Lipase-catalyzed glycerolysis in ionic liquids directed towards diglyceride synthesis

This work examined the lipase-catalyzed glycerolysis of triglycerides (TG) in a list of commercially available ionic liquids (ILs) with varied cations and anions for the purpose of developing an efficient reaction protocol for diglyceride (DG) production and to understand whether ILs could assist the reaction systems. The reaction performances (reaction rate, TG conversion and DG yield) were found to be greatly dependent on the structure and property of ILs. The reactions in [TOMA·Tf₂N] and Ammoeng 120 produced comparable yield of DG to those most efficient conventional systems but with less by-products. Temperature enhancement generally yields positive effect on the conversion of TG, which was much more significant for the ILs with high viscosity. Unusually, increasing substrate concentration in many types of ILs led to enhanced conversion and yield; whereas the increase of glycerol/TG ratio resulted in a dramatic improvement of the reactions in the ILs with strong acidic anions. This work also sorted out some promising IL candidates, namely the ILs with good DG formation selectivity and the ones being able to achieve high TG conversion, which offered possibility to design binary IL systems. Overall, this study presented the first attempt concerning evaluation and characterization of lipase-catalyzed glycerolysis of TG for DG production in IL-based systems.     

Enzyme activity in dialkyl phosphate ionic liquids

The activity of four metagenomic enzymes and an enzyme cloned from the straw mushroom, Volvariella volvacea were studied in the following ionic liquids, 1,3-dimethylimidazolium dimethyl phosphate, [mmim][dmp], 1-ethyl-3-methylimidazolium dimethyl phosphate, [emim][dmp], 1-ethyl-3-methylimidazolium diethyl phosphate, [emim][dep] and 1-ethyl-3-methylimidazolium acetate, [emim][OAc]. Activity was determined by analyzing the hydrolysis of para-nitrobenzene carbohydrate derivatives. In general, the enzymes were most active in the dimethyl phosphate ionic liquids, followed by acetate. Generally speaking, activity decreased sharply for concentrations of [emim][dep] above 10% v/v, while the other ionic liquids showed less impact on activity up to 20% v/v.     

Investigation of 1,3-Dipolar Cycloaddition Reactions of Unsaturated Nitrones in Ionic Liquids

The effect of the media (achiral and chiral ionic liquids) on the stereochemistry of intramolecular 1,3-dipolar cycloaddition reactions of D-galactose-derived ω-unsaturated nitrones, leading to bicyclic isoxazolidines, has been investigated.     

Ionic Liquid Mediated Synthesis of 5-Halouracil Nucleosides: Key Precursors for Potential Antiviral Drugs

Synthesis of antiviral 5-halouracil nucleosides, also used as key precursors for the synthesis of other potential antiviral drugs, has been demonstrated using ionic liquids as convenient and efficient reaction medium.     

Thermal Stability and Activity Regain of Horseradish Peroxidase in Aqueous Mixtures of Imidazolium-Based Ionic Liquids

Thermal deactivation kinetics of horseradish peroxidase (HRP) were studied from 45 to 90 °C in phosphate buffer and 5–25% (v,w/v) 1-butyl-3-methylimidazolium tetrafluoroborate [BMIM][BF₄] and 1-butyl-3-methylimidazolium chloride [BMIM][Cl]. HRP activity at 25 °C was not affected by the presence of ionic liquids up to 20% (v,w/v). Increasing the ionic liquids concentration up to 25% (v,w/v) changed the biphasic character of deactivation kinetics to an apparent single first-order step. The presence of 5–10% (v/v) [BMIM][BF₄] significantly improved HRP thermal stability with lower activation energies for the deactivation second phase (83–87 kJ mol⁻¹). After deactivation, enhanced activity regain of the enzyme, up to 70–80% of the initial activity, was found in 25% (v/v) [BMIM][BF₄] and 10% (w/v) [BMIM][Cl] and correlated to prevalence of the deactivation first phase.     

Enzymatic selective acylation of glycosides in ionic liquids: significantly enhanced reactivity and regioselectivity

The enzymatic selective acylations of carbohydrates in ionic liquids were explored in both organic solvents and ionic liquids to see any significant differences in terms of reactivity and regioselectivity between two different classes of reaction media. Monoprotected glycosides (methyl-6-O-trityl-glucosides and galactosides) were chosen as the substrates with Candida rugosa lipase as an acylation enzyme. Two organic solvents, THF and chloroform, and two ionic liquids, [BMIM]⁺PF₆⁻ ([BMIM]⁺ = 1-butyl-3-methylimidazolium) and [MOEMIM]⁺PF₆⁻ ([MOEMIM]⁺ = 1-methoxyethyl-3-methylimidazolium), were employed as reaction media. The enzymatic reactions were performed in the presence of vinyl acetate at room temperature. It was observed that the reactions in ionic liquids took place more rapidly and more selectively than those in conventional organic solvents.     

