固定化Candida sp.99-125脂肪酶催化大豆油合成脂肪酸乙酯

探讨了酶法合成脂肪酸乙酯作为生物柴油的可行性. 以大豆油和乙醇为原料,利用本实验室自制的固定化Candida sp. 99-125脂肪酶催化反应,深入研究水含量、溶剂量、脂肪酶量及反应温度等因素对酶法合成脂肪酸乙酯的影响. 结果表明,以大豆油质量为基准,在水含量为 12.5%(w)、溶剂正己烷为3 mL/g、脂肪酶量为20%(w)、温度40℃的优化反应条件下,3次流加乙醇,170 r/min摇瓶反应,12 h后可以达到96.8%的最高酯得率. 进一步研究表明,在此优化反应条件下,连续使用14批脂肪酶酯得率可保持70%以上.     

Fatty Acid and Position Specificities of an Intracellular Lipase from Geotrichum candidum

An intracellular lipase was isolated and purified to homogeneity from mycelia of Geotrichum candidum. The lipase showed maximum activity at 35° C and pH 7.5. The enzyme preferentially hydrolyzed oleic acid glycerol ester bonds on using mono-acid triglycerides (C_10:0, C_18:0) as substrates. However the results indicated that the enzyme is position unspecific.     

Anticancer Potential of Small-Molecule Inhibitors of Fatty Acid Amide Hydrolase and Monoacylglycerol Lipase

Recently fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL) inhibitors have been in the limelight due to their anticancer potential. Both FAAH and MAGL are the endocannabinoid degrading enzymes that hydrolyze several endogenous ligands, mainly anandamide (AEA) and 2-arachidonic glycerol (2-AG), which regulate various pathophysiological conditions in the body such as emotion, cognition, energy balance, pain sensation, neuroinflammation, and cancer cell proliferation. FAAH and MAGL inhibitors block the metabolism of AEA and 2-AG, increase endogenous levels of fatty acid amides, and exert various therapeutic effects including chronic pain, metabolic disorders, psychoses, nausea and vomiting, depression, and anxiety disorders. FAAH and MAGL are primarily neurotherapeutic targets, but their contribution to various types of carcinomas are significant. Inhibitors of these enzymes either alone or as multitarget agents, or with supra-additive effects show the potential effect in ovarian, breast, prostate, and colorectal cancers. Besides highlighting the role of FAAH and MAGL in cancer progression, this review provides an update on the anticancer capabilities of known and newly discovered FAAH and MAGL inhibitors and also provides further directions to develop FAAH and MAGL inhibitors as new candidates for cancer therapy.     

表面活性剂包衣Candida rugosa脂肪酶在异辛烷中的稳定性

The stability of Candida rugosa lipase coated with glutamic acid didodecyl ester ribitol amide was investigated taking esterification of lauryl alcohol and lauric acid in isooctane as a model reaction.At 30℃,the half-life of the activity of the coated lipase was ca 10h,the enzyme activity became less changed after 12h and the residual activity was 39% of the initial value ,The coated lipase obeyed a first-order deactivation model with a deactivation energy of 29.9 J.mol^-1.     

Lipase production in continuous culture of Candida rugosa

The yeast Candida rugosa produces multiple extracellular lipases. The production of extra‐ and intracellular lipases was investigated in continuous cultures using a sole or different mixtures of carbon sources. Also, the effect of different C:N ratios was tested. Lipase productivity in continuous cultures increased by 50% compared with data obtained from batch fermentations and depended on the dilution rate applied. Maximum yields relative to consumed substrate were obtained with oleic acid at low dilution rate. It was found that during nitrogen limitation, lipase activity was suppressed. All carbon source mixtures tested allowed both cell growth and lipase production, but extra‐ and intracellular lipase activities were affected by the combination of substrates used. Maximum extracellular lipolytic productivity was attained with lactic and oleic acid mixtures, probably due to the non‐repressor effect of these carbon sources. The chemical composition of the biomass also depended on the type of substrate used and was related to the accumulation of lipidic compounds as intracellular inclusions, which were observed when oleic acid was used as the carbon source. The results obtained were compared with previous data from batch and fed‐batch cultures in order to select the best process strategies for the lipase production with C rugosa. The best lipase yields were obtained in fed‐batch fermentations using oleic acid. Copyright © 2003 Society of Chemical Industry     

