脂肪酶在丙二醇溶液中的活性变化及其机理

目的:研究猪胰脂肪酶(porcine pancreas lipase,PPL)在丙二醇单相共溶体系中催化橄榄油水解反应的活性变化及其作用机理.方法:测定PPL在不同体积浓度的丙二醇溶液中催化橄榄油水解的活性、动力学参数变化及紫外光谱、紫外差示光谱、荧光光谱的变化情况.结果:体积分数为17%的丙二醇使PPL活性提高了55%;丙二醇处理后,PPL的νmax基本不变,而km值则明显下降;光谱分析发现,经丙二醇处理后,PPL的紫外吸收光谱发生蓝移,而紫外差示光谱则出现了两个明显的负峰,荧光发射峰强度显著降低.结论:体积分数为17%的丙二醇对PPL有一定的激活作用,该激活作用可能是由于在丙二醇溶液中PPL发生构型和构象的变化,提高了酶分子对底物的亲和力,从而引起酶活性的升高.     

二氧化硅纳米材料固定中性脂肪酶的条件优化及其特性

以二氧化硅纳米材料为载体,采用吸附法对脂肪酶进行固定化,研究了不同条件对固定化脂肪酶的催化活性的影响,得到最佳的固定化条件:给酶量为28300U/g,固定化温度为45oC,pH值为7.5,时间为10h,此时固定化酶的活力约为3867U/g载体。固定化酶的最适反应温度为45oC,比游离酶的反应温度高5oC,最适pH下降到5.5,低于游离酶的反应pH(pH7)。固定化酶的热稳定性和pH稳定性较游离酶有了很大的提高,其在70oC以下能保持70%以上的酶活力,而游离酶在50oC下残余酶活力仅为30%。在pH5～8的范围内,固定化酶的酶活力能保持50%以上,而游离酶只能保持20%左右。用固定化的中性脂肪酶催化不同的油品,即大豆油、菜籽油及泔水油生产生物柴油,菜籽油的酯化率最高。     

器壁固定化脂肪酶Pseudomonas cepacia lipase的非水活性与稳定性

许多脂肪酶在有机体系中表现出催化作用,可用于绿色有机合成. 但其催化活性和稳定性明显低于水/油（有机相）界面上的表现. 为了提高脂肪酶在有机反应体系中的活性和稳定性,依据脂肪酶的界面活化机制,以水为酶蛋白构象优化剂、羧甲基纤维素为赋形剂,通过物理吸附的方式,将典型的假单胞菌脂肪酶（Pseudomonas cepacia lipase）固定在锥形瓶的内壁上,形成简易的生物反应器. 为方便检测器壁固定化酶促反应动力学,选择特征吸收为640 nm的生化指示剂2,6-二氯靛酚为反应底物,乙酸乙烯酯为酰化试剂,丙酮为溶剂. 光谱检测表明,催化反应0.5 h后,器壁固定化脂肪酶转化底物的能力是脂肪酶粉的6倍. 在每次催化5 h共10次的循环催化中,器壁固定化脂肪酶的催化活性平均每次仅降低3.2%,而酶粉降低11.8%. 结果表明,该器壁固定化脂肪酶的活性和稳定性相对于酶粉明显提高,这将为通过固定化有效提高脂肪酶的非水催化作用提供重要的参考.     

固定化脂肪酶催化毛油合成生物柴油

本研究开发了一种用石油醚提取毛油的工艺,研究了以提取的毛油和甲醇为原料,用固定化Candida sp.99-125脂肪酶催化合成脂肪酸甲酯(FAMEs)的可行性。同时考察了磷脂对固定化酶活性、反应起始速率、固定化酶使用批次的影响以及毛油和精炼油对固定化酶使用批次等的影响。研究结果表明,用磷脂质量分数为1%的石油醚悬液浸泡过的脂肪酶比仅用石油醚浸泡过的脂肪酶初始转酯化速率显著下降。当大豆油中无磷脂时,15min时FAMEs的产率为26.2%;磷脂质量分数为5%时,FAMEs降为12.4%。当大豆油中磷脂质量分数小于1%时,固定化酶使用10个批次,FAMEs产率无明显变化。固定化脂肪酶催化石油醚浸提得到的大豆和小桐子毛油,经过10个批次反应FAMEs产率都保持在70%以上,该固定化酶直接催化毛油生产生物柴油具有良好的工业化前景。     

