Dramatic solvent and hydration effects on the transition state of soybean peroxidase

Enzymes are shown to function in nonaqueous media; however, relatively little information is available on the influence of the organic solvent as well as its associated water content on the properties of the enzymatic transition states. A better understanding of these effects will be useful in developing kinetic models that can then be used to predict optimal solvent and substrate choices for enzymatic reactions in organic media. The influence of the reaction media on soybean peroxidase-catalyzed oxidation of para-substituted phenols was studied using Hammett analysis for several organic solvent systems. The catalytic activity and substrate specificity of the enzyme are influenced by the nature of the solvent and its associated hydration. These findings may allow one to draw conclusions about the reaction mechanism and the roles of solvent and solvent hydration on enzyme function.     

An Organic Soluble Lipase for Water-Free Synthesis of Biodiesel

Lipase AK was modified with short alkyl chains to form a highly organic soluble enzyme and was used to catalyze the synthesis of biodiesel from soybean oil in organic media. The effects of several key factors including water content, temperature, and solvent were examined for the solubilized enzyme in comparison with several other commercially available lipases. Whereas native lipases showed no activity in the absence of water, the organic soluble lipase demonstrated reaction rates of up to 33 g-product/g-enzyme h. The biocatalyst remains soluble in the biodiesel product, and therefore, there is no need to be removed because it is expected to be burned along with the diesel in combustion engines. This provides a promising one-pot mix-and-use strategy for biodiesel production.     

环番作为人工受体和人工酶的研究进展

本文综述了水溶液和有机溶液中环番作为人工受体包结有机分子的行为,重点综述了功能化环番作为人工酶的研究进展。     

有机相中的酶促拆分过程

总结了有机溶剂中的酶催化研究进展, 讨论了影响有机溶剂中酶促不对称转化过程对映体选择性的因素和拆分动力学。     

非水介质中酶催化的反应研究新进展

介绍了非水介质中酶催化反应有机合成中的应用及手性化合物的酶促拆分与合 成反应，分析了冷冻干燥保护剂和修饰剂对酶性质的影响，论述了固定化酶在有机 溶剂中的应用，并讨论了影响固定化酶性质的因素。     

酵母脂肪酶催化拆分外消旋2-甲基-1-丁醇

近年来,水相中酶催化拆分外消旋酯制备具有光学活性的手性酸或醇,其酶在非水体系的研究取得了重要进展,已发展到用含有微水或几乎无水的有机溶剂取代最初的水相,这样既可避免水参与有关的副反应,又有利于酶的回收。而且酶在某些有机溶剂中比在水中稳定,不会由于微生物污染而使酶失活。脂肪酶能够催化酯的     

异辛醇中酶催化高效合成阿莫西林的研究

通过比较6种有机溶剂作为反应介质时对阿莫西林合成的影响,发现反应介质在保持酶的催化活性和稳定性方面发挥着非常重要的作用,确定异辛醇为酶催化合成阿莫西林的反应介质.通过研究不同温度下异辛醇中酶催化合成阿莫西林的时间曲线,确定了最佳反应温度和反应时间,通过对底物浓度和酶浓度进行响应面优化,最终得到阿莫西林合成的最优反应条件,在最优条件下可得到91.37%的最大阿莫西林产率.     

Enantioselectivity of alcohol-treated Candida rugosa lipase in the kinetic resolution of racemic methyl 2-aryloxypropionates in water and aqueous organic media

Racemic methyl 2-aryloxypropionates (±)-1 were subjected to hydrolysis in water and in a series of two-phase aqueous organic media in the presence of Candida rugosa lipase (CRL). The biocatalytic material used was the enzyme of commercial CRL purified by treatment with different alcohols. The purification of CRL and the reaction medium play an important role in the enantioselection of racemates (±)-1. While it is not possible to use the same protocol for all substrates, by combining the different ways of purifying the enzyme with the various reaction media, it is possible to achieve high enantioselectivities of racemic esters.     

