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1.
The effects of a water-miscible ionic liquid, 1-butyl-3-methylimidazolium tetrafluoroborate ([BMIM][BF4]), on both thermodynamics and kinetic mechanism of the horseradish peroxidase (HRP)-catalyzed oxidation of guaiacol (2-methoxyphenol) by H2O2 were investigated. The ionic liquid stabilized the ground state of guaiacol by causing an 8-fold increase of Km from 3 to 23 mM upon the addition of 25% (v/v) [BMIM][BF4]. In addition, the effect of [BMIM][BF4] in decreasing the kcat value of HRP catalysis was described by a non-competitive inhibition mechanism. The value of the inhibition constant of [BMIM][BF4] was 2.9 M indicating that the ionic liquid plays the role of a weak non-competitive inhibitor for HRP catalysis.  相似文献   

2.
The inhibition mechanism of a water-miscible ionic liquid, N-butyl-3-methypyridinium tetrafluoroborate ([BMPy][BF4]), on the catalysis of horseradish peroxidase (HRP) was investigated. The K m value for the oxidation of guaiacol (2-methoxyphenol) with H2O2 catalyzed by HRP increased from 2.8 mM in 100% water to 12.6 mM in 25% (v/v) [BMPy][BF4]. This increase of K m by the ionic liquid was elucidated to be caused by the strong stabilization of the ground state of guaiacol by the ionic liquid. On the contrary, the k cat value for the HRP-catalyzed reaction decreased from 13.8/sec in 100% water to 6.7/sec in 25% (v/v) [BMPy][BF4]. Such decrease of k cat value of HRP catalysis by the increasing content of [BMPy][BF4] was described using the noncompetitive inhibition of the enzyme by the ionic liquid. The value of the inhibition constant of [BMPy][BF4] was 1.48 M indicating that the ionic liquid exerts a weak noncompetitive inhibition effect on the HRP catalysis.  相似文献   

3.
The activity and stability of free and immobilized D-amino acid oxidase (DAAO, EC 1.4.3.3) from Trigonopsis variabilis CBS 4095 in different water-soluble and water-insoluble ionic liquids (ILs) as well as in organic solvents were studied for comparison. The most promising ILs ([BMIM][BF(4)] and [MMIM][MMPO(4)]) were investigated in detail. The kinetic parameters (v(max) = 187 nkat/g dry weight, K(M) = 1.38 mM) with D-phenylalanine as substrate were calculated in 40% [BMIM][BF(4)]. Bioconversions of D/L-phenylalanine in 40% [BMIM][BF(4)] and 20% [MMIM][MMPO(4)] on a 3 ml scale using immobilized DAAO were performed by addition of free catalase from Micrococcus lysodeikticus. After total conversion of substrate in presence of 20% [MMIM][MMPO(4)] the residual activity of the immobilized DAAO was 79% and 100% of the free catalase.  相似文献   

4.
Park JH  Yoo IK  Kwon OY  Ryu K 《Biotechnology letters》2011,33(8):1657-1662
The ionic liquid, 1-butyl-3-methylimidazolium methylsulfate ([BMIM][MeSO4]), was used to investigate the catalytic mechanism of horseradish peroxidase (HRP). The ionic liquid decreased both Km and kcat values for the HRP-catalyzed oxidation of guaiacol (2-methoxyphenol) by H2O2. These studies imply that [BMIM][MeSO4] inhibits the enzyme in an uncompetitive manner. The incorporation of substrate stabilization effects measured by a thermodynamic method into the partial uncompetitive inhibition scheme successfully describes HRP-catalysis in the presence of [BMIM][MeSO4], which participates as the inhibitor. The inhibition constant of the ionic liquid was 0.051 M. The turn-over number of the native HRP was almost 14-times higher than that of the HRP-ionic liquid complex indicating that [BMIM][MeSO4] does not form a dead-end complex with HRP.  相似文献   