溶胶凝胶法固定化黑曲霉脂肪酶的性质研究

近年来溶胶-凝胶法固定脂肪酶已成为研究热点。选用TMOS、MTMS、ETMS和PTMS 4种硅烷试剂对黑曲霉脂肪酶进行了固定化研究。固定化的最佳配方为ETMS/TMOS=5：1、水与硅烷试剂分子比为8;固定化脂肪酶的固定率为80.2%、相对活性为136.3%;以乳化橄榄油作为底物,在50℃和pH4.0的条件下,固定化脂肪酶与游离脂肪酶K_m分别为1.899×10^-4M和2.789×10^-4M;最适反应pH均为pH4.0,固定化脂肪酶在pH4.0~pH5.5之间其活性能保持95%以上;固定化脂肪酶最适反应温度为60℃,较游离脂肪酶提高了10℃;固定化脂肪酶的酸碱稳定性和热稳定性较非固定化酶有显著的提高。固定化脂肪酶的使用寿命和保存稳定性良好,使用12次后仍能够保留71.7%活性,在室温避光条件下保存180天后仍可保留79.2%活性。     

钙离子对辣根过氧化物酶同工酶C的溶液构象的影响

运用荧光光谱方法研究了脱辅基的辣根过氧化物酶同工酶Ｃ。结果表明：（１）ａｐｏ－ＨＲＰ（Ｃ）与其他“Ｂ”类蛋白质不同,有明显的Ｔｙｒ荧光;（２）低浓度变性剂（〈２ｍｏｌ／Ｌ脲或０．２ｍｏｌ／Ｌ盐酸胍）能增强酶的Ｔｒｐ荧光,但并不改变光谱特性;（３）进一步增加变性剂浓度则使Ｔｒｐ残基暴露于水溶液中,荧光强度略有降低,荧光谱红移;（４）Ｃａ＾２＋络合剂ＥＤＴＡ对色氨酸荧光的影响与低浓度变性剂相同。有意思     

荧光假单胞菌脂肪酶固定化及催化制备生物柴油的工艺研究

研究了不同因素对制备固定化荧光假单胞菌脂肪酶的影响及固定化酶的酶学性质,并初步探讨了利用该固定化酶制备生物柴油的工艺。以海藻酸钠明胶为复合载体,采用包埋法制备固定化荧光假单胞菌脂肪酶,考察了载酶量、颗粒直径等因子对固定化效果的影响,并用制备的固定化酶进行了酶促酯交换合成生物柴油的工艺研究,考察了反应条件如酶量、反应温度、甲醇流加方式、醇油比等因素对甲酯得率的影响。试验结果表明,制备固定化荧光假单胞菌脂肪酶的最优条件为:每克载体给酶量为300 IU,选用6号注射器针头(内径为0.5 mm);通过酯交换,催化大豆油合成生物柴油的最佳反应工艺参数为:固定化酶25%,醇油比4:1,含水量6%,反应温度40℃;此条件下反应35 h后,甲酯的最高得率可达82%。     

一些理化因子对α淀粉酶构象与活力的影响

用荧光光谱,紫外差示光谱和ＣＤ谱研究了一些理化因子对枯草芽孢杆菌８６３１５α淀粉酶的构象与活力的影响,实验表明,酸变性和碱变性所引起的酶构象变化是不同的;乙醇不降低α淀粉酶活力,但使其构象发生较大变化,α螺旋度从天然酶的２６,１％降到２１．８％,其构象变化不引起活性中心的改变;酶在７０℃处理１０ｍｉｎ后,由原来紧密构象变为松散构象,α螺旋度从２６．１％降到９．０％,酶活性完全丧失;而在０．０２ｍｏｌ／ＬＣａＣｌ_２和０．０２ｍｏｌ／ＬＮａＣｌ的共同存在下,７０℃处理１０ｍｉｎ,酶活性不变,其荧光光谱和ＣＤ谱接近于天然酶,所以,ＣａＣ_ｌ２和ＮａＣｌ能保护α淀粉酶的构象,使之不受热变性。     