Specific anion effects on enzymatic activity in nonaqueous media

The present work shows that salt anions affect the activity of Pseudomonas cepacia lipase both in aqueous and in nonaqueous media (NAM) according to a Hofmeister series. The biocatalytic assay in water was the hydrolysis of p-nitrophenyl acetate, whereas the esterification between 1-hexyl-beta-D-galactopyranoside and palmitic acid was followed in an organic solvent. The solid lipase preparations to be used in NAM were obtained through lyophilization in the presence of concentrated solutions of Hofmeister salts (Na2SO4, NaH2PO4/Na2HPO4, NaCl, NaBr, NaI, NaSCN). Salts affect enzyme activity in organic media through two mechanisms: (1) enzyme protection during lyophilization; (2) enzyme activation during the reaction. At least in our case, the latter seems to be more important than the former. The decrease of the activation energy caused by the stabilization of the transition state due to "kosmotropic" anions might be the driving force of enzyme activation. According to the most recent findings, dispersion forces may be responsible of specific anion enzyme activation/deactivation in NAM.     

Enhanced activity of α-chymotrypsin in organic media using designed molecular staples

We report the enhancement of α-chymotrypsin activity in organic solvents using modified peptides bearing two crown ethers. The transesterification of N-acetyl-l-phenylalanine ethyl ester with 1-propanol was used as model reaction. Co-lyophilization of crown ether modified peptides with α-chymotrypsin prior to use resulted in an increase of enzyme catalytic activity in non-aqueous media. The efficiency of enzyme activation is dependent on the amino acid sequence of peptidic additives and on the positions of the amino acids bearing the crown ligand.     

离子液体中的酶催化反应

离子液体作为酶催化反应的介质正越来越多的受到关注,因为与传统有机溶剂和水相比,酶在离子液体中表现出了更好的活性、热稳定性、立体选择性、对映体选择性和可循环性。本文综述了近几年来脂肪酶、氧化还原酶、蛋白酶在离子液体中的催化反应。     

Activity and Enantioselectivity of the Hydroxynitrile Lyase MeHNL in Dry Organic Solvents

Water concentration affects both the enantioselectivity and activity of enzymes in dry organic media. Its influence has been investigated using the hydrocyanation of benzaldehyde catalyzed by hydroxynitrile lyase cross‐linked enzyme aggregate (MeHNL‐CLEA) as a model reaction. The enzyme displayed higher enantioselectivity at higher water concentration, thus suggesting a positive effect of enzyme flexibility on selectivity. The activity increased on reducing the solvent water content, but drastic dehydration of the enzyme resulted in a reversible loss of activity.     

Unique role of water content in enzymatic synthesis of ethyl lactate using ionic liquid as solvent

Enzymatic synthesis of ethyl lactate was studied in organic solvents and in ionic liquids to determine optimal media for the reaction, and to investigate the effect of water content on the ester yield. Experiments proved that Cyphos 202 ionic liquid is the best solvent affording the highest ethyl lactate yields. Furthermore, 20 times less enzyme sufficed to carry out the reaction in this ionic liquid compared to organic solvents; ionic liquid could be applied as solvent. Using water removal, the ester yield decreased since a side reaction, dimerization of lactic acid, occurred as well. In contrast to these results, without water removal, the produced water was partly consumed by the decomposition of lactoyllactic acid to free lactic acid, increasing thus the substrate concentration of the mixture and enhancing the ester yield.     

Enantioselective transacetylation of (R,S)-β-citronellol by propanol rinsed immobilized Rhizomucor miehei lipase

Background  

Use of enzymes in low water media is now widely used for synthesis and kinetic resolution of organic compounds. The frequently used enzyme form is the freeze-dried powders. It has been shown earlier that removal of water molecules from enzyme by rinsing with n-propanol gives preparation (PREP) which show higher activity in low water media. The present work evaluates PREP of the lipase (from Rhizomucor miehei) for kinetic resolution of (R,S)-β-citronellol. The acylating agent was vinyl acetate and the reaction was carried out in solvent free media.     

Optimization of Chlorophyllase-catalyzed Hydrolysis of Chlorophyll in Monophasic Organic Solvent Media

The effects of selected reaction parameters, including solvent hydrophobicity, initial water activity, agitation speed, temperature and enzyme concentration, on the biocatalytic efficiency of a chlorophyllase enzymatic extract from Phaeodactylum tricornutum in neat organic solvent media were investigated. The highest chlorophyllase specific activity of 322 nmol hydrolyzed chlorophyll per gram of protein per minute and bioconversion yield of 91% were obtained in the reaction mixture of hexane/2-octanone (98.3:1.7, v/v), at a controlled initial water activity of 0.90. R _O/A value, which is the ratio of the specific activity in the organic solvent to that in the aqueous/miscible organic solvent medium, was 1.5 × 10⁻³. To reduce the substrate diffusional limitations, the appropriate agitation speed and enzyme concentration were determined. The optimum temperature for maximal enzymatic activity and activation energy were 35°C and 105.0 kJ/mol, respectively. Although the catalytic efficiency of chlorphyllase in the neat organic solvent mixture was lower than that in the aqueous medium, its half-life time in the first environment at temperature ranging from 35 to 50°C was increased by 5.0 to 15.0 times.     

