固定化脂肪酶催化制备L-薄荷醇

用大孔树脂NKA固定高选择性的脂肪酶,催化有机相中转酯化反应,从而拆分八异构体消旋薄荷醇来制备L-薄荷醇。研究pH、载体与酶比例对固定化酶制备的影响及固定化酶的反应稳定性;考察温度、转酯化过程醇酯比例、及底物醇异构组成变化对拆分效果的影响。结果表明:固定化酶的最适pH为8,载体与酶的比例为5∶1时,所得固定化酶的反应稳定性比游离酶的反应稳定性提高了约50%;转酯化反应的最优温度为40℃,醇酯比例为1.5∶1时最佳,改进八异构体消旋薄荷醇组分比例后,非对映体选择率dep达到了95.1%。     

神经网络优化非水相脂肪酶催化合成抗坏血酸油酸酯的条件研究

抗坏血酸油酸酯具有强抗氧化作用．为了获得脂肪酶催化合成抗坏血酸油酸酯的最适条件,主要研究了反应温度、脂肪酶量、油酸量对抗坏血酸油酸酯合成效果的影响．采用中心组合设计和动量梯度下降神经网络对反应条件网络进行训练仿真,并利用训练好的网络对催化酯化工艺条件进行预测．研究结果表明：经过训练的网络可以很好的模拟反应条件,得到了脂肪酶催化反应的最佳工艺参数．当抗坏血酸0．8g时,反应温度56℃,油酸量0．95g,固定化脂肪酶量0．74g,添加分子筛条件下,抗坏血酸油酸酯的转化率为46.5％．该方法为抗坏血酸酯化催化效果的预测提供了一条可行的途径．     

由扩展青霉PF86产生脂肪酶催化油脂水解的研究

研究了由扩展青霉ＰＦ８６８产生脂肪酶催化水解三种油脂的影响因素与工艺条件,其中包括：水解时间、温度、ＰＨ、酶量、油水比及添加剂,并用气相色谱对产品脂肪酸进行了分析鉴定,初步分析其催化水解的脂肪酸的特异性。     

脂肪酶催化制备生物柴油的研究进展

生物柴油作为一种可再生的清洁能源,以其良好的环境效应受到越来越多的关注。酶法生产生物柴油具有化学催化法不可比拟的优越性,是工业化生产的发展方向。本文综述了利用固定化脂肪酶、游离酶、全细胞生物催化剂制备生物柴油的研究与应用进展,并探讨了我国生物柴油产业化发展的困境和对策。     

固定化假丝酵母1619脂肪酶催化油酸油醇酯的合成

比较了14种不同来源的脂肪酶催化油酸油醇酯的合成。其中,假丝酵母(Candidasp．)1619脂肪酶酯化能力最强,以硅藻土为载体,分别按0．1％添加椰子油、吐温80．按l％添加MgSO43种共固定物,醇化反应初速度提高了1．5倍。此固定化酶催化油酸油醇酯合成的最适温度为30℃,0～60℃下反应24h的酯化率均在90％以上,100℃下还有10．25％的酯化率。最适酯化pH6．0。反应中去水,可使终酯化率提高到99％。在添加的23种有机溶剂中,以异辛烷促进酯化的效果最好．正壬烷和正己烷次之。此固定化酶在28℃下批式重复反应的半衰期为990h,柱式固定床反应器中28℃连续运转1000h后酯化率为78％。     

脂肪酶水解植物油的研究

本文报导了薄层层析法及高压液相色谱法定性定量地测定植物油甘油酯的组成,对不同来源的脂肪酶对植物油的水解进行了研究。实验结果表明,不同来源的脂肪酶对植物油(橄榄油)的水解性不同,同一脂肪酶水解不同种类的植物油,脂肪酶的水解率也不同,脂肪酶水解植物油有最适pH和最适温度。     