5.
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.  相似文献   

6.
The reactivity of horseradish peroxidase (HRP) with water insoluble phenolic compounds has been studied in 1-butyl-3-methylimidazolium tetrafluoroborate ([BMIM][BF4])/water mixtures. The enzyme retained some catalytic activity up to 90% ionic liquid in water at 25 °C only at pH values higher than 9.0. Activity steadily decreased towards neutral and acidic conditions, as judged by 4-aminoantypirin/phenol activity tests. Inhibition of horseradish peroxidase under neutral acidic condition was due to the binding of fluoride anions released from tetrafluoroborate anion to the heme iron as demonstrated by the sharp UV–visible absorption transition diagnostic of the conversion from a five coordinated to a six coordinated high spin ferric heme iron. Thus, reactions with water insoluble phenols were carried out under alkaline reaction conditions and 75% [BMIM][BF4]/water mixture. Under these conditions, the distribution of the reaction products was much narrower with respect to that observed in aqueous buffers or water/dimethylformamide or water/dimethylsulfoxide mixtures, and polymeric species other than dimers were not observed. Technical scale preparations of a novel 4-phenylphenol ortho dimer [2,2′-bi-(4-phenylphenol)] with a high yield of the desired product were obtained.  相似文献   

7.
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  相似文献   

8.
The effect of ionic liquid 1-butyl-3-methylimidazolium hexafluorophosphate ([BMIM][PF6]) on the asymmetric reduction of ethyl 2-oxo-4-phenylbutyrate (EOPB) to synthesize optical active ethyl 2-hydroxy-4-phenylbutyrate (EHPB) catalyzed by Saccharomyces cerevisiae was investigated. (R)-EHPB [70.4%, e.e.(R)] is obtained using ethyl ether or benzene as the solvent. The main product is (S)-EHPB [27.7%, e.e.(S)] in [BMIM][PF6]. However, in ionic liquid-water (10:1, v/v) biphasic system, the enantioselectivity of the reduction is shifted towards (R)-side, and e.e.(R) is increased from 6.6 to 82.5% with the addition of ethanol (1%, v/v). The effect of the use of [BMIM][PF6] as an additive in relatively small amounts on the reduction was also studied. We find that there is a decline in the enantioselectivity of the reduction in benzene. In addition, a decrease in the conversion of EOPB and the yield of EHPB with increasing [BMIM][PF6] concentrations occurs in either organic solvent–water biphasic systems or benzene.  相似文献   

9.
The activity of a lipase from a newly isolated Pseudomonas sp. was investigated in the presence of organic solvents and imidazolium chloride‐based ionic liquids (IL) such as BMIM[Cl] and HMIM[Cl]. The lipase activity in the presence of IL was higher compared to that in common organic solvents such as methanol and 2‐propanol. A possible explanation for the enzyme activation might be the structural changes induced in the protein in organic systems. Since IL quench the intensity of fluorescence emission, it was not possible to investigate the major factor that influences the enzyme behavior in these new organic salts. Furthermore, the enzyme exhibited excellent activity in buffer mixtures containing both organic solvent and IL. The stability of the lipase at 50°C was considerably increased in the presence of 20% BMIM[Cl] compared with the untreated lipase in aqueous medium. The light scattering method clearly showed that prevention of aggregation could be the reason for thermal stabilization at 50°C in reactions containing IL. Kinetic analysis of the enzyme in the presence of different concentrations of IL showed that the Km value increased from 0.45 mM in aqueous buffer to 2.4 mM in 50% v/v BMIM[Cl]/buffer. The increase in Km indicates that IL can significantly reduce the binding affinity of the substrate to the enzyme. Also, a linear correlation was observed between the BMIM[Cl] concentration and Vmax of the enzyme. As the concentration of BMIM[Cl] increased from 10 to 50% v/v, the Vmax value increased from 1.8 to 46 μM/min.  相似文献   

