亚油酸异构酶及其性质

共轭亚油酸（conjugated linoleic acids简称CLA）是一种具有多种生理活性的天然脂肪酸。亚油酸异构酶可将亚油酸转化成CLA,亚油酸异构酶可以特异性地转化合成CLA,克服了化学法催化合成CLA的缺点。本文介绍了亚油酸异构酶的来源、分离纯化;比较了Butyrivibrio fibrisolvens、Lactobacillus reuteri、Propionibacterium acnes、Clostridium sporogenes等来源的亚油酸异构酶的性质。     

植物乳杆菌ZS2058生物转化共轭亚油酸的反应动力学

摘要：【目的】对本实验室从泡菜中筛选到的植物乳杆菌ZS2058完整细胞生物转化共轭亚油酸的反应动力学进行研究。【方法】探讨底物浓度、细胞浓度、反应体系pH值等因素对生物转化共轭亚油酸反应速度的影响,并通过双倒数和Hanes-Woolf作图法拟合反应初始阶段的速度方程。【结果】生物转化共轭亚油酸时存在明显的底物抑制现象,当亚油酸浓度为0.4 mg/mL时产c9, t11-共轭亚油酸的反应速度达最大值15.99 μg/(mL?h);反应速度随细胞浓度增加而上升,当细胞浓度为5×1010 cfu/mL时反应速度达到最高;最适pH值和最适反应温度分别为6.5和40 ℃。利用双倒数和Hanes-Woolf作图法求得米氏常数和最大反应速度,在低底物浓度下,反应初始阶段的反应规律与经典的米氏方程相符,而在高底物浓度下,存在明显的底物抑制现象。【结论】通过对植物乳杆菌ZS2058完整细胞催化合成共轭亚油酸各因素的考察,在得到最佳反应条件的同时建立了不同底物浓度范围内的反应速度方程,这对于实现共轭亚油酸的生产和研究其生理功能具有十分重要的理论价值。     

透性化嗜酸乳杆菌细胞转化亚油酸为共轭亚油酸的反应动力学

本实验旨在研究透性化嗜酸乳杆菌细胞生物转化共轭亚油酸的反应动力学。探讨了细胞浓度、底物浓度、反应体系pH值和温度等因素对生物转化共轭亚油酸反应速度的影响;建立了透性化嗜酸乳杆菌细胞生物转化共轭亚油酸的动力学模型。结果表明,透性化嗜酸乳杆菌细胞有利于共轭亚油酸的生物转化,最适细胞浓度、pH值和反应温度分别为10×1010ufc/mL、4.5和45℃;生物转化共轭亚油酸存在底物抑制现象,当亚油酸的浓度为0.6mg/mL时,反应速度达到最大值17.8μg/(mL·min)。在低亚油酸浓度下,反应初始阶段的反应规律与经典米氏方程相符,而在高亚油酸浓度下,存在底物抑制现象。在最适反应条件下建立了动力学模型,模型基本反映了共轭亚油酸的生物转化特性。     

曲霉菌油制备共轭亚油酸的研究

从南方红豆杉树皮形成层分离纯化得到一株产油曲霉E1.3,摇瓶发酵培养,索氏提取得曲霉油.采用气质联用分析脂肪酸成分,测得亚油酸含量最高达31.6%.本文首次以曲霉油作为原料,选用乙二醇单丁醚作为溶剂,KOH作催化剂,碱催化异构化法制备共轭亚油酸.通过紫外光谱跟踪反映进程,研究多种因素对亚油酸转化率的影响,对反应条件进行优化:温度140℃,反应周期3 h,强碱催化剂与原料质量比为0.2:1,原料与溶剂质量比为1:3.在此条件下,亚油酸的转化率达到86%.结果表明:曲霉油可以替代植物油作为碱催化异构化法制备共轭亚油酸的原料.     

