Activation of enzymes for nonaqueous biocatalysis by denaturing concentrations of urea

Urea is one of the most commonly used denaturants of proteins. However, herein we report that enzymes lyophilized from denaturing concentrations of aqueous urea exhibited much higher activity in organic solvents than their native counterparts. Thus, instead of causing deactivation, urea effected unexpected activation of enzymes suspended in organic media. Activation of subtilisin Carlsberg (SC) in the organic solvents (hexane, tetrahydrofuran, and acetone) increased with increasing urea concentrations up to 8 M. Active-site titration results and activity assays indicated the presence of partially unfolded but catalytically active SC in 8 M urea; however, the urea-modified enzyme retained high enantioselectivity and was ca. 80 times more active than the native enzyme in anhydrous hexane. Likewise, the activity of horseradish peroxidase (HRP) lyophilized from 8 M urea was ca. 56 times and 350 times higher in 97% acetone and water-saturated hexane, respectively, than the activity of HRP lyophilized from aqueous buffer. Compared with the native enzyme, the partially unfolded enzyme may have a more pliant and less rigid conformation in organic solvents, thus enabling it to retain higher catalytic activity. However, no substantial activation was observed for alpha-chymotrypsin lyophilized from urea solutions in which the enzyme retained some activity, illustrating that the activation effect is not completely general.     

Lipase‐catalyzed enantioselective transesterification of prochiral 1‐((1,3‐dihydroxypropan‐2‐yloxy)methyl)‐5,6,7,8‐tetrahydroquinazoline‐2,4(1H,3H)‐dione in ionic liquids

The application of ionic liquids as solvents for transesterification of prochiral pirymidine acyclonucleoside using lipase (EC 3.1.1.3) Amano PS from Burkholderia cepacia (BCL) is reported. The effect of using medium reaction, acyl group donor, and temperature on the activity and enantioselectivity of BCL was studied. From the investigated ionic solvents, the hydrophobic ionic liquid [BMIM]PF₆] was the preferred medium for enzymatic reactions. However, the best result was obtained in the mixture [BMIM][PF₆]:TBME (1:1 v/v) at 50°C. Enzyme activity and selectivity in [BMIM][PF₆]:TBME (1:1 v/v) was slightly higher in than in conventional organic solvents (for example, TBME), and in this condition, good activity and enantioselectivity were associated with unique properties of ionic liquid such as hydrophobicity and high polarity. Independently of solvents, monester of (R)‐configuration was obtained in excess. Under optimal conditions, desymmetrization of the prochiral compound using different acyl donors was performed. If vinyl butyrate was used as the acylating agent, BCL completely selectively acylated enantiotopic hydroxyl groups.     

溶剂稳定性蛋白酶产生菌的筛选和鉴定

自油污土样等样品中分离获得一株具有产胞外溶剂稳定性蛋白酶的耐有机溶剂极端微生物,经BIOLOG系统鉴定及16S rDNA序列(GenBank,EF105377)分析,该菌株为Bacillus licheniformis YP1。该菌株能耐受中浓度盐、强碱性环境(pH12)及多种不同浓度的有机溶剂,但对多种抗生素敏感。在YP1产蛋白酶发酵过程中添加各种有机溶剂结果表明,丙酮虽抑制了菌体生物量的生长却促进了单位菌体的蛋白酶分泌,而长链烷醇如辛醇、十二醇等能强烈抑制该蛋白酶的分泌。该菌株所产蛋白酶经11种50%(V/V)有机溶剂处理后均能保留高活力。该溶剂稳定性蛋白酶在有机相生物催化等领域具有良好的应用前景。     

Biocatalysis using an organic-soluble enzyme for the preparation of poly(lactic acid) in organic solvents