10.
An ionic liquid-tolerant bacterium, Bacillus amyloliquefaciens CMW1, was isolated from a Japanese fermented soybean paste. Strain CMW1 grew in the presence of 10 % (v/v) 1-butyl-3-methylimidazolium chloride ([BMIM]Cl), a commonly used ionic liquid. Additionally, strain CMW1 grew adequately in the presence of the hydrophilic ionic liquids 10 % (v/v) 1-ethyl-3-methylimidazolium trifluoromethanesulfonate ([EMIM]CF3SO3) or 2.5 % (v/v) 1-butyl-3-methylimidazolium trifluoromethanesulfonate ([BMIM]CF3SO3). Strain CMW1 produced an extracellular protease (BapIL) in the culture medium. BapIL was stable in the presence of 80 % (v/v) ionic liquids, [EMIM]CF3SO3, [BMIM]Cl, [BMIM]CF3SO3, 1-butyl-3-methylimidazolium tetrafluoroborate, 1-butyl-3-methylimidazolium hexafluorophosphate, and 1-butyl-3-methylimidazolium bis(trifluoromethylsulfonyl)imide, and functioned in 10 % (v/v) these ionic liquids. BapIL was stable at pH 4.0–12.6 or in 4004 mM NaCl solution, and exhibited activity in the presence of 50 % (v/v) hydrophilic or hydrophobic organic solvents. BapIL was completely inhibited by 1 mM PMSF and partially by 5 mM EDTA. BapIL belongs to the true subtilisins according to analysis of the deduced amino acid sequence. We showed that BapIL from the ionic liquid-tolerant B. amyloliquefaciens CMW1 exhibited tolerance to ionic liquid and halo, alkaline, and organic solvents.  相似文献   

11.
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.  相似文献   

12.
The interactions between dibenzothiophene (DBT) and N-butyl-N-methylimidazolium tetrafluoroborate ([BMIM][BF4]), N-butyl-N-methylmorpholinium tetrafluoroborate ([Bmmorpholinium][BF4]), N-butyl-N-methylpiperdinium tetrafluoroborate ([BMPiper][BF4]), N-butyl-N-methylpyrrolidinium tetrafluoroborate ([BMPyrro][BF4]), and N-butylpyridinium tetrafluoroborate ([BPY][BF4]) were investigated using density functional theory approach. Geometric, electron, and topological properties were analyzed using natural bond orbital, atoms in molecules theory, and noncovalent interaction methods in order to understand intermolecular interactions between DBT and ionic liquids. The result shows that hydrogen bond and van der Waals interactions are widespread in all the ionic liquids-DBT systems. Ion-π interactions between DBT and cation or anion are also observed, while π+-π interactions are only found in the [BMIM][BF4]-DBT and [BPY][BF4]-DBT systems. The order of interaction energy is [BPY][BF4]-DBT > [BMIM][BF4]-DBT >> [BMPiper][BF4]-DBT > [BMPyrro][BF4]-DBT > [BMmorpholinum][BF4]-DBT. The energies between DBT and the two ionic liquids containing aromatic cations are significantly higher.  相似文献   

13.
K. Ryu  Y. Kim 《Biotechnology letters》1997,19(10):1019-1022
A thermostable alkaline peroxidase was partially purified from rice hulls by precipitation in 70% (v/v) isopropanol, anion exchange chromatography on a DEAE cellulose column (eluted by 50 mM potassium phosphate, pH 6.0), and gel filtration on a Sephacryl S-200 column. The peroxidase (RHP) showed a maximum activity at a slightly alkaline condition, between pH 7 and 8, for the oxidation of guaiacol in the presence of 0.2 mM H O . The half life time for the inactivation of RHP at 68°C was 168 min nearly six times that of horseradish peroxidase (HRP) at the same temperature. Dioxane enhanced the activity of RHP but decreased that of HRP.  相似文献   

14.
Selective lipase-catalyzed synthesis of glucose fatty acid esters in two-phase systems consisting of an ionic liquid (1-butyl-3-methyl imidazolium tetrafluoroborate [BMIM][BF4] or 1-butyl-3-methyl imidazolium hexafluorophosphate [BMIM][PF6]) and t-butanol as organic solvent was investigated. The best enzyme was commercially available lipase B from Candida antarctica (CAL-B), but also lipase from Thermomyces lanuginosa (TLL) gave good conversion. After thorough optimization of several reaction conditions (chain-length and type of acyl donor, temperature, reaction time, percentage of co-solvent) conversions up to 60% could be achieved using fatty acid vinyl ester as acyl donors in [BMIM][PF6] in the presence of 40% t-BuOH with CAL-B at 60 °C.  相似文献   

