The Use of Salt Hydrates as Water buffers To Control Enzyme Activity in Organic Solvents

The conversion of o-diphenols to o-quinones was carried out in chloroform using a dry powder prepared from mushrooms as the catalyst. Several salt hydrates proved effective in supplying the small amount of water necessary for catalysis. The efficacy of the hydrates was related to their water activities and their use provided a convenient method for controlling water activity in nearly non-aqueous conditions.     

Preliminary Studies on the Denaturation of Cross-Linked Lysozyme Crystals

When monoclinic lysozyme crystals are fully cross-linked with glutaraldehyde, and then the protein molecules are denatured while in the crystalline state, a single crystal-gel is formed which is a jelly-like crystal of a denatured protein molecule. It is highly disordered, but has crystalline optical and morphological properties and can be renatured to a cross-linked crystal resembling the original crystal as determined from the X-ray diffraction pattern. Experiments with the following denaturants are described: guanidinium chloride, bromoethanol, urea, and lithium chloride.     

Dispersibility of Nanocrystalline Cellulose in Organic Solvents

Russian Journal of Bioorganic Chemistry - Aqueous suspensions of nanocrystalline cellulose (NCC) were obtained by sulfuric acid hydrolysis using the standard procedure. Suspensions, films, and...     

低共熔溶剂对酶的影响及应用研究*

低共熔溶剂是由一定化学计量比的氢键受体和氢键供体组合而成的新型绿色溶剂,具有成本低、易制备、环境友好等特点,可以作为普通有机溶剂和离子液体的替代溶剂。酶作为生物催化剂时反应条件温和,对反应底物专一性高,并且具有极高的催化效率和反应速度。酶促反应通常发生在水溶液体系,但近年来发现在低共熔溶剂中酶促反应也能有效进行。综述酶与低共熔溶剂共同作用的机理以及低共熔溶剂在酶促反应中的应用,展望未来的研究方向,为酶促反应体系的进一步开发奠定理论基础。     

Biodesulfurization in Biphasic Systems Containing Organic Solvents

Biphasic systems can overcome the problem of low productivity in conventional media and have been exploited for biocatalysis. Solvent-tolerant microorganisms are useful in biotransformation with whole cells in biphasic reactions. A solvent-tolerant desulfurizing bacterium, Pseudomonas putida A4, was constructed by introducing the biodesulfurizing gene cluster dszABCD, which was from Rhodococcus erythropolis XP, into the solvent-tolerant strain P. putida Idaho. Biphasic reactions were performed to investigate the desulfurization of various sulfur-containing heterocyclic compounds in the presence of various organic solvents. P. putida A4 had the same substrate range as R. erythropolis XP and could degrade dibenzothiophene at a specific rate of 1.29 mM g (dry weight) of cells⁻¹ h⁻¹ for the first 2 h in the presence of 10% (vol/vol) p-xylene. P. putida A4 was also able to degrade dibenzothiophene in the presence of many other organic solvents at a concentration of 10% (vol/vol). This study is a significant step in the exploration of the biotechnological potential of novel biocatalysts for developing an efficient biodesulfurization process in biphasic reaction mixtures containing toxic organic solvents.     

Mixed Organic Solvents Induce Renal Injury in Rats

To investigate the injury effects of organic solvents on kidney, an animal model of Sprague-Dawley (SD) rats treated with mixed organic solvents via inhalation was generated and characterized. The mixed organic solvents consisted of gasoline, dimethylbenzene and formaldehyde (GDF) in the ratio of 2∶2:1, and were used at 12,000 PPM to treat the rats twice a day, each for 3 hours. Proteinuria appeared in the rats after exposure for 5–6 weeks. The incidences of proteinuria in male and female rats after exposure for 12 weeks were 43.8% (7/16) and 25% (4/16), respectively. Urinary N-Acetyl-β-(D)-Glucosaminidase (NAG) activity was increased significantly after exposure for 4 weeks. Histological examination revealed remarkable injuries in the proximal renal tubules, including tubular epithelial cell detachment, cloud swelling and vacuole formation in the proximal tubular cells, as well as proliferation of parietal epithelium and tubular reflux in glomeruli. Ultrastructural examination found that brush border and cytoplasm of tubular epithelial cell were dropped, that tubular epithelial cells were partially disintegrated, and that the mitochondria of tubular epithelial cells were degenerated and lost. In addition to tubular lesions, glomerular damages were also observed, including segmental foot process fusion and loss of foot process covering on glomerular basement membrane (GBM). Immunofluorescence staining indicated that the expression of nephrin and podocin were both decreased after exposure of GDF. In contrast, increased expression of desmin, a marker of podocyte injury, was found in some areas of a glomerulus. TUNEL staining showed that GDF induced apoptosis in tubular cells and glomerular cells. These studies demonstrate that GDF can induce both severe proximal tubular damage and podocyte injury in rats, and the tubular lesions appear earlier than that of glomeruli.     