植物乳杆菌ZS2058的亚油酸异构酶基因在乳酸克鲁维酵母中的克隆表达

目的：将植物乳杆菌ZS2058（Lactobacillus plantarum ZS2058）的亚油酸异构酶基因在乳酸克鲁维酵母（Kluyveromyces lactis）中进行克隆表达。方法：根据NCBI中已报道亚油酸异构酶（linoleate isomerase,LAI）基因的序列特征,设计引物对筛得的植物乳杆菌ZS2058进行PCR扩增,得到亚油酸异构酶全基因序列,克隆至乳酸克鲁维酵母表达载体pKLAC1,电转化得重组菌pKLAC1-LAI /Kluyveromyces lactis GG799。结果：SDS-PAGE检测,重组菌进行分泌表达获得目的蛋白,大小约为67 kDa;气相色谱（Gas Chromatogram,GC）检测到共轭亚油酸（conjugated linoleic acids,CLA）典型峰。结论：植物乳杆菌ZS2058中的亚油酸异构酶基因在乳酸克鲁维酵母中得到分泌表达,重组酶转化效率约为26%。     

反刍动物体内共轭亚油酸生物合成途径研究进展

共轭亚油酸是一组共轭双键具有不同位置和几何构象的亚油酸异构体,主要存在于反刍动物的肉及乳中。本文就其中2种具有重要生理功能的共轭亚油酸(c-9,t-11CLA和t-10,c-12CLA)的生物合成途径的研究进行综述,以期了解共轭亚油酸代谢途径及其调控路径,为今后利用合成生物学指导共轭亚油酸的合成奠定基础。     

共轭亚油酸降血脂及抗动脉粥样硬化作用的研究

目的:探讨共轭亚油酸降血脂及抗动脉粥样硬化形成的作用机制。方法:选用大鼠随机分为正常对照组,高脂模型组,c9,t11CLA:t10,c12CLA=2:1、1:1、1:3、1:6,只含t10,c12CLA共7组。实验至第8周末取血,检测血清胆固醇(TC)、三酰甘油(TG)、高密度脂蛋白(HDL)、低密度脂蛋白(LDL)和丙二醛(MDA)、超氧化物歧化酶(SOD)活性。结果:与高脂模型组比较,共轭亚油酸组大鼠血清TC、TG、MDA含量明显降低,HDL、SOD含量明显提高(P<0.05)。结论:共轭亚油酸具有降低血脂和抗动脉粥样硬化的作用。     

共轭亚油酸降血脂及抗动脉粥样硬化作用的研究

目的：探讨共轭亚油酸降血脂及抗动脉粥样硬化形成的作用机制。方法：选用大鼠随机分为正常对照组,高脂模型组,c9,t11CLA：t10,c12CLA=2：1、1：1、1：3、1：6,只含t10,c12CLA共7组。实验至第8周末取血,检测血清胆固醇（TC）、三酰甘油（TG）、高密度脂蛋白（HDL）、低密度脂蛋白（LDL）和丙二醛（MDA）、超氧化物歧化酶（SOD）活性。结果：与高脂模型组比较,共轭亚油酸组大鼠血清TC、TG、MDA含量明显降低,HDL、SOD含量明显提高（P〈0.05）。结论：共轭亚油酸具有降低血脂和抗动脉粥样硬化的作用。     

植物乳杆菌P8产共轭亚油酸条件的优化

以植物乳杆菌P8菌株为研究对象,系统研究了发酵条件对共轭亚油酸生成的影响。分别研究了培养条件和培养基成分对共轭亚油酸产量的影响。通过单因素和正交试验表明:植物乳杆菌P8产共轭亚油酸的最佳条件为:培养时间为22 h、亚油酸(LA)添加量为0.9 mg/mL、接种量为1.5%、氮源采用2 g/L的胰蛋白胨,碳源采用3 g/L的葡萄糖。     