Enzymatic Analysis of Positional Distribution of Fatty Acids in Solid Fat by 1,3-Selective Transesterification with Candida antarctica Lipase B

A protocol for the analysis of the positional distribution of fatty acids (FA) in solid triacylglycerols (TAG) was developed using sn-1(3) selective alcoholysis catalyzed by immobilized Candida antarctica lipase B (CALB). One part by weight of solid fat and ten parts by weight of ethanol (99.5 %) were warmed to liquefy the fat. After adding 0.44 parts by weight of CALB, the mixture was shaken at 50 °C for 10 min then at 30 °C for 2.8 h. The recovery of 2-MAG after the 3-h transesterification reaction was ca. 85 % of the maximum theoretical yield (33 mol%), with the loss of 15 % attributable to the acyl migration from sn-2 to sn-1(3). The recovery was similar to that of the solvent-free alcoholysis of structured lipids, 1,3-dipalmitoyl, 2-oleoyl glycerol and 1,3-dioleoyl, 2-palmitoyl glycerol, conducted at 30 °C for 3 h. In contrast, the acyl migration from sn-1(3) to sn-2 was hardly observed. Because the detected acyl migration was only in the direction of sn-2 to sn-1(3), and not vice versa, it is proposed to determine the FA composition of the sn-2 position of TAG by the gas chromatographic analysis of 2-MAG fraction recovered from the enzymatic reaction mixture, and the FA composition of sn-1(3) position by a mass balance using the FA composition of TAG and of the sn-2 position as inputs. The procedure was successfully applied to palm oil and shea butter, and docosahexaenoic acid (DHA)-rich single cell oil from Aurantiochytrium sp. KH105 for the first time.     

假丝酵母99-125脂肪酶促酯化合成生物柴油的研究

1 INTRODUCTION Biodiesel, that is long-chain fatty acid short-chain alcohol esters (methyl, ethyl, propyl and butyl ester), is produced by esterification of fatty acids or inter- esterification of oils and fats. These fatty acid alcohol esters are not only used as important industrial addi- tives and surfactants, but also used for biofuel. The biodiesel is a biodegradable, environmental friendly, renewable substitute of diesel fuel[1]. The traditional production of biodiesel is by chem- i…     

Investigation of Structural Features of Two Related Lipases and the Impact on Fatty Acid Specificity in Vegetable Fats

One of the indispensable applications of lipases in modification of oils and fats is the possibility to tailor the fatty acid content of triacylglycerols (TAGs), to meet specific requirements from various applications in food, nutrition, and cosmetic industries. Oleic acid (C18:1) and stearic acid (C18:0) are two common long fatty acids in the side chain of triglycerides in plant fats and oils that have similar chemical composition and structures, except for an unsaturated bond between C9 and C10 in oleic acid. Two lipases from Rhizomucor miehei (RML) and Rhizopus oryzae (ROL), show activity in reactions involving oleate and stearate, and share high sequence and structural identity. In this research, the preference for one of these two similar fatty acid side chains was investigated for the two lipases and was related to the respective enzyme structure. From transesterification reactions with 1:1 (molar ratio) mixed ethyl stearate (ES) and ethyl oleate (EO), both RML and ROL showed a higher activity towards EO than ES, but RML showed around 10% higher preference for ES compared with ROL. In silico results showed that stearate has a less stable interaction with the substrate binding crevice in both RML and ROL and higher tendency to freely move out of the substrate binding region, compared with oleate whose structure is more rigid due to the existence of the double bond. However, Trp88 from RML which is an Ala at the identical position in ROL shows a significant stabilization effect in the substrate interaction in RML, especially with stearate as a ligand.     

