A review focusing on mechanisms and ecological risks of enrichment and propagation of antibiotic resistance genes and mobile genetic elements by microplastic biofilms.

Microplastics (MPs) are emerging ubiquitous pollutants in aquatic environment and have received extensive global attention. In addition to the traditional studies related to the toxicity of MPs and their carrier effects, their unique surface-induced biofilm formation also increases the ecotoxicity potential of MPs from multiple perspectives. In this review, the ecological risks of MPs biofilms were summarized and assessed in detail from several aspects, including the formation and factors affecting the development of MPs biofilms, the selective enrichment and propagation mechanisms of current pollution status of antibiotic resistance genes (ARGs) and mobile genetic elements (MGEs) in MPs biofilms, the dominant bacterial communities in MPs biofilms, as well as the potential risks of ARGs and MGEs transferring from MPs biofilms to aquatic organisms. On this basis, this paper also put forward the inadequacy and prospects of the current research and revealed that the MGEs-mediated ARG propagation on MPs under actual environmental conditions and the ecological risk of the transmission of ARGs and MGEs to aquatic organisms and human beings are hot spots for future research. Relevant research from the perspective of MPs biofilm should be carried out as soon as possible to provide support for the ecological pollution prevention and control of MPs.

Genetic and epidemic characteristics of porcine parvovirus 7 in the Fujian and Guangdong regions of southern China.

Porcine parvovirus (PPV) is the primary cause of reproductive disorders in pigs. The porcine parvovirus 7 (PPV7) subtype was first identified in the United States in 2016. In this study, PPV7 was detected in different porcine samples, including serum, feces, saliva, and milk, from 69 pig farms in the Fujian and Guangdong regions of South China, and its coinfection with porcine circovirus 2 (PCV2), porcine circovirus 3 (PCV3), and porcine reproductive and respiratory syndrome virus (PRRSV) was determined. Whole-genome sequencing, phylogenetic analysis, and recombination analysis were performed on seven isolates, with each selected isolate originating from a different farm. There was a high rate of PPV7 positivity in blood, stool, and saliva but PPV7 DNA was absent from breast milk. The findings also showed that PPV7-positive samples had a high rate of coinfection with PCV2, PCV3, and PRRSV. Real-time PCR was used to determine the viral copy numbers of PCV2, PCV3, PRRSV, and PPV7 in serum samples and to assess whether PPV7 affected PCV2, PCV3, and PRRSV viral loads. Phylogenetic analysis showed that PPV7e and PPV7f were the most prevalent and widespread subtypes in the Fujian and Guangdong regions, respectively. While the PPV7a, PPV7b, PPV7c, and PPV7f subtypes were most prevalent in Fujian Province, PPV7a-e subtypes were prevalent in Guangdong, indicating that PPV7 has rich genetic diversity in these regions. A putative recombinant strain, 21FJ09, was identified using SimPlot and the Recombination Detection Program 4 software.

Electropolymerization of EDOT in an anionic surfactant-stabilized hydrophobic ionic liquid-based microemulsion.

Here, an anionic surfactant [AOT]- (bis-(2-ethylhexyl) sulfosuccinate)-stabilized H2O/[Omim][PF6] (1-octyl-3-methylimidazolium hexafluorophosphate) microemulsion has been tested for the first time as a medium for the electropolymerization of 3,4-ethylenedioxythiophene (EDOT). To formulate AOT-stabilized [Omim][PF6]-based microemulsions of different water contents, the phase triangle was determined at 35 °C. Measurements of the conductivities of the microemulsions, their solubilization capacities toward EDOT and their catalytic effects on EDOT electrooxidation show that the present [AOT]--stabilized ionic liquid microemulsion is a good medium for EDOT electropolymerization. Studies on the process of the electropolymerization of EDOT in this [Omim][PF6]-based microemulsion indicate that the water content (i.e., microstructure) of the microemulsion medium is an important factor affecting the onset potential and the deposition rate of the PEDOT. The morphology and the doping level of the as-prepared PEDOT are also found to be correlated with the water content of the ionic liquid microemulsion. The microemulsion with higher water content results in a PEDOT with better electroactivity and higher doping levels. FTIR spectra and XPS analysis show that the PEDOT electrosynthesized in the microemulsion is co-doped by both [AOT]- and [PF6]-. Compared with the neat [Omim][PF6], the use of the ionic liquid microemulsions can reduce not only the consumption of the expensive ionic liquid, but also the onset potential for the electrooxidation of EDOT. Moreover, by tuning the water content of the medium, the electropolymerization of PEDOT and its electrochemical properties could be regulated accordingly. Under the identical deposited charge, the PEDOT originated from the high water content microemulsion (50% H2O µE) has a higher specific capacitance (124 F g-1) than that from neat [Omim][PF6] (117 F g-1). It follows that the present ionic liquid microemulsion is a good medium for EDOT electropolymerization. The present study opens up a new route for the green and low-cost electrochemical preparation of high-performance PEDOT.