15.
An effective means of relieving the toxicity of furan aldehydes, furfural (FFA) and 5-hydroxymethylfurfural (HMF), on fermenting organisms is essential for achieving efficient fermentation of lignocellulosic biomass to ethanol and other products. Ari1p, an aldehyde reductase from Saccharomyces cerevisiae, has been shown to mitigate the toxicity of FFA and HMF by catalyzing the NADPH-dependent conversion to corresponding alcohols, furfuryl alcohol (FFOH) and 5-hydroxymethylfurfuryl alcohol (HMFOH). At pH 7.0 and 25°C, purified Ari1p catalyzes the NADPH-dependent reduction of substrates with the following values (k(cat) (s(-1)), k(cat)/K(m) (s(-1)mM(-1)), K(m) (mM)): FFA (23.3, 1.82, 12.8), HMF (4.08, 0.173, 23.6), and dl-glyceraldehyde (2.40, 0.0650, 37.0). When acting on HMF and dl-glyceraldehyde, the enzyme operates through an equilibrium ordered kinetic mechanism. In the physiological direction of the reaction, NADPH binds first and NADP(+) dissociates from the enzyme last, demonstrated by k(cat) of HMF and dl-glyceraldehyde that are independent of [NADPH] and (K(ia)(NADPH)/k(cat)) that extrapolate to zero at saturating HMF or dl-glyceraldehyde concentration. Microscopic kinetic parameters were determined for the HMF reaction (HMF+NADPH?HMFOH+NADP(+)), by applying steady-state, presteady-state, kinetic isotope effects, and dynamic modeling methods. Release of products, HMFOH and NADP(+), is 84% rate limiting to k(cat) in the forward direction. Equilibrium constants, [NADP(+)][FFOH]/[NADPH][FFA][H(+)]=5600×10(7)M(-1) and [NADP(+)][HMFOH]/[NADPH][HMF][H(+)]=4200×10(7)M(-1), favor the physiological direction mirrored by the slowness of hydride transfer in the non-physiological direction, NADP(+)-dependent oxidation of alcohols (k(cat) (s(-1)), k(cat)/K(m) (s(-1)mM(-1)), K(m) (mM)): FFOH (0.221, 0.00158, 140) and HMFOH (0.0105, 0.000104, 101).  相似文献   

16.
Biocatalytic trans-sialylation is relevant for the design of biomimetic oligosaccharides such as human milk oligosaccharides. t-Butanol and ionic liquids, EAN (ethylammonium nitrate), [MMIm][MeSO4] (1,3-dimethylimidazolium methyl sulfate), and [C2OHMIm][PF6] (1-(2-hydroxyethyl)-3-methylimidazolium hexafluorophosphate), were examined as co-solvents for the improvement of the synthesis versus hydrolysis ratio in the trans-sialylation of lactose, catalysed by an engineered sialidase from Trypanosoma rangeli. The use of 25 % (v/v) t-butanol as co-solvent significantly increased 3′-sialyllactose production by 40 % from 1.04 ± 0.09 to 1.47 ± 0.01 mM. The synthesis versus hydrolysis ratio increased correspondingly by 1.2-times. 1–2.5 % (v/v) EAN or [C2OHMIm][PF6] improved the synthesis versus hydrolysis ratio up to 2.5-times but simultaneously decreased the 3′-sialyllactose yield, probably due to enzyme inactivation caused by the ionic liquid. [MMIm][MeSO4] had a detrimental effect on the trans-sialylation yield and on the ratio between synthesis and hydrolysis.  相似文献   