固定化脂肪酶催化毛棉籽油制备生物柴油

研究了固定化脂肪酶Lipozyme TL IM和Novozym435催化毛棉籽油和乙酸甲酯制备生物柴油的过程。通过向反应体系中添加甲醇,可减少乙酸的抑制,明显提高生物柴油得率,确定最佳反应条件为:正己烷作溶剂,乙酸甲酯与油摩尔比9:1,添加油重3%的甲醇、油重10%的LipozymeTLIM和5%的Novozym435复合使用,温度55°C,反应8h,生物柴油得率达到91.83%。最后探索了酶催化毛棉籽油合成生物柴油的动力学,得到动力学方程。     

豆粕水解液为氮源发酵产脂肪酶的研究

研究了以豆粕水解液作为氮源,假丝酵母Candida sp．99—125发酵生产脂肪酶的过程。分析水解时间对于产酶的影响,对比豆粕水解前后作为氮源发酵时的产酶规律。在30L发酵罐中批次发酵酶活最高可达6000IU／mL,采用豆油反馈流加之后,发酵脂肪酶活力可达8500IU／mL。     

Production of Ricinoleic Acid from Castor Oil by Immobilised Lipases

Abstract

Porcine pancreatic lipase (PPL), Candida rugosa lipase (CRL), and Castor bean lipase (CBL) were immobilized on celite by deposition from aqueous solution by the addition of hexane. Lipolytic performance of free and immobilized lipases were compared and optimizations of lipolytic enzymatic reactions conditions were performed by free and immobilized derivatives using olive oil as substrate. Afterwards, the influence on lipolysis of castor oil of free lipases and immobilized lipase derivatives have been studied in the case of production of ricinoleic acid. All of the lipases performances were compared and enzyme derivative was selected to be very effective on the production of ricinoleic acid by lipolysis reaction. Various reaction parameters affecting the production of ricinoleic acid were investigated with selected the enzyme derivative.

The maximum ricinoleic acid yield was observed at pH 7–8, 50°C, for 3 hours of reaction period with immobilized 1,3-specific PPL on celite. The kinetic constants K_m and V_max were calculated as 1.6 × 10^?4 mM and 22.2 mM from a Lineweaver–Burk plot with the same enzyme derivative. To investigate the operational stability of the lipase, the three step lipolysis process was repeated by transferring the immobilized lipase to a substrate mixture. As a result, the percentange of conversion after usage decreased markedly.     

The Production of γ-Decalactone by Fermentation of Castor Oil

A microbial process for the production of optically-active γ-decalactone from the ricinoleic acid present as triglycerides in castor oil has been developed, γ-decalactone (γDL) is a component of some fruit flavours, being an important organoleptic component of peach flavours. Screening showed two red yeast microorganisms, Rhodotorula glutinis and Sporobolomyces odortts to be especially suitable for this biotransformation. The process involves lipase-mediated hydrolysis of the castor oil to give free ricinoleic acid, uptake of the acid by the cells and aerobic fermentation to achieve abbreviated β-oxidation of the ricinoleic acid (12-hydroxyoleic acid) into 4-hydroxydecanoic acid (4HDA), lactonisation of the acid into γ-DL, followed by solvent extraction and distillation. γ-DL broth concentrations of 0.5-1.2g · 1^-t were obtained after 3-5 days from fermentation media containing 10 g · 1^-1 castor oil, representing an 8.3-20.0% theoretical yield. Intermediates detected were consistent with the operation of the β-oxidation pathway. Appreciable amounts of novel metabolites identified as cis and trans isomers of a tetrahydrofuran (C₁₀) were also produced. Their formation from 4HDA appeared to be non-enzymic and was favoured by anaerobic conditions. Yields of γ-DL were inversely proportional to the concentration of castor oil present in the medium, indicating that substrate inhibition takes place. The highest yields of γ-DL were obtained when castor oil was present from the beginning of the fermentation, rather than when added once the fermentation had become established, demonstrating that the β-oxidation pathway and/or transport system require continual induction. Significant amounts of γ-DL were not produced from other fatty acids, including ricinelaidic acid, the trans isomer of ricinoleic acid. γ-DL formation was dramatically inhibited by antibiotic inhibitors of oxidative phosphorylation, indicating the importance of intact β-oxidation pathways, whereas inhibitors of protein synthesis and cell-wall synthesis had much less marked effects. Selective extraction of 4HDA from the fermentation broths, and of γDL from broth lactonised by heating at low pH, could be achieved by adsorption to Amberlite XAD-1 and XAD-7 resins respectively. Some product could be recovered from the exit gases of the fermenter by passing through propylene glycol traps. This pathway is unusual in that it is a rare example of the truncated β-oxidation of a fatty acid by microorganisms. This effect probably occurs because of partial inhibition of one or more enzymes of the β-oxidation pathway by the C₁₀ hydroxylated fatty acid intermediate(s) allowing intracellular accumulation of the 4HDA, followed by leakage out of the cell; although further metabolism of this C₁₀ intermediate does take place slowly.     