Good's buffer ionic liquid tunes the phase behavior of an anionic surfactant SDBS-stabilized n-octane-water microemulsion and the stability of the solubilized horseradish peroxidase.

A Good's buffer ionic liquid (GB-IL) composed of quaternary ammonium cations and Good's buffer anions is first introduced into a microemulsion system as a self-buffering and biocompatible electrolyte. The effects of the constituting ions of a GB-IL and their concentrations on the phase behavior of the anionic surfactant SDBS stabilized n-octane-H2O microemulsion system were studied for the first time using the ε-ß fish-like phase diagram method. The result indicates that the phase behavior of the above microemulsion system is greatly affected by GB-IL cations with a longer alkyl chain on the cation being more favorable for phase inversion. Compared with NaCl, a GB-IL of the same concentration is more efficient for achieving phase inversion, due to the dual role of an electrolyte and a co-alcohol. In addition to the phase behavior, the stability of horseradish peroxidase (HRP) solubilized in an SDBS stabilized bicontinuous microemulsion is also affected by a GB-IL. It is found that the variation of the cationic alkyl chain has a negligible effect on the microemulsion microstructure, but has a significant influence on the stability of the solubilized HRP. At a fixed concentration of the GB-IL, the quaternary ammonium cation with a longer alkyl chain is better for the stabilization of the HRP activity. For a given GB-IL, a higher level of the GB-IL results in a better HRP stability. More importantly, the GB-IL-buffered microemulsion, at the same level of the buffering salt, is more advantageous than the phosphate-buffered one for the stabilization of the HRP activity. This advantage is more pronounced for higher concentrations of the GB-IL. This difference in the HRP stability, caused by the buffering salts, should be ascribed to the microemulsion microstructure effect as well as the Hofmeister effect. The present study provides a guideline for the construction of a bicontinuous microemulsion with a simplified composition and stabilizing effect on the solubilized enzyme.

Anionic-Surfactant-Stabilized Hydrophobic Ionic-Liquid-Based Bicontinuous Microemulsion as a Medium for Enzymatic Oxidative Polymerization of Aniline.

The hydrophobic ionic liquid [C8mim][PF6] (1-octyl-3-methylimidazolium hexafluorophosphate)-based bicontinuous microemulsion stabilized by the anionic surfactant [C4mim][AOT] (1-butyl-3-methylimidazolium bis(2-ethylhexyl) sulfosuccinate) was first tried as a medium for horseradish peroxidase (HRP)-triggered oxidative polymerization of aniline. The effects of the mass ratio of [C8mim][PF6]-to-water (α), the mass fraction of [C4mim][AOT] in the total mixture (γ), and temperature (T) on the enzymatic polymerization were investigated using UV-vis-NIR absorption, electron spin resonance, and small-angle X-ray scattering spectroscopy techniques. The bicontinuous microemulsion is demonstrated to play a template role in the biosynthesis of polyaniline (PANI). The conductivity of the resulting PANI depends on the microemulsion microstructure and the microstructure- and T-dependent catalytic properties of the solubilized HRP. With the increase in α, the conductivity of the synthesized PANI decreases due to the increase in the template curvature (decrease of the microdomain size) and the decrease in the activity and stability of HRP. Compared with α, γ has little effect on the microdomain size of the template; so, the γ-dependent change in the conductivity of PANI is mainly caused by the changes of the microstructure-dependent activity and stability of HRP. Over the range of 20-35 °C, T has little effect on the microdomain size, but it greatly changes the activity and stability of HRP. With the increase in T, the activity of HRP increases steadily, but its stability decreases significantly, which should be one of the reasons why the conductivity of PANI decreases with increasing T. In conclusion, lower values of α, γ, and T are favorable for the biosynthesis of conductive PANI. The present study not only deepens the insight into the role of the template in the process of PANI synthesis, but also opens up a green new way for the biosynthesis of the conducting polymer.

The physicochemical properties of a room-temperature liquidus binary ionic liquid mixture of [HNMP][CH3SO3]/[Bmim]Cl and its application for fructose conversion to 5-hydroxymethylfurfural.