Proleather from Bacillus sp. was chemically modified with decanoyl chloride for enhanced activity for the preparation of poly(lactic acid) in organic solvents. The modified enzyme was highly soluble (up to 44 mg-protein/ml) and active in various organic solvents including chloroform, tetrahydrofuran (THF), pyridine and acetone. The organic-soluble proleather efficiently catalyzed the polymerization of ethyl lactate. The reaction rate was 4-22 times that of native proleather, depending upon the solvent applied. The solubilized enzyme showed a highest activity at 50 degrees C, the same optimum temperature for both the native proleather and an immobilized enzyme, Novozyme-435. Denaturation of the enzymes' protein structures appeared to be the critical factor regulating the optimum activity temperature. Differential scanning calorimetry (DSC) analyses of the enzymes showed endothermic peaks around 55 degrees C, indicating the proteins' structures altered in that temperature range. Interestingly, the activity of the solubilized enzyme showed a more complicated water dependence as compared to native proleather.     

Laboratory evolution of cytochrome p450 BM-3 monooxygenase for organic cosolvents

Cytochrome p450 BM-3 (EC 1.14.14.1) catalyzes the hydroxylation and/or epoxidation of a broad range of substrates, including alkanes, alkenes, alcohols, fatty acids, amides, polyaromatic hydrocarbons, and heterocycles. For many of these notoriously water-insoluble compounds, p450 BM-3's K(m) values are in the millimolar range. Polar organic cosolvents are therefore added to increase substrate solubility and achieve high catalytic efficiency. Using p450 BM-3 as a catalyst for these important transformations requires that we improve its ability to tolerate the cosolvents. By directed evolution, we improved the activity of p450 BM-3 in the presence of dimethylsulfoxide (DMSO) and tetrahydrofuran (THF), achieving increases in specific activity up to 10-fold in 2% (v/v) THF and 6-fold in 25% (v/v) DMSO. The engineered p450 BM-3's are also significantly more resistant to acetone, acetonitrile, dimethylformamide, and ethanol as cosolvents in the reaction.     

Preparation and kinetic behavior of immobilized whole cell biocatalysts

Actinoplanes missouriensis (for glucose isomerase), Kluyveromyces fragilis (for beta-galactosidase), and Saccharomyces cerevisiae (for invertase) cells were successfully entrapped within cellulose and cellulose di- and triacetate beads employing several carried solvent systems. Cellulose beads prepared using a melt of dimethylsulfoxide (DMSO) and N-ethylpyridinium chloride (NEPC), or cellulose diacetate using a mixture of acetone and DMSO as solvent, were found to be promising as carriers for the invertase system, cellulose triacetate beads with DMSO as solvent for yeast beta-galactosidase, and cellulose beads with a melt of DMSO and NEPC as solvent for glucose isomerase. The kinetic behavior of A. missouriensis glucose isomerase whole cell cellulose beads in a plug-flow column reactor was studied as an example system in greater detail.     

Lipase in aqueous‐polar organic solvents: Activity,structure, and stability

Studying alterations in biophysical and biochemical behavior of enzymes in the presence of organic solvents and the underlying cause(s) has important implications in biotechnology. We investigated the effects of aqueous solutions of polar organic solvents on ester hydrolytic activity, structure and stability of a lipase. Relative activity of the lipase monotonically decreased with increasing concentration of acetone, acetonitrile, and DMF but increased at lower concentrations (upto ～20% v/v) of dimethylsulfoxide, isopropanol, and methanol. None of the organic solvents caused any appreciable structural change as evident from circular dichorism and NMR studies, thus do not support any significant role of enzyme denaturation in activity change. Change in 2D [15N, 1H]‐HSQC chemical shifts suggested that all the organic solvents preferentially localize to a hydrophobic patch in the active‐site vicinity and no chemical shift perturbation was observed for residues present in protein's core. This suggests that activity alteration might be directly linked to change in active site environment only. All organic solvents decreased the apparent binding of substrate to the enzyme (increased K_m); however significantly enhanced the k_cat. Melting temperature (T_m) of lipase, measured by circular dichroism and differential scanning calorimetry, altered in all solvents, albeit to a variable extent. Interestingly, although the effect of all organic solvents on various properties on lipase is qualitatively similar, our study suggest that magnitudes of effects do not appear to follow bulk solvent properties like polarity and the solvent effects are apparently dictated by specific and local interactions of solvent molecule(s) with the protein.     