乙醛对假丝酵母脂肪酶催化活性的影响

采用紫外可见分光光度法和荧光发射光谱法研究了乙醛溶液对假丝酵母脂肪酶水解三油酸甘油酯的催化活性和构象的影响。结果表明,低浓度乙醛能提高酶的催化水解活力,当乙醛浓度为0.221 5 mmol/L时,酶活力提高了18.84%;高浓度乙醛对酶活力有抑制作用,当乙醛浓度为3.322 5 mmol/L时,酶活力降低了25.85%。低浓度乙醛使酶催化反应的最适p H向碱性方向偏移,紫外吸收光谱和荧光发射光谱均有显著增强和光谱峰偏移现象。动力学分析表明,加入乙醛后,酶的V_(max)增大,K_m减小。     

Utilization of rice hull ash as a support material for immobilization of Candida cylindracea lipase

Rice hull ash was heated in a muffle furnace at 700°C for 2 h and metallic oxides were leached with 10% sulfuric acid. The acid-activated ash was then examined for immobilization of Candida cylindracea lipase. Immobilization was carried out by direct addition of the enzyme solution to the activated ash suspended in hexane. The immobilized lipase retained 30% of its hydrolytic activity, but thermal stability was greatly increased. Half-lives of the immobilized enzyme at 50, 60, and 70°C were 45, 17, and 4 min, respectively. Optimal pH of the immobilized enzyme was 7.2. The apparent K_m and V_max for olive oil were 41 mM and 99.5 μmol/h-mg solid, respectively.     

Study of a Lipase from Candida rugosa Diddens and Lodder

Lipasic system of Candida rugosa (CBS 613) strain was studied. The enzyme was purified in one step by hydrophobic chromatography. The properties of this lipase were determined. It is an oligomeric enzyme composed of five identical monomers of 46 kg · mol^?1. Its optimum reaction conditions are pH = 7 and temperature = 40°C. This enzyme presents a rapid thermal denaturation and then a more stable form. It is a cell-bound lipase which is induced by triacyl glycerols. This enzyme presents a high specificity for external positions on glycerol.     

Lipoprotein Lipase,Tissue Expression and Effects on Genes Related to Fatty Acid Synthesis in Goat Mammary Epithelial Cells

Lipoprotein lipase (LPL) serves as a central factor in hydrolysis of triacylglycerol and uptake of free fatty acids from the plasma. However, there are limited data concerning the action of LPL on the regulation of milk fat synthesis in goat mammary gland. In this investigation, we describe the cloning and sequencing of the LPL gene from Xinong Saanen dairy goat mammary gland, along with a study of its phylogenetic relationships. Sequence analysis showed that goat LPL shares similarities with other species including sheep, bovine, human and mouse. LPL mRNA expression in various tissues determined by RT-qPCR revealed the highest expression in white adipose tissue, with lower expression in heart, lung, spleen, rumen, small intestine, mammary gland, and kidney. Expression was almost undetectable in liver and muscle. The expression profiles of LPL gene in mammary gland at early, peak, mid, late lactation, and the dry period were also measured. Compared with the dry period, LPL mRNA expression was markedly greater at early lactation. However, compared with early lactation, the expression was lower at peak lactation and mid lactation. Despite those differences, LPL mRNA expression was still greater at peak, mid, and late lactation compared with the dry period. Using goat mammary epithelial cells (GMEC), the in vitro knockdown of LPL via shRNA or with Orlistat resulted in a similar degree of down-regulation of LPL (respectively). Furthermore, knockdown of LPL was associated with reduced mRNA expression of SREBF1, FASN, LIPE and PPARG but greater expression of FFAR3. There was no effect on ACACA expression. Orlistat decreased expression of LIPE, FASN, ACACA, and PPARG, and increased FFAR3 and SREBF1 expression. The pattern of LPL expression was similar to the changes in milk fat percentage in lactating goats. Taken together, results suggest that LPL may play a crucial role in fatty acid synthesis.     