Via heating first and then cooling, binary ionic liquid (IL) mixture of N-methyl-2-pyrrolidonium methylsulfonate ([HNMP][CH3SO3]) and 1-butyl-3-methylimidazolium chloride ([Bmim]Cl) could form a liquid at room temperature. The glass-transition temperature (T g) characterized by DSC depends on its composition with T g being as low as -63 °C. The physicochemical properties of the binary IL mixtures also vary with the composition. With the increase of the mole fraction of [Bmim]Cl, the hydrogen-bond accepting ability (ß) of the binary IL mixture increases, but the hydrogen-bond donating ability (α) deceases. In this binary IL mixture, fructose could be effectively converted into 5-hydroxymethylfurfural (HMF) at room temperature. The HMF yields at a given time are found to be well correlated with the physicochemical properties of the binary mixture, especially the α and ß values. Under specified conditions, the present IL mixture as medium for fructose dehydration into HMF is comparable to the medium formed by ILs and alcohol, where the alcohols have negative effect on the HMF formation.

Enhancing fructooligosaccharides production by genetic improvement of the industrial fungus Aspergillus niger ATCC 20611.

Aspergillus niger ATCC20611 is one of the most potent filamentous fungi used commercially for production of fructooligosaccharides (FOS), which are prospective components of functional food by stimulating probiotic bacteria in the human gut. However, current strategies for improving FOS yield still rely on production process development. The genetic engineering approach hasn't been applied in industrial strains to increase FOS production level. Here, an optimized polyethylene glycol (PEG)-mediated protoplast transformation system was established in A. niger ATCC 20611 and used for further strain improvement. The pyrithiamine resistance gene (ptrA) was selected as a dominant marker and protoplasts were prepared with high concentration (up to 108g-1 wet weight mycelium) by using mixed cell wall-lysing enzymes. The transformation frequency with ptrA can reach 30-50 transformants per µg of DNA. In addition, the efficiency of co-transformation with the EGFP reporter gene (egfp) was high (approx. 82%). Furthermore, an activity-improved variant of ß-fructofuranosidase, FopA(A178P), was successfully overexpressed in A. niger ATCC 20611 by using the transformation system. The transformant, CM6, exhibited a 58% increase in specific ß-fructofuranosidase activity (up to 507U/g), compared to the parental strain (320U/g), and effectively reduced the time needed for completion of FOS synthesis. These results illustrate the feasibility of strain improvement through genetic engineering for further enhancement of FOS production level.

Electrochemical synthesis of poly(3-aminophenylboronic acid) in ethylene glycol without exogenous protons.

A non-aqueous solution of tetra-n-butylammonium fluoride (TBAF) in ethylene glycol has been tried for the first time as a supporting electrolyte for the electropolymerization of 3-aminophenylboronic acid (APBA). Unlike the traditional acidic aqueous solution, the present medium needs no exogenous protons; moreover, the presence of CF3COOH is found to be unfavorable for the polymerization. The protons are in situ generated by the reaction between the boronic acid group on APBA and 1,2-dihydroxyl on ethylene glycol. So, ethylene glycol serves as not only the solvent but also the proton source. As a part of the supporting electrolyte, F(-) is found to be involved in the electrochemical synthesis of poly(3-aminophenylboronic acid) (PAPBA), but it is not indispensable. Studies on the electropolymerization process indicate that the size of the ions in the electrolyte affects the rate of the doping/dedoping process. The smaller the cation, the easier the doping/dedoping process, and the better the stability of the grown film. As demonstrated by Fourier transform infrared spectra, UV-vis spectra, and scanning electron microscopy, the obtained PAPBA is a cross-linked nanoporous polymer membrane that has a good adherence to the glassy carbon electrode.

Study on the catalytic performance of laccase in the hydrophobic ionic liquid-based bicontinuous microemulsion stabilized by polyoxyethylene-type nonionic surfactants.