Enantioselective synthesis of (S)‐naproxen using immobilized lipase on chitosan beads

S‐naproxen by enantioselective hydrolysis of racemic naproxen methyl ester was produced using immobilized lipase. The lipase enzyme was immobilized on chitosan beads, activated chitosan beads by glutaraldehyde, and Amberlite XAD7. In order to find an appropriate support for the hydrolysis reaction of racemic naproxen methyl ester, the conversion and enantioselectivity for all carriers were compared. In addition, effects of the volumetric ratio of two phases in different organic solvents, addition of cosolvent and surfactant, optimum pH and temperature, reusability, and inhibitory effect of methanol were investigated. The optimum volumetric ratio of two phases was defined as 3:2 of aqueous phase to organic phase. Various water miscible and water immiscible solvents were examined. Finally, isooctane was chosen as an organic solvent, while 2‐ethoxyethanol was added as a cosolvent in the organic phase of the reaction mixture. The optimum reaction conditions were determined to be 35 °C, pH 7, and 24 h. Addition of Tween‐80 in the organic phase increased the accessibility of immobilized enzyme to the reactant. The optimum organic phase compositions using a volumetric ratio of 2‐ethoxyethanol, isooctane and Tween‐80 were 3:7 and 0.1% (v /v/v), respectively. The best conversion and enantioselectivity of immobilized enzyme using chitosan beads activated by glutaraldehyde were 0.45 and 185, respectively.     

Effects of organic solvents on the enzyme activity of Trypanosoma cruzi glyceraldehyde-3-phosphate dehydrogenase in calorimetric assays

In drug discovery programs, dimethyl sulfoxide (DMSO) is a standard solvent widely used in biochemical assays. Despite the extensive use and study of enzymes in the presence of organic solvents, for some enzymes the effect of organic solvent is unknown. Macromolecular targets may be affected by the presence of different solvents in such a way that conformational changes perturb their active site structure accompanied by dramatic variations in activity when performing biochemical screenings. To address this issue, in this work we studied the effects of two organic solvents, DMSO and methanol (MeOH), in the isothermal titration calorimetry (ITC) kinetic assays for the catalyzed reaction of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from Trypanosoma cruzi. The solvent effects on T. cruzi GAPDH had not yet been studied. This enzyme was shown here to be affected by the organic solvents content up to 5.0% for MeOH and up to 7.5% for DMSO. The results show that when GAPDH is assayed in the presence of DMSO (5%, v/v) using the ITC experiment, the enzyme exhibits approximately twofold higher activity than that of GAPDH with no cosolvent added. When MeOH (5%, v/v) is the cosolvent, the GAPDH activity is sixfold higher. The favorable effects of the organic solvents on the Michaelis-Menten enzyme-substrate complex formation ensure the consistency of the biological assays, structural integrity of the protein, and reproducibility over the measurement time. The reaction was also kinetically monitored by standard spectrophotometric assays to establish a behavioral performance of T. cruzi GAPDH when used for screening of potential inhibitors.     

Guanine nucleotide regulation of adenylate cyclase in permeabilized cells of Saccharomyces cerevisiae