植物乳杆菌ZS2058在磷酸盐缓冲液体系中生物转化共轭亚油酸

植物乳杆菌ZS2058是从泡菜中筛选到一株具有转化共轭亚油酸能力的乳酸菌。该菌株在MRS培养基中经0.5mg/mL的亚油酸诱导培养后,所获得的菌体细胞具有较强的转化能力。文中就植物乳杆菌ZS2058水洗细胞在磷酸盐缓冲液体系中生物转化共轭亚油酸进行了深入研究。在非厌氧条件下,植物乳杆菌ZS2058在亚油酸浓度为1mg/mL,湿细胞质量浓度约为150mg/mL,120r/min、37℃的条件下反应24h后,能将亚油酸转化为共轭亚油酸和羟基脂肪酸,其中c9,t11-CLA占所产生的CLA总量的96.4%,产量可高达312.4μg/mL,说明该菌株有很强的专一性。随着反应进一步进行,反应至36h时,c9,t11-CLA含量逐渐减少,伴随着大量羟基脂肪酸的产生;并且,以CLA(c9,t11-CLA和t10,c12-CLA的混合样品)为底物进行反应时,c9,t11-CLA被转化为羟基脂肪酸。由此可知,c9,t11-CLA可能是该菌株生物转化LA过程中的一个中间产物。     

Isolation of a novel strain of Butyrivibrio fibrisolvens that isomerizes linoleic acid to conjugated linoleic acid without hydrogenation, and its utilization as a probiotic for animals

AIM: Isolation of a new strain of Butyrivibrio fibrisolvens possessing great capacity to produce conjugated linoleic acid (CLA) in order to utilize as a probiotic for animals. METHODS AND RESULTS: A novel strain (MDT-5) was isolated from the goat rumen, which exclusively converted linoleic acid (LA) to CLA, because of its high LA isomerase activity with virtually no CLA reductase activity. MDT-5 also converted linolenic acid to conjugated linolenic acid that may be more bioactive than CLA. The oral administration of MDT-5 every other day to mice for 2 weeks resulted in increased amounts of CLA in the contents of the large intestine (2.5-fold), as well as in adipose tissue (threefold). Feeding a high-LA diet, as well as prolonging the period of MDT-5 administration, further increased the CLA content in body fat. CONCLUSIONS: MDT-5 has by far greater ability to produce CLA than any other known bacteria. Administration of MDT-5 to mice increases CLA production in the large intestine, which results in increased CLA absorption. SIGNIFICANCE AND IMPACT OF THE STUDY: MDT-5 may be useful in pet animals as a probiotic to provide CLA continuously.     

Linoleic acid isomerase in Lactobacillus plantarum AKU1009a proved to be a multi-component enzyme system requiring oxidoreduction cofactors

Linoleic acid isomerase in Lactobacillus plantarum was found to be a novel multi-component enzyme system widespread in membrane and soluble fractions. The isomerization reaction involved a hydration step, 10-hydroxy-12-octadecenoic acid production from linoleic acid, as part of the reaction, and the hydration reaction was catalyzed by the membrane fraction. Both membrane and soluble fractions were required for the whole isomerization reaction, i.e., conjugated linoleic acid (CLA) production from linoleic acid, and for CLA production from 10-hydroxy-12-octadecenoic acid, a reaction intermediate. The multi-component enzyme system was inhibited by o-phenanthroline, and divalent metal ions such as Ni(2+) and Co(2+) restored activity. Metal oxides such as VO(4)(3+), MoO(4)(2+), and MnO(4)(2+) enhanced activity. The multi-component enzyme systems required oxidoreduction cofactors such as NADH together with FAD or NADPH for total activity.     

Linoleate isomerase activity occurs in lactic acid bacteria strains and is affected by pH and temperature

