Lipase-Catalyzed Interesterification of Schizochytrium sp. Oil and Medium-Chain Triacylglycerols for Preparation of DHA-Rich Medium and Long-Chain Structured Lipids

DHA-rich medium and long-chain structured lipids (MLSL) were successfully synthesized by lipase-catalyzed interesterification of microbial oil from Schizochytrium sp. with medium-chain triacylglycerols (MCT) containing 99% of caprylic acid. Parameters that affected the reaction process were investigated and the conditions were selected as follows: lipase from Aspergillus oryzae, NS40086; reaction time, 8 hours; substrate molar ratio (MCT/microbial oil), 1:1; lipase load, 8 wt%; reaction temperature, 60 °C. Under these conditions, the proportions of MCT, MLSL, and long-chain triacylglycerols (TAG) in the final products were 12.5%, 62.8%, and 24.6%, respectively. The final product was then subjected to UPLC-MS/MS. Eighty-three types of TAG were identified, in which 54 types contained MCFA and MLSL species with relatively high contents were 22:6–8:0–8:0 (6.8%), 8:0–8:0–16:0 (7.5%), and 16:0–16:0–8:0 (7.5%). This product rich in MLSL with DHA and MCFA in the same TAG molecule is beneficial for fat digestion and absorption in infant and thus can increase the bioavailability of DHA at the molecular level.     

Lipase-mediated acidolysis of tristearin with CLA in a packed-bed reactor: A kinetic study

Solvent-free acidolysis of tristearin with CLA has been carried out in a packed-bed reactor. An immobilized lipase from Thermomyces lanuginosa (Lipozyme TL IM) was employed as the biocatalyst. Elevated temperatures (75°C) were utilized to eliminate solid substrates. The reaction kinetics were modeled by using a rate equation of the general Michaelis-Menten form. Both the extent of incorporation of CLA and the extent to which FFA were released were investigated. Positional analysis of the purified TAG obtained after a pseudo space time of 0.6 h indicated that CLA was preferentially incorporated at the sn-1,3 positions of the glycerol backbone, although 10% of the sn-2 positions were occupied by CLA residues. At a pseudo space time of 0.6 h, 38% of the initial CLA was incorporated in acylglycerols; the associated extent of hydrolysis was 8.3%.     

Combination of Oxyanion Gln114 Mutation and Medium Engineering to Influence the Enantioselectivity of Thermophilic Lipase from Geobacillus zalihae

The substitution of the oxyanion Q114 with Met and Leu was carried out to investigate the role of Q114 in imparting enantioselectivity on T1 lipase. The mutation improved enantioselectivity in Q114M over the wild-type, while enantioselectivity in Q114L was reduced. The enantioselectivity of the thermophilic lipases, T1, Q114L and Q114M correlated better with log p as compared to the dielectric constant and dipole moment of the solvents. Enzyme activity was good in solvents with log p < 3.5, with the exception of hexane which deviated substantially. Isooctane was found to be the best solvent for the esterification of (R,S)-ibuprofen with oleyl alcohol for lipases Q114M and Q114L, to afford E values of 53.7 and 12.2, respectively. Selectivity of T1 was highest in tetradecane with E value 49.2. Solvents with low log p reduced overall lipase activity and dimethyl sulfoxide (DMSO) completely inhibited the lipases. Ester conversions, however, were still low. Molecular sieves employed as desiccant were found to adversely affect catalysis in the lipase variants, particularly in Q114M. The higher desiccant loading also increased viscosity in the reaction and further reduced the efficiency of the lipase-catalyzed esterifications.     

高选择性脂肪酶的筛选及其拆分布洛芬

为了实现脂肪酶在有机相中对外消旋布洛芬的高效拆分,筛选得到了一株具有高立体选择性的脂肪酶产生菌-扩展青霉TS414(Penicillium expansum TS414). 实验探讨了水分、温度、有机溶剂、酶浓度、醇结构和醇浓度对酶促拆分反应的影响,确立了酯化反应的最佳反应体系：布洛芬6.46 mmol/L,脂肪酶53.3 mg/mL,正丙醇40 mmol/L,异辛烷15 mL,水0.5%(j),60℃条件下反应50 h. 在此条件下,拆分反应的转化率和对映体选择率分别为42%和429.63. 结果表明,Penicillium expansum TS414脂肪酶是一种较为理想的用于外消旋布洛芬拆分的工具酶,在布洛芬手性拆分方面具有广阔的应用前景.     