To formulate a compatible green medium for the conversion of a hydrophobic substrate by a hydrophilic enzyme, we investigated the phase behavior of pseudo ternary hydrophobic ionic liquid (HIL)/buffer/polyoxyethylene-type nonionic surfactant (CnEm)/n-alcohol system and the effects of the components on the formulation of the HIL-based bicontinuous microemulsion. It is found that small head group of the surfactant, high concentration of n-alcohol (medium/long alkyl chain) and low cohesive energy density of the HIL result in low phase transition temperature. In the CnEm stabilized compatible bicontinuous microemulsion, the kinetics of laccase catalyzed oxidation of 2,6-dimethoxyphenol were also investigated. It is found that in addition to temperature, n-alcohol is the key parameter affecting the catalytic performance of laccase, and the optimum n-alcohol depends on the type of HIL as an oil phase. All the kinetic parameters, such as Km, kcat, kcat/Km, and Ea (apparent activation energy), indicate that the bicontinuous microemulsion consisting of [Omim]NTf2/buffer/CnEm/n-hexanol is a suitable medium for the laccase-catalyzed reaction. To the best of our knowledge, this is the first report on the formulation of HIL-based bicontinuous microemulsion for enzyme catalysis.

Effect of the physicochemical properties of binary ionic liquids on lipase activity and stability.

In the present study, the lipase-catalyzed hydrolysis of p-nitrophenyl butyrate is used as a model reaction to determine the activity and stability of Candida rugosa lipase in binary ionic liquids (ILs). The binary ILs consist of hydrophobic 1-butyl-3-methylimidazolium hexafluorophosphate ([Bmim]PF6) and a small amount of hydrophilic 1-butyl-3-methylimidazolium nitrate ([Bmim]NO3) or 1-butyl-3-methylimidazolium trifluoromethanesulfonate ([Bmim]CF3SO3) or 1-butyl-3-methylimidazolium tetrafluoroborate ([Bmim]BF4). The activity and the stability of lipase are first correlated with the physicochemical properties of the binary ILs. In the three binary IL systems, both the hydrophilicity and the polarity of the systems increase with the increase of the content of hydrophilic ILs (HILs). At a fixed concentration of HIL, they vary in a descending order of [Bmim]PF6/[Bmim]NO3>[Bmim]PF6/[Bmim]CF3SO3>[Bmim]PF6/[Bmim]BF4. This order is in contrast with the order of the lipase conformation stability, i.e., the higher the polarity of ILs, the more unstable the lipase conformation. However, both the activity and the stability of lipase depend on the type and the content of the HIL in binary ILs, showing a complex dependency. Analysis shows that the catalytic performance of lipase in the binary ILs is affected not only by the direct influence of the ILs on lipase conformation, but also through their indirect influence on the physicochemical properties of water. The present study helps to explore binary IL mixtures suitable for lipase-based biocatalysis.

Gold nanoparticles tune the activity of laccase in anionic reverse micelles.

The interfacial property of reverse micelles is an important factor affecting the catalytic activity of enzymes hosted in the micelles. In this article, the effect of gold nanoparticles (GNPs) on the catalytic activity of laccase (non-surface-active enzyme) and the related mechanism are reported. It was found that laccase activity was dependent on the size of the particle and its concentration as well as on the water content and the concentration of AOT. It was shown that there existed several types of micelles in the present reverse micellar system in the presence of GNPs. The population of the various micelles depended on the concentrations of both GNPs and AOT. Fluorescence and circular dichroism spectra of laccase at different water contents and GNP concentrations indicated that the conformation of laccase and its activity were tuned by GNPs via changing the structure of the reverse micelles. Analysis showed that changes in the thickness of the water layer (Lw) and in the apparent occupied area of individual AOT molecules (AAOT) caused by GNPs were the main parameters affecting the activity of laccase. The present work extends and deepens the understanding of the tuning mechanism of GNPs on enzymatic performance in reverse micelles and provides guidance for rational design of the optimal microenvironment of laccase.

Formation, characterization and enzyme activity in water-in-hydrophobic ionic liquid microemulsion stabilized by mixed cationic/nonionic surfactants.

The phase behavior of the pseudo ternary system 1-tetradecyl-3-methylimidazolium bromide ([C14mim]Br)/Triton X-100/H2O/1-butyl-3-methylimidazolium hexafluorophosphate ([Bmim]PF6) has been studied at 35°C. With the increase in the mole fraction of Triton X-100 in the mixed surfactants, the water solubilization capacity increases and the monophasic area enlarges. The H2O-in-[Bmim]PF6 (W/IL) microemulsion was identified via electrical conductivity measurement. The existence of bulk water in the W/IL microemulsion was demonstrated based on the change of the O-D vibration frequency with content of D2O added and confirmed using UV-vis technique with CoCl2 as probe. Laccase can be solubilized in the W/IL microemulsion and exhibits a catalytic activity. The interface of the W/IL microemulsion has an inhibitory effect on the expression of the laccase activity, and the inhibitory effect is varied with the molar ratio of the mixed surfactants.

