lipase-modified butterfat possesses a wider melting temperature range than regular butterfat. The total saturated triacylglycerols decreased by 2.2%, whereas triacylglycerols with 28–46 acyl carbons (which contained two or three lauric, myristic, or palmitic acid moieties) decreased by 13%. The total monoene triacylglycerols increased by 5.4%, whereas polyene triacylglycerols decreased by 2.9%. The triacylglycerols of interesterified butterfat had ca. 10.9% less lauric, 10.7% less myristic, and 13.6% less palmitic acid residues than those of the original butterfat.     

A new way to conduct enzymatic synthesis in an active membrane using ionic liquids as catalyst support

Several active membranes were prepared by immobilization of Candida antarctica lipase B (CALB) onto the surface of ceramic membranes via three different ionic liquids (ILs): [bmim⁺][PF₆⁻], [emim⁺][NTf₂⁻] and [bmim⁺][NTf₂⁻] and compared to an active membrane obtained by simple enzyme adsorption. The performances of these various membranes were evaluated by studying the synthesis of butyl laurate by the acidolysis between butyl acetate and lauric acid in a hexane/water mixture (98:2, v/v). The results obtained show that all the membranes prepared with or without ILs were active and maintained their activity after several runs and during some months.     

Hydrolysis of fish oils containing polymers of triacylglycerols by pancreatic lipasein vitro

Fish oils containing different levels of polymers of triacylglycerols formed during autoxidation were incubated with pancreatic lipase to establish whether these polymers are substrates for lipase hydrolysis. With oils containing low amounts (less than 4%) of triacylglycerol polymers as substrates, both triacylglycerols and polymers of triacylglycerols were almost completely hydrolyzed, and fatty acid monomers and monoacylglycerols were the major lipid products. Under the same incubation conditions, some triacylglycerols remained intact when highly oxidized oils containing 20 or 30% triacylglycerol polymers were the substrate. The fatty acid composition of these residual triacylglycerols was almost identical to that of triacylglycerols present at the start of the assay. When fish oil containing 30% triacylglycerol polymers was incubated with the lipase, the component triacylglycerols and polymers of triacylglycerols were hydrolyzed at similar rates, and fatty acid dimers were detected as a product. It is concluded that the high molecular weight polymers of triacylglycerols present in oxidized fish oils can be hydrolyzed by pancreatic lipasein vitro.