Adenylate cyclase in permeabilized cells of Saccharomyces cerevisiae was examined. Among various permeabilization procedures, including organic solvents, detergents and other reagents, dimethylsulfoxide (DMSO) and digitonin treatments resulted in the highest recovery of adenylate cyclase activity. Incubation of cells at 30 degrees C with digitonin at 0.01% to 0.1%, or DMSO at 20% to 40% for 15 to 30 min gave optimal adenylate cyclase activity. The enzyme activity in digitonin-permeabilized cells could be supported only by Mn2+, whereas Mg2+ with or without guanine nucleotides did not support cyclase activity. DMSO-permeabilized cells exhibit efficient Mn2+- and Mg2+/Gpp[NH]p-dependent stimulation. Furthermore, digitonin added to yeast membranes at a 1:50 detergent to protein ratio (w/w) abolishes guanyl nucleotide regulation without significantly affecting the Mn2+-supported cyclase activity. The superiority of DMSO is further supported by the fact that recovery of adenylate cyclase activity is better in the DMSO-treated cells than in the digitonin-treated cells. DMSO most probably causes less disturbance of the fabric of the native cell. We conclude that digitonin, but not DMSO, uncouples the catalytic unit of adenylate cyclase from the regulatory GTP binding (ras) proteins.     

土壤宏基因组文库来源酯酶的鉴定与表征

【目的】利用宏基因组学技术挖掘土壤微生物来源的新型酯酶。【方法】构建土壤微生物宏基因组文库,利用三丁酸甘油酯平板法对所构建的文库进行筛选,并对阳性克隆中鉴定出的酯酶基因进行异源表达和生物化学特性分析。【结果】通过筛选文库中的12万个克隆,获得了一个阳性克隆,对克隆中的DNA片段进行序列分析,发现了一个可能的酯酶基因,通过研究其表达产物,确定其最适pH为9.0,最适反应温度为56°C,在90°C下仍可保持20%的酶活性;能专一性水解短链脂类,对长链脂类无水解作用;对一定浓度范围内的有机试剂如二甲基亚砜、甲醇、乙醇有较好的耐受性,尤其当二甲基亚砜含量为10%(体积比)时,相对酶活可提高44%。【结论】不依赖于微生物可培养性的宏基因组学技术可以发现新的活性酶,本研究获得的对高温、有机试剂有较好耐受性的酯酶ESTYN1具有在工业生产中应用的潜力。     

Activity,stability and enantioselectivity of lipase-coated microcrystals of inorganic salts in organic solvents

Lipase-coated microcrystals of inorganic salts were prepared by dissolving enzymes in buffers and then mixing with 3 volumes of saturated salt solutions followed by drop-wise addition into polar precipitating organic solvents. The Mucor javanicus lipase-coated microcrystals did not show any activity for esterification of lauric acid with 1-propanol in isooctane when NaCl and Na₂SO₄ were used as the salts but showed much higher activity than the enzyme powder when KCl (10.0 times) and K₂SO₄ (5.8 times) were used as the salts and precipitated in 1-propanol. Acetonitrile was found to be the best precipitating solvent for preparing M. javanicus lipase-coated microcrystals, with enzyme activities 26.2 and 22.4 times higher than that of the enzyme powder when KCl and K₂SO₄ were used as precipitating salts, respectively. The presence of water in the precipitating solvents markedly decreased the enzyme activity. The M. javanicus lipase-coated microcrystals prepared using K₂SO₄ as the salt and acetonitrile as the precipitating solvent was as active at 80°C as at 40°C. No significant improvement in enantioselectivity of Candida rugosa lipase-coated microcrystals was observed for transesterification of 1-phenylethanol with vinyl acetate in hexane when the microcrystals were prepared by dissolving the enzymes in salt solutions containing 25% (v/v) of acetone or 2-propanol before precipitating in polar solvents.     

α‐chymotrypsin in water–acetone and water–dimethyl sulfoxide mixtures: Effect of preferential solvation and hydration