Aims: To investigate the ability of lactic acid bacteria (LAB) to convert linoleic acid (LA) and α‐linolenic acid (α‐LNA) to conjugated linoleic acid (CLA) and conjugated linolenic acid (CLNA), respectively. To assess pH and temperature influences on CLA and CLNA production by Lactobacillus sakei LMG 13558. Methods and Results: A screening of 48 LAB yielded one Lactobacillus curvatus, five Lactobacillus plantarum and four Lact. sakei strains displaying linoleate isomerase (LAI) activity. CLNA conversion percentages varied largely (1–60%). CLA conversion, occurring in three strains, was lower (2–5%). The LAI gene sequences of the ten LAI‐positive strains shared 75–99% identity with the LAI gene sequence of a Lact. plantarum AS1.555. At pH 6·2, CLA and CLNA production by Lact. sakei LMG 13558 was higher at 30°C than at 20 and 25°C. At pH 5·5 (30°C) or 37°C (pH 6·2), LA was not converted and α‐LNA only slightly converted. Conclusions: LAB show strain‐dependent LAI activity. Production of CLA and CLNA is affected by pH and temperature, as shown for Lact. sakei LMG 13558. Significance and Impact of the Study: Several LAB produce CLA and/or CLNA, as shown for Lact. sakei and Lact. curvatus for the first time. These findings offer potential for the manufacturing of fermented functional foods.     

A new strain of Butyrivibrio fibrisolvens that has high ability to isomerize linoleic acid to conjugated linoleic acid

A new strain of Butyrivibrio fibrisolvens (TH1) that has high potential to produce conjugated linoleic acid (CLA) was isolated. Strain TH1 had higher LA isomerase (LA-I) activity, and was much more tolerant to linoleic acid (LA) than other strains examined. However, high CLA reductase (CLA-R) activity resulted in the temporary accumulation of CLA and subsequent conversion to trans-vaccenic acid (t-VA). When LA was added to growing TH1 cultures in a solution with dimethylsulfoxide (LA/DMSO), CLA produced was greater than when LA was added in a mixture with bovine serum albumin (BSA). The number of viable cells decreased upon addition of LA/DMSO, but then increased as the CLA decreased upon its conversion to t-VA. This result suggests that B. fibrisolvens can resume growing by the removal of CLA from the cells. Most CLA was released from B. fibrisolvens cells by gentle washing with BSA, suggesting that CLA bound to the cells might be removed in the rumen and large intestine. Thus, CLA production by B. fibrisolvens in the digestive tract could be increased by a reduction in CLA-R activity without accompanying an overall decrease in the cell number of B. fibrisolvens. Fatty acids (FAs) with 18 carbon backbone inducted LA-I activity, whereas unsaturated FAs induced CLA-R activity, suggesting that FAs stimulate the synthesis of LA-I and CLA-R. Providing a diet with a low ratio of unsaturated to saturated FAs may favor CLA production.     

Effect of linoleic acid concentration on conjugated linoleic acid production by Butyrivibrio fibrisolvens A38

Butyrivibrio fibrisolvens A38 inocula were inhibited by as little as 15 microM linoleic acid (LA), but growing cultures tolerated 10-fold more LA before growth was inhibited. Growing cultures did not produce significant amounts of cis-9, trans-11 conjugated linoleic acid (CLA) until the LA concentration was high enough to inhibit biohydrogenation, growth was inhibited, and lysis was enhanced. Washed-cell suspensions that were incubated anaerobically with 350 microM LA converted most of the LA to hydrogenated products, and little CLA was detected. When the washed-cell suspensions were incubated aerobically, biohydrogenation was inhibited, CLA production was at least twofold greater, and CLA persisted. The LA isomerase reaction was very rapid, but the LA isomerase did not recycle like a normal enzyme to catalyze more substrate. Cells that were preincubated with CLA lost their ability to produce more CLA from LA, and the CLA accumulation was directly proportional (r(2) = 0.98) to the initial cell density. Growing cells were as sensitive to CLA as LA, the LA isomerase and reductases of biohydrogenation were linked, and free CLA was not released. Because growing cultures of B. fibrisolvens A38 did not produce significant amounts of CLA until the LA concentration was high, biohydrogenation was arrested, and the cell density had declined, the flow of CLA from the rumen may be due to LA-dependent bacterial inactivation, death, or lysis.     