Homogeneous Biocatalysis in Organic Solvents and Water-Organic Mixtures

Biocatalysis in non-aqueous media has undergone tremendous development during the last decade, and numerous reactions have been introduced and optimized for synthetic applications. In contrast to aqueous enzymology, biotransformations in organic solvents offer unique industrially attractive advantages, such as: drastic changes in the enantioselectivity of the reaction, the reversal of the thermodynamic equilibrium of hydrolysis reactions, suppression of water-dependent side reactions, and resistance to bacterial contamination. Currently, the field is dominated by heterogeneous biocatalysis based primarily on lyophilized enzyme powders, cross-linked crystals, and enzymes immobilized on inert supports that are mainly applied in enantioselective synthesis. However, low reaction rates are an inherent problem of the heterogeneous biocatalysis, while the homogeneous systems have the advantage that the elimination of diffusional barriers of substrates and products between organic and water phases results in an increase in the reaction rate. Here the discussion is focused on the correlation between activity and structure of the intact enzymes dissolved in neat organic solvents, as well as modifications of natural enzymes, which make them soluble and catalytically active in non-aqueous environment. Factors that influence conformation and stability of the enzymes are also discussed. Current developments in non-aqueous biocatalysts that combine advantages of protein modification and immobilization, i.e., HIP plastics, enzyme chips, ionic liquids, are introduced. Finally, engineering enzymes for biotransformations in non-conventional media by directed evolution is summarized.     

Storage of Pollen Grains in Organic Solvents: Effect of Organic Solvents on Leaching of Phospholipids and its Relationship to Pollen Viability

In vitro germinability and membrane integrity (as revealed bythe fluorochromatic reaction (FCR) test) of pollen grains ofCrotalaria retusa L. stored in various organic solvents forsix months at –20±2 °C were studied and correlatedwith leaching of lipids, phospholipids, sugars and free aminoacids from pollen grains into organic solvents during storage.Pollen grains stored in organic solvents with low dielectricconstants (a measure of their non-polar nature), such as hexane,cyclohexane and diethyl ether, showed high scores for germinationand FCR and very little leaching of phospholipids, sugars andamino acids. Pollen grains stored in solvents with high dielectricconstants (a measure of their polar nature) such as isopropanoland methanol did not show germination or positive FCR scores,but showed extensive leaching of phospholipids, sugars and freeamino acids. The viability of pollen grains stored in organicsolvents seems to be determined largely by the effect of theorganic solvents on pollen phospholipid composition, which inturn affects membrane integrity and consequently pollen viability. Crotalaria retusa, organic solvents, pollen storage, viability, phospholipids     

Modification of the Microenvironment of Enzymes in Organic Solvents. Substitution of Water by Polar Solvents

Enzyme catalysis in water-immiscible organic solvents is strongly influenced by the amount of water present in the reaction mixture. Effects of substitution of part of the water by other polar solvents were studied. In an alcoholysis reaction catalyzed by chymotrypsin deposited on celite, it was possible to exchange half of the water by formamide, ethylene glycol or dimethyl sulfoxide with often increased initial reaction rate. Furthermore, these substitutions caused the suppression of the competing hydrolysis reaction. However, formamide caused enzyme inactivation, and ethylene glycol participated as a reactant in the alcoholysis to some extent, hence dimethyl sulfoxide was considered the best water substitute among the solvents tested. These effects were noted for chymotrypsin catalyzed alcoholysis in several water immiscible solvents and also for interesterification reactions catalyzed by Candida cylindracea lipase on celite. In the latter case a change in the stereoselectivity was observed. At a low water content a high stereoselectivity was observed; when the amount of polar solvent was increased, either by doubling the water content or adding an equal amount of DMSO, the stereoselectivity decreased.     

Polyethylene Glycol-Modified Lipase Soluble and Active in Organic Solvents

A new approach in biotechnological processes is to use lipase modified with polyethylene glycol(PEG) which has both hydrophilic and hydrophobic properties. The PEG-lipase is soluble in organic solvents such as benzene and chlorinated hydrocarbons and exhibits high enzymic activity in organic solvents. The PEG-lipase catalyses the reverse reaction of hydrolysis in organic solvents; ester synthesis and ester exchange reactions. The PEG-lipase can also be conjugated to magnetite (Fe₃O₄). The magnetic lipase catalyses ester synthesis in organic solvents and can be readily recovered by magnetic force without loss of enzymic activity.     