We investigated water/organic solvent sorption and residual enzyme activity to simultaneously monitor preferential solvation/hydration of protein macromolecules in the entire range of water content at 25°C. We applied this approach to estimate protein destabilization/stabilization due to the preferential interactions of bovine pancreatic α‐chymotrypsin with water‐acetone (moderate‐strength H‐bond acceptor) and water‐DMSO (strong H‐bond acceptor) mixtures. There are three concentration regimes for the dried α‐chymotrypsin. α‐Chymotrypsin is preferentially hydrated at high water content. The residual enzyme activity values are close to 100%. At intermediate water content, the dehydrated α‐chymotrypsin has a higher affinity for acetone/DMSO than for water. Residual enzyme activity is minimal in this concentration range. The acetone/DMSO molecules are preferentially excluded from the protein surface at the lowest water content, resulting in preferential hydration. The residual catalytic activity in the water‐poor acetone is ～80%, compared with that observed after incubation in pure water. This effect is very small for the water‐poor DMSO. Two different schemes are operative for the hydrated enzyme. At high and intermediate water content, α‐chymotrypsin exhibits preferential hydration. However, at intermediate water content, in contrast to the dried enzyme, the initially hydrated α‐chymotrypsin possesses increased preferential hydration parameters. At low water content, no residual enzyme activity was observed. Preferential binding of DMSO/acetone to α‐chymotrypsin was detected. Our data clearly demonstrate that the hydrogen bond accepting ability of organic solvents and the protein hydration level constitute key factors in determining the stability of protein–water–organic solvent systems.     

不同实验方法检测常用有机溶剂对细菌活性的影响及其安全使用限量

【目的】采用不同实验方法测定常用有机溶剂二甲基亚砜(DMSO)、丙酮和乙醇对细菌活性的影响,以指导抗菌类药物体外抑菌实验所用溶剂的选择和添加限量。【方法】采用常规体外抑菌实验方法(纸片扩散法、肉汤稀释法),并参照生长曲线法检测有机溶剂DMSO、丙酮和乙醇对大肠杆菌(Escherichia coli)及金黄色葡萄球菌(Staphylococcus aureus)的抑菌作用,采用扫描电子显微镜(SEM)观察溶剂作用后的细菌形态变化。【结果】3种溶剂对E.coli和S.aureus抑菌率达到20%时,在肉汤稀释法下,DMSO、丙酮、乙醇的浓度(体积比)分别为1.00%、0.25%、2.00%和1.00%、1.00%、0.50%;在生长曲线法下,溶剂浓度(体积比)分别为0.50%、1.00%、0.50%和1.00%、0.50%、0.50%;而在纸片扩散法下,32%(体积比)DMSO和32%(体积比)乙醇对E.coli产生明显抑菌圈,但3种溶剂对S.aureus均无抑菌圈出现。3种方法比较后得出:当3种溶剂的抑菌率达到20%时,溶剂浓度(体积比)均低于0.5%,对细菌整体生长活性影响较小。SEM结果表明控制溶剂使用限量可有效减少其对E.coli生长过程的影响。【结论】相对于DMSO和丙酮,乙醇对微生物生长繁殖能力的影响更加明显;采用相同浓度有机溶剂时,液态条件下(肉汤稀释法和生长曲线法)微生物受到有机溶剂的影响较大。     

Purification and characterization of the enantioselective esterase from Kluyveromyces marxianus CBS 1553

An intracellular esterase from the yeast Kluyveromyces marxianus CBS 1553 with interesting enantioselective hydrolytic activity towards racemic esters of 1,2-O-isopropylidene glycerol (IPG) was purified and characterized. Optimal culture conditions for the obtainment of the enantioselective esterase on a 5 l-fermentation scale were investigated. Two esterase activities (EST1 and EST2) in the crude cell extract were identified by native PAGE with specific activity staining and separated from each other by anion-exchange chromatography. EST1 showed higher activity and enantioselectivity than EST2 in the resolution of racemic IPG acetate and was further purified by hydrophobic interaction chromatography and preparative electrophoresis (final specific activity approximately = 300 U mg(-1), showing a main protein band with a molecular mass of 29 kDa. EST1 showed optimal activity between pH 8.0 and 10.0 and was stable in the pH range 7.0-10.0. Moreover, it was rather thermostable and active up to 80 degrees C, and retained most of its activity in the presence of 15% (v/v) of various organic solvents. The enzyme showed similar Vmax in the hydrolysis of the acetate esters of IPG, whereas the Km value towards (S)-IPG acetate was significantly lower than the one towards the (R)-enantiomer (5.3 and 70 microM, respectively). Finally, comparison of EST1 activity in the presence of different glycerol esters and synthetic substrates with different chain lengths showed a strong preference of this biocatalyst for short-chain substrates.     