活性部位的柔性

比较酶在变性过程中构象和活力变化,发现在活性完全丧失时尚无可察 觉的整体构象变化。排除变性剂抑制和寡聚酶解聚等可能性之后,提出了酶活性部位柔性假说。随后用多种实验方法直接证实了活性部位的构象变化先于分子整体构象变化,并与活性丧失同步,根据催化过程中活性部位构旬变化,以及限制活性部位构象变化对酶活性的影响,提出了酶活性部位柔性为酶充分表现其催化活性所必需的设想。     

3-磷酸甘油醛脱氢酶胍变性时的活力及构象变化

酵母3-磷酸甘油醛脱氢酶在盐酸胍溶液中的内源荧光及剩余活力的变化结果提示:apo酶及holo酶的活力在胍浓度为0.5M左右可完全丧失.同时伴有内源荧光强度的下降,光谱宽度的增加和335nm最大发射峰的红移(提示了色氨酸残基的暴露).与已经报导的肌肉酶(内源荧光强度在胍浓度为0.4—1.2M范围相对稳定)不同,酵母酶内源荧光在此浓度范围内表现为逐渐降低.在0.7M胍溶液中,内源荧光变化动力学过程只能测出一相,而酶失活动力学过程为快慢两相,快相动力学速度常数至少大于内源荧光降低速度常数三个数量级以上.以上结果提示:低浓度胍可引起该酶的完全失活,活性部位的空间构象比酶分子的构象更易受到变性剂的扰乱;有一个色氨酸残基位于或靠近酶的活性部位.     

Covalent immobilization of lipase in organic solvents

Lipase from Rhizopus sp. has been immobilized covalently on tresyl activated silica. Three different coupling media were evaluated: aqueous buffer, n-hexane, and a microemulsion based on n-hexane, aqueous buffer, and the nonionic surfactant triethylene glycol monododecyl ether. In addition, coupling via a very long, hydrophilic spacer arm, polyethylene glycol 1500 (PEG 1500), was compared with attachment to the silica via a short silane bridge only. The enzyme preparations were tested in hydrolysis and transesterification reactions. In the hydrolysis no marked differences in activity were found between the coupling media used. In the transesterification, on the other hand, the choice of immobilization medium had a very large effect on lipase activity, the preparation from microemulsion being the most active one. The use of the hydrophilic spacer had a large effect on activity in the hydrolysis reaction. Whereas direct coupling gave an activity of immobilized lipase of 26-34% of that of free enzyme, depending on the reaction medium, lipase bound via the spacer exhibited 56-67% activity. The latter values are considerably higher than previously reported in the literature for covalently immobilized lipase. The hydrophilic spacer had no effect on enzyme activity in the transesterification, however, a fact which is attributed to the hydrophobic medium of this reaction. The spacer is incompatible with the reaction medium and will, therefore, adsorb on the particles rather than stretch out into the bulk phase. The stability of the bound lipase was extremely good, no loss in activity being observed after a period of three weeks in aqueous solution of 37 degrees C.     

Novel surfactant-coated enzymes immobilized in poly(ethylene glycol) microcapsules

Summary Novel surfactant-coated enzymes immobilized in poly(ethylene glycol) microcapsules have been developed for the re-use of an oil-soluble enzyme in organic media. The esterification rate of the surfactant-coated lipase immobilized in the microcapsules was thirty times that of the powder lipase. More than 90% of the enzymatic activity of the capsulated lipases has been maintained after recycling six times.     