Genetic and functional aspects of linoleate isomerase in Lactobacillus acidophilus

While the remarkable health effects of conjugated linoleic acid (CLA) catalyzed from α-linoleic acid by the enzyme linoleate isomerase (LI, EC 5.2.1.5) are well recognized, how widely this biochemical activity is present and the mechanisms of its regulation in lactic acid bacteria are unknown. Although certain strains of Lactobacillus acidophilus can enrich CLA in fermented dairy products, it is unknown if other strains share this capacity. Due to its immense economic importance, this work aimed to investigate genetic aspects of CLA production in L. acidophilus for the first time. The genomic DNA from industrial and type strains of L. acidophilus were subjected to PCR and immunoblot analyses using the putative LI gene of L. reuteri ATCC 55739 as probe. The CLA production ability was estimated by gas chromatography of the biomass extracts. The presumptive LI gene from L. acidophilus ATCC 832 was isolated and sequenced. The resulting sequence shared 71% identity with that of L. reuteri and at least 99% with reported sequences from other L. acidophilus strains. All the strains accumulated detectable levels of CLA and tested positive by PCR and immunoblotting. However, no apparent correlation was observed between the yields and the hybridization patterns. The results suggest that LI activity might be common among L. acidophilus and related species and provide a new tool for screening potential CLA producers.     

Effect of Linoleic Acid Concentration on Conjugated Linoleic Acid Production by Butyrivibrio fibrisolvens A38

Butyrivibrio fibrisolvens A38 inocula were inhibited by as little as 15 μM linoleic acid (LA), but growing cultures tolerated 10-fold more LA before growth was inhibited. Growing cultures did not produce significant amounts of cis-9, trans-11 conjugated linoleic acid (CLA) until the LA concentration was high enough to inhibit biohydrogenation, growth was inhibited, and lysis was enhanced. Washed-cell suspensions that were incubated anaerobically with 350 μM LA converted most of the LA to hydrogenated products, and little CLA was detected. When the washed-cell suspensions were incubated aerobically, biohydrogenation was inhibited, CLA production was at least twofold greater, and CLA persisted. The LA isomerase reaction was very rapid, but the LA isomerase did not recycle like a normal enzyme to catalyze more substrate. Cells that were preincubated with CLA lost their ability to produce more CLA from LA, and the CLA accumulation was directly proportional (r² = 0.98) to the initial cell density. Growing cells were as sensitive to CLA as LA, the LA isomerase and reductases of biohydrogenation were linked, and free CLA was not released. Because growing cultures of B. fibrisolvens A38 did not produce significant amounts of CLA until the LA concentration was high, biohydrogenation was arrested, and the cell density had declined, the flow of CLA from the rumen may be due to LA-dependent bacterial inactivation, death, or lysis.     

Conjugated linoleic acid accumulation via 10-hydroxy-12-octadecaenoic acid during microaerobic transformation of linoleic acid by Lactobacillus acidophilus

Specific isomers of conjugated linoleic acid (CLA), a fatty acid with potentially beneficial physiological and anticarcinogenic effects, were efficiently produced from linoleic acid by washed cells of Lactobacillus acidophilus AKU 1137 under microaerobic conditions, and the metabolic pathway of CLA production from linoleic acid is explained for the first time. The CLA isomers produced were identified as cis-9, trans-11- or trans-9, cis-11-octadecadienoic acid and trans-9, trans-11-octadecadienoic acid. Preceding the production of CLA, hydroxy fatty acids identified as 10-hydroxy-cis-12-octadecaenoic acid and 10-hydroxy-trans-12-octadecaenoic acid had accumulated. The isolated 10-hydroxy-cis-12-octadecaenoic acid was transformed into CLA during incubation with washed cells of L. acidophilus, suggesting that this hydroxy fatty acid is one of the intermediates of CLA production from linoleic acid. The washed cells of L. acidophilus producing high levels of CLA were obtained by cultivation in a medium containing linoleic acid, indicating that the enzyme system for CLA production is induced by linoleic acid. After 4 days of reaction with these washed cells, more than 95% of the added linoleic acid (5 mg/ml) was transformed into CLA, and the CLA content in total fatty acids recovered exceeded 80% (wt/wt). Almost all of the CLA produced was in the cells or was associated with the cells as free fatty acid.