棉油酸二乙醇酰胺

由于椰子油价格飞涨,本文介绍了一种以棉籽油为原料制备烷醇酰胺的方法。将精炼棉籽油所剩皂脚进行皂化、盐析、酸解、蒸馏等工艺制得棉油酸,经酯化、缩合得到棉油酸二乙醇酰胺。产品性能大体上优于椰油酸二乙醇酰胺,而成本降低了约2000元/t。     

Enzymatic esterification of DL‐menthol with propionic acid by lipase from Candida cylindracea

This work deals with the resolution of DL ‐menthol with propionic acid by Candida cylindracea lipase (Ccl) in organic solvent reaction systems and a reverse micelles system of sodium 1,4‐bis (2‐ethylhexyl) sulfosuccinate (AOT). The activity and stability as well as enantioselectivity of the lipase in two systems were studied. The results indicate that the lipase showed higher stability in reverse micelles than in organic solvent, which proved that the reverse micelles system has potential application for maintaining the activity of the enzyme for a long time. This is because lipase molecules can be entrapped in water‐containing micro‐drops of reverse micelles, avoiding direct‐contract with unfavorable organic medium. The enantioselectivity (E > 30, ee_p = 92.5) in the two systems is relatively high, although the conversion is moderate. The influence of the characteristic parameters of the two systems, such as pH, temperature, w₀ (molar ratio of water to AOT in reverse micelles systems) and water content (organic solvent) on the conversion of DL ‐menthol was also investigated. Copyright © 2005 Society of Chemical Industry     

Synthesis of Oleic Acid Esters with Immobilized Lipase

Esterification of oleic acid and alcohols with immobilized lipase from fungus Mucor miehei in a stirred batch reactor is presented in this article. The degree of conversion of oleic acid to esters was determined by measurement of acid values. The dependence of the synthesis of n-butyl oleate on the amount of enzyme, the influence of temperature on the equilibrium conversion for this reaction and the influence of temperature on initial velocities is presented. The influence of water added to reaction mixture at various reaction conditions on the activity of the enzyme preparation is also presented.     

Optimal Production and Biochemical Properties of a Lipase from Candida albicans

Lipases from microorganisms have multi-faceted properties and play an important role in ever-growing modern biotechnology and, consequently, it is of great significance to develop new ones. In the present work, a lipase gene from Candida albicans (CaLIP10) was cloned and two non-unusual CUG serine codons were mutated into universal codons, and its expression in Pichia pastoris performed optimally, as shown by response surface methodology. Optimal conditions were: initial pH of culture 6.86, temperature 25.53 °C, 3.48% of glucose and 1.32% of yeast extract. The corresponding maximal lipolytic activity of CaLIP10 was 8.06 U/mL. The purified CaLIP10 showed maximal activity at pH 8.0 and 25 °C, and a good resistance to non-ionic surfactants and polar organic solvent was noticed. CaLIP10 could effectively hydrolyze coconut oil, but exhibited no obvious preference to the fatty acids with different carbon length, and diacylglycerol was accumulated in the reaction products, suggesting that CaLIP10 is a potential lipase for the oil industry.     

Separation of Oleic Acid from Fatty Acid Impurities

Abstract

A method of oleic acid purification is described. The method consists of the following five steps: 1) cooling of the sample to 4°C for a partial separation of palmitic acid by crystallization, 2) distillation at reduced pressure (0.8 mmHg) for removal of lauric and myristic acids, 3) crystallization of stearic and palmitic acids from acetone at -25°C, 4) separation of oleic acid from palmitoleic and linoleic acids by oleic acid crystallization from aqueous methanol solutions at ?10°C, 5) reduced pressure (0.5 mmHg) distillation of the resulting oleic acid sample for removal of water and methanol. By utilizing the procedure described above, a sample containing only 82% oleic acid was refined to a product containing 98.7–98.9% oleic acid.