Expression of PD-1 Molecule on Regulatory T Lymphocytes in Patients with Insulin-Dependent Diabetes Mellitus

Type 1 diabetes is caused by autoreactive T cells that destroy pancreatic beta cells. Animal models suggested that a CD4⁺CD25⁺ population has a regulatory function capable of preventing activation and effector functions of autoreactive T cells. However, the role of CD4⁺CD25^high T cells in autoimmunity and their molecular mechanisms remain the subject of investigation. We therefore evaluated T regulatory cell frequencies and their PD-1 expression in the peripheral blood of long-standing diabetics under basal conditions and after CD3/CD28 stimulation. Under basal conditions, the percentages of T regulatory cells were significantly higher while that of T effector cells were significantly lower in patients than in controls. The ratio of regulatory to effector T cells was higher in patients than that in controls, suggesting that T regulatory cells were functional in patients. Percentages of total PD-1⁺, PD-1^low and PD-1^high expressing T regulatory cells did not change in patients and in controls. After stimulation, a defect in T regulatory cell proliferation was observed in diabetics and the percentages of total PD-1⁺, PD-1^low and PD-1^high expressing cells were lower in patients. Our data suggest a defective activation of T regulatory cells in long-standing diabetics due to a lower expression of PD-1 on their surface.     

Biodiesel Synthesis in a Solvent-Free System by Recombinant Rhizopus oryzae Lipase. Study of the Catalytic Reaction Progress

The recombinant 1,3-positional selective Rhizopus oryzae lipase (rROL) was used to synthesize biodiesel and monoacylglycerols simultaneously. The reaction was carried out in a solvent-free system with the enzyme immobilized on octadecyl-Sepabeads. Using response surface methodology, the methyl ester yield was optimized by means of the study of the effect of water, substrate molar ratio (methanol:olive oil) and methanol stepwise addition. It was concluded that in order to prevent enzyme inactivation by methanol, alcohol should be added slowly; otherwise a large amount of water would be present. Taking the best conditions, a 50.3 % yield was achieved in 3 h, which corresponds to 75.4 % of the acyl groups at the 1,3-position undergoing transesterification. It was also concluded that methyl esters result from the esterification of the free fatty acid hydrolyzed by the enzyme and also from a direct transesterification of oil. In addition, the fatty acid selectivity of rROL was found not to favor one fatty acid in olive oil over another.     

Optimized interesterification of virgin olive oil with a fully hydrogenated fat in a batch reactor: Effect of mass transfer limitations

The lipase‐catalyzed interesterification of virgin olive oil and fully hydrogenated palm oil (FHPO) was studied in a batch reactor operating at 75 °C. The reactions between olive oil {rich in OOO (32.36%), OPO (21.7%) and OLO (11.6%) [L = linoleic; O = oleic; P = palmitic acid]} and the fully hydrogenated fat {(36.5% PSP, 28.8% PPP, 23.2% SPS) [S = stearic acid]} produced semi‐solid fats. For an initial weight ratio of olive oil to FHPO of 60 : 40, the reaction product is a complex mixture of triacylglycerol (TAG) species. The TAG profile of the fat product is time dependent. Because of the high viscosity of the liquid reagent phase, it was important to determine if mass transfer effects were significant. Hence, the reaction was optimized with respect to the type and speed of agitation employed, temperature, use of solvent, and the type of biocatalyst. Three immobilized lipases [from Thermomyces lanuginosus (TL IM), Rhizomucor miehei (RM IM) and Candida antarctica B (Novozym 435)] were compared as catalysts for the interesterification reaction. Equilibrium is reached four times faster (in 1–4 h) with a magnetic stirrer to provide agitation than when agitation is not sufficient, i.e. when orbital agitation is employed. Equilibrium was reached faster with Lipozyme TL IM than with the other two lipases. The effects of all the factors investigated on the composition of the products have also been determined. Semi‐solid fats obtained with the non‐specific Novozym 435 contain levels of unsaturated fatty acid residues on sn‐2 sites that are similar to the products obtained with the 1(3)‐regiospecific enzymes Lipozyme TL IM and RM IM. The chemical properties of the product semi‐solid fat were characterized. The fat prepared using optimal reaction conditions contained 17.20% OPO, 13.61% OOO, 11.09% POP, and 10.35% OSP isomers as the primary products. The induction time obtained in the assay of the oxidative stability of the fat product was 21 h at 98 °C. The lipases Lipozyme TL IM and Novozym 435 were very stable with residual activities of 90 and 100%, respectively, after 15 batch reaction cycles.     