17.
The effects of various refolding additives, including metal cofactors, organic co‐solvents, and ionic liquids, on the refolding of horseradish peroxidase (HRP), a well‐known hemoprotein containing four disulfide bonds and two different types of metal centers, a ferrous ion‐containing heme group and two calcium atoms, which provide a stabilizing effect on protein structure and function, were investigated. Both metal cofactors (Ca2+ and hemin) and ionic liquids have positive impact on the refolding of HRP. For instance, the HRP refolding yield remarkably increased by over 3‐fold upon addition of hemin and calcium chloride to the refolding buffer as compared to that in the conventional urea‐containing refolding buffer. Moreover, the addition of ionic liquids [EMIM][Cl] to the hemin and calcium cofactor‐containing refolding buffer further enhanced the HRP refolding yield up to 80% as compared to 12% in conventional refolding buffer at relatively high initial protein concentration (5 mg/ml). These results indicated that refolding method utilizing metal cofactors and ionic liquids could enhance the yield and efficiency for metalloprotein.  相似文献   

18.
The hydrogen donor couples pyrocatechol-aniline and phenol-aminoantipyrine in the presence of hydrogen peroxide were compared as chromogens for horseradish peroxidase (HRP) assay. UV-Visible spectroscopy and high-performance liquid chromatography analysis indicated that during the HRP biocatalytic process, pyrocatechol-aniline was converted to a pink-colored reagent with a lambda(max) of 510 nm, which was used in the assay of HRP activity. Electrochemical studies revealed adequate electron transfer ability for this color reagent to serve as a proper mediator for HRP also. Using pyrocatechol-aniline a higher sensitivity and lower detection limit was obtained relative to those of the phenol-aminoantipyrine couple, which is commonly used for HRP assay. A relative standard deviation of 2.9% was obtained for 20 HRP activity measurements, indicating a satisfactory reproducibility for this method. In addition, kinetic parameters of K(m) (12.5mM) and V(max) (12.2 mM min(-1)mg(-1)) were calculated for pyrocatechol-aniline. Regarding the superiority of pyrocatechol-aniline, this couple is suggested to be a better hydrogen donor for the HRP spectrophotometric assay.  相似文献   

19.
研究疏水性离子液体1-丁基-3-甲基咪唑六氟磷酸盐([BMIM]PF6)与醋酸-醋酸钠缓冲液两相体系中,固定化产紫青霉Penicillium purpurogenum Li-3细胞转化甘草酸(GL)生成单葡萄糖醛酸基甘草次酸(GAMG)的反应,并与缓冲液单相体系作为对照.确定了在[BMIM]PF6/缓冲液两相体系中,最适离子液体加入比例、缓冲液pH、反应温度、底物浓度分别为10%、5.8、35℃和6.0mmol/L,在此条件下反应58h,甘草酸转化率为87.03%,比缓冲液单相体系提高了15.02%.离子液体循环使用8次后,回收利用率维持在85.28%.主产物GAMG和副产物甘草次酸(GA)在两相体系中得到有效分离,为后续产物分离带来便利.  相似文献   

20.
The activity and stability of horseradish peroxidase (HRP) solubilised in AOT reversed micelles in isooctane and decalin was studied using guaiacol (2-methoxyphenol) as the electron donor.

The activity of the enzyme in both reversed micellar systems increases with the water content until reaching a maximum value that remains fairly constant for water contents higher than 3.05% (v/v) in isooctane and 2.20% in decalin. The effect of pH on the activity profile was studied in the system AOT/isooctane. The enzyme is fully active at pH 7 and 8 for water contents higher than 3.05% (v/v) but it was completely deactivated at pH 9. The effect of surfactant concentration on HRP activity was also investigated. At low water contents a strong dependence was observed, whilst no further activity increase was observed for water content values higher than 2.7% (v/v).

The stability of HRP was found to be strongly dependent on the water content of the system with higher levels of stability obtained for higher values of water content. HRP stability is also affected by the presence of substrates. Whilst the stability increases markedly when the enzyme is incubated with guaiacol, it does not appear to be so strongly affected by the presence of hydrogen peroxide, at the concentrations studied.  相似文献   

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