Purification and in situ immobilization of lipase from of a mutant of Trichosporon laibacchii using aqueous two-phase systems

The crude intracellular lipase (cell homogenate) from Trichosporon laibacchii was subjected to partial purification by aqueous two-phase system (ATPS) and then in situ immobilization by directly adding diatomites as carrier to the top PEG-rich phase of ATPS. A partition study of lipase in the ATPS formed by polyethylene glycol–potassium phosphate has been performed. The influence of system parameters such as molecular weight of PEG, system phase composition and system pH on the partitioning behaviour of lipase was evaluated. The ATPS consisting of PEG 4000 (12%) and potassium phosphate (K₂HPO₄, 13%) resulted in partition of lipase to the PEG-rich phase with partition coefficient 7.61, activity recovery 80.4%, and purification factor of 5.84 at pH of 7.0 and 2.0% NaCl. Moreover, the in situ immobilization of lipase in PEG phase resulted in a highest immobilized lipase activity of 1114.6 U g^?1. The above results show that this novel lipase immobilization procedure which couples ATPS extract and enzyme immobilization is cost-effective as well as time-saving. It could be potentially useful technique for the purification and immobilization of lipase.     

Lipase immobilization on magnetic microspheres via spacer arms: Effect of steric hindrance on the activity

Poly(styrene-acrylic acid) magnetic microspheres with an average diameter of 2 μm were successfully prepared and used as carriers to immobilize lipase. Lipase immobilized on microspheres with no spacer arm exhibited low activities, which were attributed to steric hindrance on the lipase conformation. To avoid steric effects, ethylenediamine and poly(ethylene glycol) (PEG) 400/800/4000 were utilized as spacer arms to bind the lipase to the microspheres. The immobilized lipase activities were improved using PEG 800/4000 as a spacer arm. Furthermore, the influence of enzyme loading on lipase activity was investigated, and the results indicated that enzyme overloading could exert steric effect on lipase activity. The degree of PEG modification was demonstrated to affect lipase activity because excess PEG on the surface of microspheres could interact with lipase due to its mobility, consequently reducing lipase activity.     

Immobilization of nitrifying bacteria by polyethylene glycol prepolymer

The effects of immobilizing materials on the activity of nitrifying bacteria were investigated by using 11 kinds of prepolymers of polyethylene glycol. Relative respiratory activity of immobilized nitrifying bacteria with polyethylene glycol metacrylate prepolymer was higher than that of polyethylene glycol acrylate prepolymer, and there was a tendency for relative respiratory activity to be higher with a prepolymer of greater molecular weight. With the polyethylene glycol prepolymer, there was a drastic improvement over the conventional method of immobilization by acrylamide in the relative respiratory activity of the pellet. Inorganic synthetic wastewater was treated under a high loading rate of 1.14 kg-N/m³·d. Influent NH₄-N could be removed to 2 mg/l or less and the nitrogen removal was 90%.     

环氧交联剂和氨基载体固定化海洋假丝酵母脂肪酶*

游离酶经过固定化后,稳定性和环境耐受性得到提高,在食品、医药、化工、环境和皮革等领域可以很好的提高酶的利用率并降低生产成本,具有极大的应用潜力。新型交联剂在固定化酶工艺的应用极大推进了固定化酶研究的深入。借助新型交联剂聚乙二醇二缩水甘油醚(PEGDGE),利用氨基载体LX-1000HA固定化海洋假丝酵母脂肪酶,结合单因素和正交试验优化得到交联及固定化条件为:交联温度30℃,交联2h,交联剂浓度0.75%,pH7.0,加酶量800U,载体量0.5g,固定化2h,固定化温度45℃。根据上述最佳固定化工艺,制备得到固定化酶LX-1000HA-PEGDGE-CRL在最适条件下测得酶活达到160.81U/g,约为此前制备的固定化酶LX-1000HA-GA-CRL(由LX-1000HA和戊二醛交联脂肪酶得到)和LX-1000EA-PEGDGE-CRL(由短链氨基载体LX-1000EA和PEGDGE交联脂肪酶得到)酶活的2倍,发现固定化酶LX-1000HA-PEGDGE-CRL的最适反应温度相比于游离酶提高15℃;在70℃的环境中3h后酶活仍存留70%;循环使用6次后残留65%左右的酶活;酸碱耐受性和储存稳定性也表现良好,4℃保存30天后剩余约70%的初始酶活。同时,将制备的固定化酶LX-1000HA-PEGDGE-CRL与游离酶、固定化酶LX-1000HA-GA-CRL、固定化酶LX-1000EA-PEGDGE-CRL进行了比较,发现固定化酶LX-1000HA-PEGDGE-CRL在温度耐受性和重复使用性等方面具有更好的使用效果。     