Ionic liquids in biotechnology: applications and perspectives for biotransformations

Ionic liquids are considered as an alternative to organic solvents for catalysis. The literature in this field is reviewed with focus on advantageous use of ionic liquids in biocatalysis and biotransformations. The overview reveals that the exploration and mapping of ionic liquids with respect to biocatalysis is still sketchy. It is apparent that advantages can be gained in view of activity, stability and selectivity. Furthermore, integration of reaction and separation has a high potential in the field. The review presents quantitative data on the productivities, space–time yields, as well as stability as far as they can be extracted from the literature. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Enzymatic Synthesis of Ethyl-glucoside Monooleate with Lipase in Solvent-free Medium

Ethylglucoside monooleate was synthesized by esterification between ethylglucoside and oleic acid with immobilized lipase from Candida antarctica in a solvent-free system. It was shown that a stirred tank reactor was suitable for the enzymatic reaction process involving substrates with low miscibility, in which the biocatalyst was recycled five times without significant activity loss. Removal of the co-product, water, from the reaction medium by carrying out the reaction under reduced pressure benefited the esterification reaction and increased the monooleate yield up to 97% within 8 hours.     

Enzymatic Synthesis of Ethyl-glucoside Monooleate with Lipase in Solvent-free Medium

Ethylglucoside monooleate was synthesized by esterification between ethylglucoside and oleic acid with immobilized lipase from Candida antarctica in a solvent-free system. It was shown that a stirred tank reactor was suitable for the enzymatic reaction process involving substrates with low miscibility, in which the biocatalyst was recycled five times without significant activity loss. Removal of the co-product, water, from the reaction medium by carrying out the reaction under reduced pressure benefited the esterification reaction and increased the monooleate yield up to 97% within 8 hours.     

Production of extracellular enzymes in the entomopathogenic fungus Verticillium lecanii

This study investigates the mechanisms as well as strategies for purification and characterization of potential enzymes involved inpathogenesis of entomopathogenic fungi. The test strain of Verticillium lecanii that was screened, during the present investigation,proved to be an efficient producer of protein and polysaccharide degrading enzymes (amylase, protease, and lipase), henceindicating versatility in biochemical mechanisms. Halo zones produced colony growth of V. lecanii on agar confirmed activity ofprotease, amylase and lipase enzyme by the V. lecanii isolate. Enzymatic Index (EI) observed were: Protease – 2.195, Amylase-2.196, Lipase- 2.147. Spectrophotometric analysis of enzymatic activity of V.lecanii at five different pH – 3, 5, 7, 9, 11 revealed thathighest proteolytic activity of the V. lecanii isolate was reported at pH 7 and 9 whereas proteolytic activity was minimum at acidicpH 3. Maximum amylolytic activity of V. lecanii on the 7^th day of inoculation was at pH 3 i.e. in an acidic environment in contrast toneutral pH 7. Maximum lipolytic activity of V. lecanii was found at pH 7. Since enzyme production in entomopathogenic fungi isspecific and forms an important criterion for successful development as well as improvement of mycoinsecticides, hence asignificant conclusion from the present analysis is the degree of variation in secretion of enzymes in test strain of Verticillium lecanii.     

Growth of Escherichia coli, Pichia pastoris and Bacillus cereus in the Presence of the Ionic Liquids [BMIM][BF4] and [BMIM][PF6] and Organic Solvents

The influence of the two most commonly used ionic liquids (1-butyl-3-methyl imidazolium tetrafluoroborate, [BMIM][BF₄], 1-butyl-3-methyl imidazolium hexafluorophosphate, [BMIM][PF₆]) and three selected organic solvents (dimethylsulfoxide, ethanol, methanol) on the growth of Escherichia coli, Pichia pastoris and Bacillus cereus was investigated. [BMIM][BF₄] was toxic at 1% (v/v) on all three microorganisms. The minimal inhibitory concentration (MIC) of [BMIM][BF₄] on E. coli growth was between 0.7 and 1% (v/v). In contrast, [BMIM][PF₆] was less toxic for P. pastoris and B. cereus, whereas E. coli was not able to tolerate [BMIM][PF₆] (MIC value: 0.3–0.7% v/v). Growth of P. pastoris was unaffected by [BMIM][PF₆] at 10% (v/v). Similar results were found for dimethylsulfoxide. Thus, ionic liquids (ILs) can have substantial inhibitory effects on the growth of microorganisms, which should be taken into account for environmental reasons as well as for the use of ILs as co-solvents in biotransformations. Revisions requested 2 November 2005; Revisions received 20 December 2005     