Epoxide hydrolase activity of Streptomyces strains

The discovery of epoxide hydrolases within a Streptomyces sp. strain collection is described. Screening was performed in 96 well microtiter plates using a modified 4-(p-nitrobenzyl)pyridine assay with styrene oxide, 1,2-epoxy-hexane or 3-phenyl ethylglycidate (3-PEG) as substrates. Out of 120 strains investigated, S. antibioticus Tü4, S. arenae Tü495 and S. fradiae Tü27 exhibited epoxide hydrolase activity. These strains were further investigated by performing laboratory-scale biotransformations utilizing styrene oxide, 1,2-epoxy-hexane and 3-PEG followed by subsequent quantitative analysis employing chiral gas chromatography. The highest conversions were achieved with whole cells from S. antibioticus Tü4 in the presence of 10% (v/v) DMSO. However, enantioselectivity was only satisfying (E = 31) in the presence of 5% (v/v) acetone, which allowed isolation of optically pure non-hydrolyzed (R)-styrene oxide (99% enantiomeric excess (ee)) and (S)-phenyl-1,2-ethandiol (72% ee) at 55% conversion after 24 h. The resolution of 3-PEG proceeded with slightly lower enantioselectivity albeit higher reaction rates. With S. fradiae Tü27 and S. arenae Tü495 enantioselectivity towards styrene oxide was only E = 3-4.     

The effects of organic solvents on the membrane-induced fibrillation of human islet amyloid polypeptide and on the inhibition of the fibrillation

The organic solvent dimethylsulphoxide (DMSO) and 1,1,1,3,3,3-hexafluoro-2-isopropanol (HFIP) have been widely used as a pre-treating agent of amyloid peptides and as a vehicle for water-insoluble inhibitors. These solvents are left in many cases as a trace quantity in bulk and membrane environments with treated amyloid peptides or inhibitors. In the present work, we studied the effects of the two organic solvents on the aggregation behaviors of human islet amyloid polypeptide (hIAPP) and the performances of an all-D-amino-acid inhibitor D-NFGAIL in preventing hIAPP fibrillation both in bulk solution and at phospholipid membrane. We showed that the presence of 1% v/v DMSO or HFIP decreases the rate of fibril formation of hIAPP at the lipid membrane rather than accelerates the fibril formation as what happened in bulk solution. We also showed that the presence of 1% v/v DMSO or HFIP impairs the activity of the inhibitor at the lipid membrane surface dramatically, while it affects the efficiency of the inhibitor in bulk solution slightly. We found that the inhibitor inserts into the lipid membrane more deeply or with more proportion in the presence of the organic solvents than it does in the absence of the organic solvents, which may hinder the binding of the inhibitor to hIAPP at the lipid membrane. Our results suggest that the organic solvents should be used with caution in studying membrane-induced fibrillogenesis of amyloid peptides and in testing amyloid inhibitors under membrane environments to avoid incorrect evaluation to the fibrillation process of amyloid peptides and the activity of inhibitors.     

Improvement of hydrogenase enzyme activity by water-miscible organic solvents

Both stability and catalytic activity of the HynSL Thiocapsa roseopersicina hydrogenase in the presence of different water-miscible organic solvents were investigated. For all organic solvents under study the substantial raise in hydrogenase catalytic activity was observed. The stimulating effect of acetone and acetonitrile on the reaction rate rose with the increase in solvent concentration up to 80%. At certain concentrations of acetonitrile and acetone (60–80%, v/v in buffer solution) the enzyme activity was improved even 4–5 times compared to pure aqueous buffer. Other solvents (aliphatic alcohols, dimethylsulfoxide and tetrahydrofuran) improved the enzyme activity at low concentrations and caused enzyme inactivation at intermediate concentrations. The long-term incubation of the hydrogenase with aliphatic alcohols, dimethylsulfoxide and tetrahydrofuran at intermediate concentrations of the latter caused enzyme inactivation. The reduced form of hydrogenase was found to be much more sensitive to action of these organic solvents than the enzyme being in oxidized state. The hydrogenase is rather stable at high concentrations of acetone or acetonitrile during long-term storage: its residual activity after incubation in these solvents upon air within 30 days was about 50%, and immobilized enzyme remained at the 100% of its activity during this period.     