Novel lipase isolated from a Pseudomonas strain and its application in the synthesis of S(+)‐2‐O‐benzylglycerol‐1‐acetate

Isolation of a novel microbial lipase (EC 3.1.1.3) having specific catalytic activity for the synthesis of optically pure 2‐O‐benzylglycerol‐1‐acetate, the building block for the preparation of many β‐blockers, phospholipase A2 inhibitors and other biologically active compounds was the aim of this investigation. A Pseudomonas (strain G6), recently isolated from soil, produced an extracellular lipase. SDS–PAGE analysis showed that the lipase protein was a hexamer. The molecular weight of the sub‐units of the lipase protein were 10, 19, 29, 30, 47 and 53. The catalytic activity of the lipase was exploited for the synthesis of 2‐O‐benzylglycerol‐1‐acetate from 2‐O‐benzylglycerol through transesterification using vinyl acetate as acylating agent. High selectivity of the lipase towards the monoacetate product was demonstrated. A 97% enantiomeric excess (ee) of S(+)‐2‐O‐benzylglycerol‐1‐acetate was obtained when the reaction was carried out at room temperature with shaking. The lipase was highly active in anhydrous organic microenvironments and in non‐polar organic solvents with log P values above 2.5. © 2002 Society of Chemical Industry     

Alpha-Lipoic Acid Inhibits Spontaneous Diabetes and Autoimmune Recurrence in Non-Obese Diabetic Mice by Enhancing Differentiation of Regulatory T Cells and Showed Potential for Use in Cell Therapies for the Treatment of Type 1 Diabetes

Type 1 diabetes (T1D) is caused by the destruction of β cells in pancreatic islets by autoimmune T cells. Islet transplantation has been established as an effective treatment for T1D. However, the survival of islet grafts is often disrupted by recurrent autoimmunity. Alpha-lipoic acid (ALA) has been reported to have immunomodulatory effects and, therefore, may have therapeutic potential in the treatment of T1D. In this study, we investigated the therapeutic potential of ALA in autoimmunity inhibition. We treated non-obese diabetic (NOD) mice with spontaneous diabetes and islet-transplantation mice with ALA. The onset of diabetes was decreased and survival of the islet grafts was extended. The populations of Th1 cells decreased, and regulatory T cells (Tregs) increased in ALA-treated mice. The in vitro Treg differentiation was significantly increased by treatment with ALA. The adoptive transfer of ALA-differentiated Tregs into NOD recipients improved the outcome of the islet grafts. Our results showed that in vivo ALA treatment suppressed spontaneous diabetes and autoimmune recurrence in NOD mice by inhibiting the Th1 immune response and inducing the differentiation of Tregs. Our study also demonstrated the therapeutic potential of ALA in Treg-based cell therapies and islet transplantation used in the treatment of T1D.     

Synthesis of Urethane Derivatives of Mono- and Diacylglycerols for Use as HPLC Standards in the Enantiomeric Separation

This paper presents a convenient method for the preparation of reference standards for high-performance liquid chromatography (HPLC) used in stereospecific analysis of triacyl-sn-glycerols via monoacylglycerol or diacylglycerol intermediates. In the analysis, these partial acylglycerols are separated into their respective positional and enantiomeric isomer classes by chiral HPLC as their 3,5-dinitrophenylurethane derivatives or by silicic acid HPLC as their (S)- or (R)-1-(1-naphthyl)ethyl urethane derivatives. In this study, these urethane derivative standards were synthesized by the following novel procedure: first, partial urethane derivatives of glycerol were prepared by carbamoylation of glycerol with isocyanates; secondly, the products were separated into positional isomer classes by silicic acid HPLC, and; finally, a fatty acid was added to the partial urethanes using N,N′-dicyclohexylcarbodiimide. The identities of the resulting urethane derivatives of glycerol were verified by mass spectrometry and HPLC. This new procedure is advantageous in that standard urethane derivatives of partial acylglycerols can be synthesized from no more than 50 μg of fatty acids. This benefit is especially important in the case of rare and expensive fatty acids, such as very long chain polyunsaturated fatty acids, tetracosahexaenoic acid, and hexacosaheptaenoic acid, found in marine lipids.     