Magnetic lipase active in organic solvents

Magnetic lipase (magnetite particles coated with polyethylene glycol-modified lipase) was prepared in two steps: Lipase was coupled with 2,4-bis(O-methoxypolyethylene glycol)-6-chloro-s-triazine, activated PEG2, to obtain polyethylene glycol-modified lipase, PEG-lipase. The PEG-lipase was added to the solution of ferrous (Fe2+)- and ferric(Fe3+)-ions with the pH value adjusted to 8.0-8.5 to obtain magnetic lipase. The magnetic lipase was dispersed in organic solvents such as benzene and 1,1,1-trichloroethane with the particle size of 120 +/- 60 nm. The colloidal solution was very stable and no aggregation occurred even after 5 days. A high enzymic activity (11.6 mumol/min/mg protein) for lauryl laurate synthesis was observed in 1,1,1-trichloroethane. The magnetic lipase was readily recovered from the organic solvents in a magnetic field of 6000 Oe without loss of the enzymic activity.     

Porcine pancreatic lipase partition in potassium phosphate-polyethylene glycol aqueous two-phase systems

This research study examined porcine pancreatic lipase partition in aqueous two-phase systems formed by polyethylene glycol-potassium phosphate at pH 6.0, 7.0 and 8.0, the effect of polymer molecular mass, and NaCl concentration. The enzyme was preferentially partitioned into the polyethylene glycol rich phase in systems with molecular mass 4000-8000, while with polyethylene glycol of 10,000 molecular mass it was concentrated in the phosphate rich phase. The enthalpic and entropic changes found due to the protein partition were negative for all the polyethylene glycol molecular mass systems assessed. Both thermodynamic functions were shown to be associated by an entropic-enthalpic compensation effect suggesting that the water structure ordered in the ethylene chain of polyethylene glycol plays a role in the protein partition. The addition of NaCl increased the lipase affinity to the top phase and this effect was most significant in the system polyethylene glycol 2000-NaCl 3%. This system yielded an enzyme recovery more than 90% with a purification factor of approximately 3.4.     

Modification of Lipase by Polyethylene Glycol

Lipase was modified using polyethylene glycol activated by p-nitrochloroformate. The hydrolytic activity of the polyethylene glycol-derivatised lipase (PEG-lipase) was relatively low compared with that of the unmodified enzyme in aqueous system. The esterification activity, however, was enhanced following the modification. The rate of esterification of butyric acid was higher than that of oleic acid. Benzene was the best solvent for the esterification reaction.     

Ester synthesis at extraordinarily low temperature of -3 degrees C by modified lipase in benzene

The lipoprotein lipase from Pseudomonas fluorescens was modified with 2,4-bis(O-methoxypolyethylene glycol)-6-chloro-s-triazine. The modified lipase in which 55% of the amino groups in the enzyme molecule were coupled with polyethylene glycol was found to be soluble in benzene and catalyzed the reactions of ester synthesis, ester exchange, aminolysis and ester hydrolysis in benzene. The modified lipase had an extraordinary temperature-dependency: enzymic activity for methyl laurate synthesis from methyl alcohol and lauric acid increased with decreasing temperature and attained the maximum at the extremely low temperature of -3 degrees C. The optimum temperature for hydrolysis of methyl laurate was as low as -4 degrees C.