Synthesis of a highly fluorescent N,N‐dimethyl benzylamine–palladium(II) curcuminate complex and its application for determination of trace amounts of water in organic solvents

In this work a new highly fluorescent N,N‐dimethyl benzylamine–palladium(II) yu complex was synthesized by the reaction of [Pd₂{(C,N–C₆H₄CH₂N(CH₃)₂}₂(μ‐OAc)]₂] with curcumin. The structure of the synthesized complex was characterized using Fourier transform infra‐red (FT‐IR) spectroscopy, ¹H nuclear magnetic resonance spectroscopy, and elemental analysis. Fluorescence quantum yield (Φ_F) values of the synthesized complex in dimethyl sulfoxide (DMSO), acetonitrile, ethanol, and methanol were 0.160, 0.104, 0.068, and 0.061, respectively. The fluorescence signal of the complex in the organic solvents was very sensitive to the water content of the organic solvent. The quenching effect of water was used to determine trace amounts of water in the heteroatom‐containing organic solvents (ethanol, methanol, acetonitrile) and redox‐active solvents (DMSO). The linear ranges for determination of water (v/v %) in ethanol, DMSO and acetonitrile were found to be 0.03–14.5, 0.08–13.8, and 0.07–18.8, respectively. Two linear ranges were found for determination of water (v/v %) in methanol (0.1–1.2 and 4.7–25.0). Detection limit (DL) values were calculated to be 0.001, 0.05, 0.004, and 0.01 (v/v %) in ethanol, methanol, acetonitrile, and DMSO, respectively. The proposed method overcomes the problems of the standard Karl Fischer method for determination of water in DMSO. In addition, it gave the best DL value for determination of water in ethanol compared with all published papers to date.     

A new method for extraction of iron-molybdenum cofactor (FeMoco) from nitrogenase adsorbed to DEAE-cellulose. 2. Solubilization of FeMoco in a wide range of organic solvents

While the iron-molybdenum cofactor (FeMoco) of nitrogenase, a constituent of the active site for nitrogen reduction, can be extracted into N-methylformamide (NMF) and pyrrollidinone, the inability to solubilize it in any other organic solvents has hampered further understanding of its structure and chemical properties. A method to solubilize FeMoco, prepared in N,N-dimethylformamide (DMF) with Bu4N+ as counterion [McLean, P. A., Wink, D. A., Chapman, S. K., Hickman, A. B., McKillop, D. M., & Orme-Johnson, W. H. (1989) Biochemistry (preceding paper in this issue)], in acetonitrile, acetone, methylene chloride, tetrahydrofuran, and benzene is reported. FeMoco evaporated to dryness in vacuo dissolves readily in good yield (55-100%) and with no significant loss in specific activity. In addition, FeMoco can be extracted directly into these solvents from MoFe protein bound to a DEAE-Sepharose column if the protein is pretreated with DMF. Methods have also been developed to extract fully active FeMoco into acetone and acetonitrile in the absence of any amide solvents (NMF or DMF). Extraction of FeMoco into acetone (30% yield) involves only pretreatment of column-bound protein with methanol, while extraction into acetonitrile (22% yield) requires pretreatment with methanol followed by THF. We conclude that the presence of a suitable soluble cation confers solubility to the cofactor in many common organic solvents and that the solubility of FeMoco in a given solvent may be independent of the ability of that solvent to extract the cofactor from column-bound protein.