Tuning lipase enantioselectivity in organic media using solid-state buffers.

The enantioselectivity exhibited by Candida antarctica lipase B (CALB) in predominantly organic media has been studied for different enzyme protonation states. Alcoholysis of (+/-)-2-phenyl-4-benzyloxazol-5(4H)-one (1) using butan-1-ol as the nucleophile in low-water organic solvents was used as a model reaction. Using either organo-soluble bases or the newly introduced solid-state buffers of known pK(a), the protonation state of the lipase was altered. By choice of the appropriate solid-state buffer or organic base, the enantioselectivity could be selectively tuned. Both Et(3)N and the solid-state buffer pair CAPSO/CAPSO.Na were found to increase the enantioselectivity of the reaction catalyzed by CALB and that of another lipase (Mucor miehei). Significant differences to both the enantioselectivity and catalytic rate were observed, especially under hydrated conditions where byproduct acid was formed.     

离子液体中脂肪酶催化酯类合成的新进展

作为21世纪最具发展潜力的绿色溶剂,离子液体用于酶促合成的反应介质具有得天独厚的优势.与传统的有机溶剂相比,离子液体能够提高脂肪酶的稳定性和选择性,减少有机合成中的副反应和有毒气体的产生,后处理简单,可重复利用.参考10年来的文献,从脂肪酶催化酯类合成的常用离子液体种类、过程因素及场/反应器强化等方面进行了综述,同时展望了离子液体在未来酯类酶促合成领域的发展趋势.     

Effect of Organic Solvents on the Rate of Oxidation of Sulfoxides with Peroxy Acids

The reaction of sulfoxides with peroxy acids in various organic media was studied. The reaction mechanism involves the rapid formation of a sulfoxide­–peroxy acid intermediate which decomposes in the second stage to form carboxylic acid and the corresponding sulfone. The second stage is the rate-limiting step. The reaction medium significantly affects the rate of oxidation. The calculated activation parameters of the oxidation process indicate a compensation effect in the investigated reaction. Correlations between the main physicochemical parameters of solvents and the effective rate constants (k) of dimethyl sulfoxide oxidation with peroxy acids were found. Depending on the reaction conditions, the main factors affecting the k values are specific and nonspecific solvation of the reactants and structural factors.     

一种凝血酶亲和色谱介质的制备方法

对凝血酶-琼脂糖亲和色谱介质的制备方法进行了研究。首先使用凝血酶和溴化氰活化的琼脂糖制备凝血酶-琼脂糖亲和色谱介质,然后用生色底物法考察亲和色谱介质上凝血酶的活性,以凝血酶活性为指标对最佳偶联条件进行了优化。结果表明最佳条件为使用pH 8.3的Na₂CO₃-NaHCO₃溶液(含0.5 mol/L NaCl)为缓冲溶液,凝血酶用量为每1 g色谱介质加入凝血酶200 U,室温反应10 h。在最佳条件下所制备的色谱介质有较好的稳定性,在4℃条件下存放40天,亲和介质上的凝血酶活性仍有70.6%保留。该亲和色谱介质可广泛用于含凝血酶抑制剂的天然药物筛选和分离纯化。     

Enzymatic Reactions using Ionic Liquids for Green Sustainable Chemical Process; Stabilization and Activation of Lipases

The enzymatic reaction is highly respected from an environmentally-friendly point-of-view. Optimization of the reaction media and supporting materials of enzymes must be investigated in parallel with the effort to develop new enzymes. Lipases are frequently used for organic syntheses as synthetic tools even industry because of their acceptance of having a broad range of substrates, stability, and availability. We have investigated the possibility of ILs as both a solvent and activating or stabilization agent of enzymes, in particular, lipase as a model enzyme. ILs allowed recyclable use of a lipase and significant acceleration of transesterification, and also enhanced the stability and reaction activity of a lipase by immobilization through a lyophilization process. We discuss how we enhanced the enzyme capability using the IL engineering focusing on lipase-catalyzed reactions, i. e., realization of the recyclable use of an enzyme, how ILs mediated the enhanced reaction rate, and improved the stability of the enzyme.