Simultaneous Use of ROCK Inhibitors and EP2 Agonists Induces Unexpected Effects on Adipogenesis and the Physical Properties of 3T3-L1 Preadipocytes

To elucidate the additive effects of an EP2 agonist, omidenepag (OMD) or butaprost (Buta) on the Rho-associated coiled-coil-containing protein kinase (ROCK) inhibitor, ripasudil (Rip) on adipose tissue, two- or three-dimension (2D or 3D) cultures of 3T3-L1 cells were analyzed by lipid staining, the mRNA expression of adipogenesis-related genes, extracellular matrix (ECM) molecules including collagen (Col) -1, -4 and -6, and fibronectin (Fn), and the sizes and physical properties of 3D organoids, as measured by a micro-squeezer. The results indicate that adipogenesis induced (1) an enlargement of the 3D organoids; (2) a substantial enhancement in lipid staining as well as the expression of the Pparγ, Ap2 and Leptin genes; (3) a significant softening of the 3D organoids, the effects of which were all enhanced by Rip except for Pparγ expression; and (4) a significant downregulation in Col1 and Fn, and a significant upregulation in Col4, Col6, the effects of which were unchanged by Rip. When adding the EP2 agonist to Rip, (1) the sizes of the 3D organoids were reduced substantially; (2) lipid staining was increased (OMD), or decreased (Buta); (3) the stiffness of the 3D organoids was substantially increased in Buta; (4-1) the expression of Pparγ was suppressed (2D, OMD) or increased (2D, Buta), and the expressions of Ap2 were downregulated (2D, 3D) and Leptin was increased (2D) or decreased (3D), (4-2) all the expressions of four ECM molecules were upregulated in 2D (2D), and in 3D, the expression of Col1, Col4 was upregulated. The collective findings reported herein indicate that the addition of an EP2 agonist, OMD or Buta significantly but differently modulate the Rip-induced effects on adipogenesis and the physical properties of 2D and 3D cultured 3T3-L1 cells.     

不同晶粒大小Silicalite-1分子筛的合成及其MTP反应性能

采用不同模板剂和碱源合成一系列Silicalite-1分子筛.所得样品通过X射线衍射仪(XRD),氮气吸脱附仪,电子扫描电镜(SEM),氨气程序升温脱附仪(NH3-TPD)和傅里叶红外光谱仪(FT-IR)表征.结果表明:TPA+具有稳定的正四面体结构,在合成Silicalite-1分子筛的过程中导向作用最强;通过改变碱源调节凝胶碱度,碱度降低,晶粒尺寸增大.将最佳条件下制备的Silicalite-1催化剂应用于甲醇制丙烯(MTP)反应中,与工业lurgi催化剂相比, Silicalite-1催化剂具有丙烯选择性高、失活速率慢的特点.     

Gut Microbiota Functional Traits,Blood pH,and Anti-GAD Antibodies Concur in the Clinical Characterization of T1D at Onset

Alterations of gut microbiota have been identified before clinical manifestation of type 1 diabetes (T1D). To identify the associations amongst gut microbiome profile, metabolism and disease markers, the 16S rRNA-based microbiota profiling and ¹H-NMR metabolomic analysis were performed on stool samples of 52 T1D patients at onset, 17 T1D siblings and 57 healthy subjects (CTRL). Univariate, multivariate analyses and classification models were applied to clinical and -omic integrated datasets. In T1D patients and their siblings, Clostridiales and Dorea were increased and Dialister and Akkermansia were decreased compared to CTRL, while in T1D, Lachnospiraceae were higher and Collinsella was lower, compared to siblings and CTRL. Higher levels of isobutyrate, malonate, Clostridium, Enterobacteriaceae, Clostridiales, Bacteroidales, were associated to T1D compared to CTRL. Patients with higher anti-GAD levels showed low abundances of Roseburia, Faecalibacterium and Alistipes and those with normal blood pH and low serum HbA_1c levels showed high levels of purine and pyrimidine intermediates. We detected specific gut microbiota profiles linked to both T1D at the onset and to diabetes familiarity. The presence of specific microbial and metabolic profiles in gut linked to anti-GAD levels and to blood acidosis can be considered as predictive biomarker associated progression and severity of T1D.     

LFA-1基因对胶原诱导小鼠关节炎关节局部引流淋巴结T细胞活性的影响

目的探讨淋巴细胞功能相关抗原1(LFA-1)在类风湿性关节炎形成中的作用。方法采用Ⅱ型胶原诱导小鼠关节炎模型,观察LFA-1基因敲除小鼠引流淋巴结T细胞增殖和细胞因子水平。结果LFA-1基因敲除小鼠在应用Ⅱ型胶原免疫后,引流淋巴结和关节局部Th1型细胞因子IFN-γ和IL-12p40mRNA水平明显低于野生型对照小鼠,Th2型细胞因子IL-4mRNA水平无明显变化。LFA-1基因敲除小鼠引流淋巴结来源的CD4+T细胞在体外受Ⅱ型胶原刺激后,其特异性增殖和细胞因子IFN-γ的产生也明显低于野生型对照小鼠。结论LFA-1基因缺失抑制了辅助性T细胞的活化和向Th1方向的分化,进而抑制关节炎的发生。