Enhanced Reactivity of Rhizopus oryzae Lipase Displayed on Yeast Cell Surfaces in Organic Solvents: Potential as a Whole-Cell Biocatalyst in Organic Solvents

Immobilization of enzymes on some solid supports has been used to stabilize enzymes in organic solvents. In this study, we evaluated applications of genetically immobilized Rhizopus oryzae lipase displayed on the cell surface of Saccharomyces cerevisiae in organic solvents and measured the catalytic activity of the displayed enzyme as a fusion protein with α-agglutinin. Compared to the activity of a commercial preparation of this lipase, the activity of the new preparation was 4.4 × 10⁴-fold higher in a hydrolysis reaction using p-nitrophenyl palmitate and 3.8 × 10⁴-fold higher in an esterification reaction with palmitic acid and n-pentanol (0.2% H₂O). Increased enzyme activity may occur because the lipase displayed on the yeast cell surface is stabilized by the cell wall. We used a combination of error-prone PCR and cell surface display to increase lipase activity. Of 7,000 colonies in a library of mutated lipases, 13 formed a clear halo on plates containing 0.2% methyl palmitate. In organic solvents, the catalytic activity of 5/13 mutants was three- to sixfold higher than that of the original construct. Thus, yeast cells displaying the lipase can be used in organic solvents, and the lipase activity may be increased by a combination of protein engineering and display techniques. Thus, this immobilized lipase, which is more easily prepared and has higher activity than commercially available free and immobilized lipases, may be a practical alternative for the production of esters derived from fatty acids.     

Modification Effects of Organic Solvents on Microenvironment of the Enzyme in Thermolysin-catalyzed Peptide Synthesis of N-(Benzyloxycarbonyl)-l-phenylalanyl-l-phenylalanine Methyl Ester

Thermolysin-catalyzed peptide synthesis using N-benzyloxycarbonyl)-l-phenylalanine (Z-Phe) and l-phenylalanine methyl ester (Phe-OMe) as substrates was done mainly in a water-organic one phase solvent system. The organic solvent content used was less than the saturation concentration in buffer. With organic solvents with high log P values, the enzymatic activity increased as the organic solvent content increased; but further increases in the organic solvent content decreased the enzymatic activity, showing an “organic activity” profile. On the other hand, with organic solvents of low log P values, the enzymatic reaction was inhibited even by the initial addition of organic solvents. When a correlation between maximum activities and logP values or Hildebrand solubility parameters was investigated, a linear correlation was obtained among the same category of organic solvents, but not between categories. This suggests that the direct effect of organic solvents on the microenvironment of the enzyme largely depends on the molecular structure of the solvents.     

低水有机介质中的酶催化

酶不仅能在水溶液里催化化学反应,而且能在有机介质中显示催化活性．其中低水溶剂体系对有机合成最为有利．文章就低水溶剂体系中影响酶催化的三要素（水、溶剂和载体）以及酶在该体系表现出来的一些特殊性质进行了讨论,并列举了低水溶剂体系中的酶催化在有机合成,化学分析,和高分子化学等方面的应用．     

Biocatalysis in Organic Solvents with a Polymer-Bound Horseradish Peroxidase

This paper describes the preparation of polyethyleneglycol-bound horseradish peroxidase. Coupling with the polymer occurs via the glycolic moiety of the protein after an optimised oxidation process with periodate. Analysis of the modified enzyme shows that three chains of polymer are attached to the protein, which then becomes soluble and active in both chloroform and toluene.     

Biocatalysis in Organic Solvents with a Polymer-Bound Horseradish Peroxidase

This paper describes the preparation of polyethyleneglycol-bound horseradish peroxidase. Coupling with the polymer occurs via the glycolic moiety of the protein after an optimised oxidation process with periodate. Analysis of the modified enzyme shows that three chains of polymer are attached to the protein, which then becomes soluble and active in both chloroform and toluene.     

有机溶剂可溶的超氧化物歧化酶的制备及其性质

本文报道用谷氨酸、十二醇、葡萄糖酸内酯合成了一种精脂（２Ｃ（１２）ＧＥ）,并制备了ＳＯＤ－糖脂复合体．所得的ＳＯＤ－糖脂复合体是脂溶性的而不是水溶性的,它在乙醇等有机溶剂中的活性比在水中高,而且存在一最适有机溶剂浓度。其对温度、ｐＨ、蛋白酶水解的稳定性比天然ＳＯＤ明显增强。     

Microbial Oxidation of Tetradecanols and Related Substances in Organic Solvents

Microbial oxidations of n-tetradecane, tetradecanols and tetradecanoic acid were investigated by using intact cells of Corynebacterium equi, a hydrocarbon-assimilating bacterium, in an aqueous phase and organic solvents. The bacterial cells were hydrophobic and could be well dispersed in all organic solvents employed to give homogeneous reaction mixtures, and among them, isooctane was found to be the best for the reaction. n-Tetradecane and tetradecanoic acid were completely oxidized in the aqueous phase, but not in isooctane. In contrast, 1-tetradecanol was oxidized much more readily in isooctane than in the aqueous phase, and an oxidation product identified as myristyl myristate was accumulated in isooctane at the conversion rate of 80%. 2-Tetradecanol was also readily oxidized in isooctane, and 2-tetradecanone was obtained at the conversion rate of nearly 100%. Similar results were obtained when toluene and n-hexane were used as the solvent in place of isooctane, while no reaction was observed when chloroform was employed.