酶促红花油水解中的时间效应

目的:研究无溶剂体系中脂肪酶催化红花油水解反应的时间效应。方法:采用单因素实验法,确定不同反应时间时各因素的最佳水平。结果:反应1h时的最适条件为35℃、n(水)/n(油)比35p、H 7.0、缓冲液浓度0.05mol/L、添加0.2?Cl2;反应24h时,最适条件为26℃、n(水)/n(油)比60p、H 7.6,缓冲液浓度及CaCl2的影响不显著。结论:水解最佳条件随反应时间变化,应根据反应时间长短确定各因素的最佳水平。     

超声波辅助下脂肪酶催化高酸值废油脂制备生物柴油

探讨了超声波辅助条件下脂肪酶催化高酸值废油脂转化为生物柴油的反应。来源于Aspergillus oryzae和Candida antarctica的固定化脂肪酶,在超声波辅助下,对高酸值废油脂转化为生物柴油具有高的催化活性。以来自于C.antarctica的固定化脂肪酶Novozym435为催化剂,以酸价为157mg KOH/g的高酸值废油脂为原料在超声波辅助下与丙醇反应,在脂肪酶用量为油质量的8%、初始醇油摩尔比为3∶1、反应温度控制在40~45℃、超声波频率和功率分别采用28kHz和100W的条件下,反应50min转化率达到94.86%。在此条件下,不同碳原子数(C1~C5)的直链和支链醇均有较高的转化率,在短链醇的选择上具有宽广的适应性。超声波还减少了反应产物和反应体系中其他黏性杂质在固定化脂肪酶表面的吸附,回收的Novozym435相较单纯机械搅拌条件下回收的外观干净、分散良好无结块现象、易于洗涤和再次利用,具有良好的操作稳定性。     

Lipase applications in oil hydrolysis with a case study on castor oil: a review

Lipase (triacylglycerol acylhydrolase) is a unique enzyme which can catalyze various types of reactions such as hydrolysis, esterification, alcoholysis etc. In particular, hydrolysis of vegetable oil with lipase as a catalyst is widely studied. Free lipase, lipase immobilized on suitable support, lipase encapsulated in a reverse micelle and lipase immobilized on a suitable membrane to be used in membrane reactor are the most common ways of employing lipase in oil hydrolysis. Castor oil is a unique vegetable oil as it contains high amounts (90%) of a hydroxy monounsaturated fatty acid named ricinoleic acid. This industrially important acid can be obtained by hydrolysis of castor oil. Different conventional hydrolysis processes have certain disadvantages which can be avoided by a lipase-catalyzed process. The degree of hydrolysis varies widely for different lipases depending on the operating range of process variables such as temperature, pH and enzyme loading. Immobilization of lipase on a suitable support can enhance hydrolysis by suppressing thermal inactivation and estolide formation. The presence of metal ions also affects lipase-catalyzed hydrolysis of castor oil. Even a particular ion has different effects on the activity of different lipases. Hydrophobic organic solvents perform better than hydrophilic solvents during the reaction. Sonication considerably increases hydrolysis in case of lipolase. The effects of additives on the same lipase vary with their types. Nonionic surfactants enhance hydrolysis whereas cationic and anionic surfactants decrease it. A single variable optimization method is used to obtain optimum conditions. In order to eliminate its disadvantages, a statistical optimization method is used in recent studies. Statistical optimization shows that interactions between any two of the following pH, enzyme concentration and buffer concentration become significant in presence of a nonionic surfactant named Span 80.     