2,4-表油菜素内酯对高温胁迫下甜瓜幼苗抗氧化酶活性和光合作用的影响

以耐热甜瓜品种红绿早脆和热敏感品种白玉香为材料,在人工气候箱内采用基质栽培法,研究了2,4-表油菜素内酯(EBR)处理对高温胁迫下甜瓜幼苗抗氧化酶活性和光合作用的影响.结果显示:EBR能有效促进高温胁迫下甜瓜幼苗的生长,增加保护酶活性,增强植株抗氧化能力,使脯氨酸和可溶性蛋白含量升高,丙二醛(MDA)含量下降,能有效缓解高温胁迫引起的膜脂过氧化伤害;同时,光合指标净光合速率(Pn)和气孔导度(Gs)升高,胞间CO2浓度(Ci)和蒸腾速率(Tr)下降;叶绿素荧光参数初始荧光(F0)下降,最大光化学效率(Fv/Fm)、光系统Ⅱ光能捕获效率(Fv′/Fm′)和实际光化学效率(ФPSⅡ)升高,且热敏感品种白玉香变化幅度大于耐热品种红绿早脆.研究结果说明,EBR有利于甜瓜幼苗在高温胁迫下抗氧化酶活性的提高和对光能的捕获与转换,促进了甜瓜幼苗的生长,降低高温胁迫对甜瓜幼苗的抑制作用,且对热敏感品种白玉香效果大于耐热品种红绿早脆.     

不同类型玉米发育籽粒中淀粉合成及相关酶活性比较

以普通玉米、爆裂玉米、甜玉米和糯玉米为试材,分析和比较不同类型的玉米品种之间籽粒发育过程中淀粉合成及相关酶活性的变化。结果表明,淀粉合成速率和蔗糖合成酶(SS)、可溶性淀粉合成酶(SSS)、束缚态淀粉合成酶(GBSS)、淀粉分支酶(SBE)、去分支酶(DBE)活性都呈单峰曲线变化。30～40 DAP,普通玉米的SS活性显著高于其他3种类型;类型间平均和最大SSS活性水平的顺序为普通玉米>糯玉米>爆裂玉米>甜玉米;30～40 DAP,普通玉米GBSS活性最高,糯玉米GBSS活性最低;20～40 DAP,糯玉米SBE活性最高;甜玉米的DBE活性很低,并且在40 DAP完全丧失。淀粉合成速率与SS、SSS、GBSS和SBE活性相关程度比较高,与腺苷二磷酸葡萄糖焦磷酸化酶(AGP酶)和DBE活性相关不显著。推测AGP酶虽然为淀粉合成提供直接前体ADPG,但可能SS活性过低致使其限速作用比AGP酶的还强,AGP酶潜在的限速作用无法表现,SS成为玉米籽粒淀粉合成的限速因子。GBSS对直链淀粉积累起重要的促进作用;SSS和SBE对支链淀粉积累起重要的促进作用。     

Recombinant Destabilase-Lysozyme: Synthesis de novo in E. coli and Action Mechanism of the Enzyme Expressed in Spodoptera frugiperda

Destabilase-lysozyme (DL) from salivary gland secretion of the medicinal leech (Hirudo medicinalis) is as a member of the invertebrate lysozyme family, which sharply differs from other lysozyme families. In this study, DL lysozyme function was confirmed during expression of a gene encoding DL in Escherichia coli. Several constructs of the expression vectors pKK OmpA and pET-3A with or without bacterial, leech, or yeast signal peptides (SP) were used. The use of a construct without signal peptide genes resulted in normal growth of the transformed cells. Transformation of E. coli cells with the constructs containing SP was accompanied by the disruption of the forming cells. The use of the expression vector pET-32 LTC-System for production of DL as a fusion protein with thioredoxin also resulted in normal cell growth. However, specific activity of DL isolated from such cells was significantly lower than that of enzyme purified from extracts of Spodoptera frugiperda cells, which were infected with the baculovirus vector carrying DL cDNA. It is shown that the action mechanism of invertebrate lysozyme does not differ from that of other families: recombinant DL from S. frugiperda extracts catalyzed cleavage of synthetic substrate, hexamer of N-acetylglucosamine, to di- and tetramers, which is typical for enzymatic function of other lysozyme families.     

Tellurium-Induced Alterations in 3-Hydroxy-3-Methylglutaryl-CoA Reductase Gene Expression and Enzyme Activity: Differential Effects in Sciatic Nerve and Liver Suggest Tissue-Specific Regulation of Cholesterol Synthesis

The demyelination of peripheral nerves that results from exposure of developing rats to tellurium is due to inhibition of squalene epoxidase, a step in cholesterol biosynthesis. In sciatic nerve, cholesterol synthesis is greatly depressed, whereas in liver, some compensatory mechanism maintains normal levels of cholesterol synthesis. This tissue specificity was further explored by examining, in various tissues, gene expression and enzyme activity of 3-hydroxy-3-methylglutaryl-CoA reductase, the rate-limiting enzyme in cholesterol biosynthesis. Exposure to tellurium resulted in pronounced increases in both message levels and enzyme activity in liver, the expected result consequent to up-regulation of this enzyme in response to decreasing levels of intracellular sterols. In contrast to liver, levels of mRNA and enzyme activity in sciatic nerve were both decreased during the tellurium-induced demyelinating period. The temporal pattern of changes in 3-hydroxy-3-methylglutaryl-CoA reductase message levels in sciatic nerve seen following exposure to tellurium was similar to the down-regulation seen for mRNA specific for PNS myelin proteins. Possible mechanisms for differential control of cholesterol biosynthesis in sciatic nerve and liver are discussed.