Biosynthesis of Castor Oil: Effect of Polyamines on the Acylation of Lysophosphatidic Acid at the sn-2 Position with Ricinoleic Acid

Polyamines with diamine structures of chain length longer than 3C were essential for the synthesis of phosphatidic acid (PA) from ricinoleoyl-CoA and lysophosphatidic acid (LPA) by the castor LPA acyltransferase reaction, suggesting that polyamines modulate enzyme affinity for the acyl-CoA substrate in vivo.     

酸水解蚕蛹制备复合氨基酸的研究

采用硫酸水解法,以蚕蛹制取复合氨基酸产品,得到氨基酸态氮分别为９．０５％和１３．４５％的食用复合氨基粉和精制复合氨基酸粉。食用复合氨基酸粉含有１８种氨基酸,其中必需氨基酸含量为３９．２％。食用复合氨基酸粉的制备方法经工厂小批量生产证实,其工艺简单易行,适合于中小企业采用,该产品的质量优良,生产成本低廉,具有市场竞争力。     

Surfactant Effects on Lipase-Catalyzed Hydrolysis of Olive Oil in AOT/ISOOCTANE Reverse Micelles

The effects of surfactant concentration on the hydrolytic activity of Candida rugosa lipase in AOT/isooctane reverse micelles with olive oil as the substrate has been investigated. A noncompetitive inhibition by the surfactant on the enzyme was observed. Strong dependences of the kinetic constants k_cat and k_M, but not k_I on the water-to-surfactant ratio (R value) have been identified. The benefits of carrying out the hydrolysis at higher surfactant and water concentrations were demonstrated from the improvement of the initial rate and time course of conversion.     

无溶剂体系中脂肪酶改造猪油制备功能性脂的研究

我国有丰富廉价的动植物油资源,但这一资源并未得到有效的利用,为了充分利用这些资源,开展了无溶剂系统脂肪酶催化猪油与辛酸酸解制备功能性脂的研究工作。脂肪酶筛选实验表明,在所选用的五种脂肪酶中,来自T .languginosa的固定化脂肪酶LiopzymeTLIM的催化效果最好。以LipozymeTLIM为催化剂,进一步研究了酶量、底物比率、反应时间、反应温度和水分添加量对猪油中辛酸插入率的影响。反应产物通过高效液相色谱法(HPLC)进行分析。研究结果表明,当脂肪酶量为20% (底物重量百分比)时,猪油中辛酸插入率最高。反应时间研究表明,当反应时间达到24h时,辛酸的插入率最高,达到38.77mol%。当猪油与辛酸的比率为1∶2 (摩尔比)时,辛酸的插入率最高,达到30 95mol%。在4 5～6 0℃范围之内,反应温度对辛酸插入率没有明显的影响,温度高于6 0℃时,辛酸插入率降低。水分添加量为2 5 %时,辛酸插入率最高,高达35 76mol%。     

Purification and characterization of a lipase from Pseudomonas aeruginosa KKA-5 and its role in castor oil hydrolysis

An extracellular lipase (triacylglycerol acylhydrolase, EC 3.1.1.3) from Pseudomonas aeruginosa KKA-5 hydrolyzed castor oil by 90%. Purification of this castor oil-hydrolyzing lipase included ammonium sulfate precipitation and successive hydroxylapatite column chromatography. The enzyme was purified 518-fold. It was homogeneous electrophoretically and its molecular weight was estimated to be 30 kDa. The enzyme was stable up to 45°C and retained its activity in the alkaline pH range. Lipase was highly stable in the presence of aqueous organic solvents like methanol and ethanol. It was weakly inhibited in the presence of acetone. The anionic surfactant, sodium dodecyl sulfate, was inhibitory while the cationic surfactants, Triton X-100 and Tween-80 appreciably enhanced activity. Lipase was stabilized significantly by Ca²⁺. Inactivation of the enzyme by EDTA was overcome by sequential CaCl₂ treatment. This finding suggests the existence of a calcium-binding site in Pseudomonas aeruginosa KKA-5 lipase. Received 22 January 1998/ Accepted in revised form 27 April 1998