Construction and direct electrochemistry of orientation controlled laccase electrode.

A laccase has multiple redox centres. Chemisorption of laccases on a gold electrode through a polypeptide tag introduced at the protein surface provides an isotropic orientation of laccases on the Au surface, which allows the orientation dependent study of the direct electrochemistry of laccase. In this paper, using genetic engineering technology, two forms of recombinant laccase which has Cys-6×His tag at the N or C terminus were generated. Via the Au-S linkage, the recombinant laccase was assembled orientationally on gold electrode. A direct electron transfer and a bioelectrocatalytic activity toward oxygen reduction were observed on the two orientation controlled laccase electrodes, but their electrochemical behaviors were found to be quite different. The orientation of laccase on the gold electrode affects both the electron transfer pathway and the electron transfer efficiency of O2 reduction. The present study is helpful not only to the in-depth understanding of the direct electrochemistry of laccase, but also to the development of laccase-based biofuel cells.

Effect of three trifluoromethanesulfonate ionic liquids on the activity, stability and conformation of laccase.

The activity, stability and conformation of laccase were first investigated in an aqueous solution of ionic liquids 1-butyl-3-methylimidazolium trifluoromethanesulfonate ([Bmim]TfO), 1-butyl-1-methylpyrrolidinium trifluoromethanesulfonate ([Bmpyr]TfO) or tetramethylammonium trifluoromethanesulfonate ([TMA]TfO). Compared with control system, high level of [Bmim]TfO or [Bmpyr]TfO destabilizes laccase while [TMA]TfO stabilizes laccase. These effects are more pronounced with the extension of the incubation time. The activity variations are well correlated with the changes of the conformation of laccase evidenced by fluorescence and circular dichroism spectra under specified conditions. The effects of the three ionic liquids on laccase are associated with the chaotropicity of the cations in Hofmeister series. For laccase, [TMA]TfO is not a good activating agent but it greatly enhances the stability of laccase in addition to maintaining the catalytic efficiency of laccase, showing its great potential in real application.

Choline acetate enhanced the catalytic performance of Candida rogusa lipase in AOT reverse micelles.

Choline acetate is an ionic liquid composed of a kosmotropic anion and a chaotropic cation. According to Hofmeister series, a kosmotropic anion and/or a chaotropic cation could stabilize an enzyme, thereby facilitating the retention of the catalytic activity of the enzyme. In this work, we first report the influence of choline acetate on the activity and stability of lipase in AOT/water/isooctane reverse micelles. The indicator reaction is the lipase-catalyzed hydrolysis of 4-nitrophenyl butyrate. The results show that a low level of choline acetate does not affect the microstructure of the AOT reverse micelles, but the ionic liquid can improve the catalytic efficiency of lipase. Fluorescence spectra show that a high level of choline acetate has an impact on the conformation of lipase, so the activation is mainly due to the influence of choline acetate on the nucleophilicity of water. Infrared spectra demonstrate that choline acetate can form stronger hydrogen bonds with water surrounding lipase, and therefore enhance the nucleophilicity of the water, which makes it easier to attack the acyl enzyme intermediate, thereby increasing the activity of the lipase-catalyzed hydrolysis of the ester. A study on the stability of lipase in AOT reverse micelles indicates that the ionic liquid is able to maintain the activity of lipase to a certain extent. The effect of choline acetate is consistent with that predicted based on Hofmeister series.

Nitrile group as infrared probe for the characterization of the conformation of bovine serum albumin solubilized in reverse micelles.

Infrared spectroscopy is a powerful technique for structure characterization. For a protein hosted in a reversed micellar medium, the spectral features of the protein are always interfered by the IR absorption bands of the medium in addition to the congestion in their IR spectra. Fortunately, there is a transparent window in the 2500-2200 cm(-1) region. Incorporation of a vibrational probe with IR absorption frequencies in this region into proteins represents a promising strategy for the study of the conformation of a protein in a reverse micelle. In the present work, we incorporated 4-cyanobenzyl group (CN) into bovine serum albumin (BSA) via cysteine alkylation reactions under mild conditions. Circular dichroism spectroscopy showed that the CN modified BSA (CNBSA) could retain its conformation. When CNBSA was hosted in AOT reverse micelle, it was found that the nitrile group on BSA was sensitive to the conformational change of BSA induced by urea as an additive in the reverse micelle. The peak splitting of nitrile group was also observed when the size of AOT reverse micelle and the concentration of an electrolyte were varied. Obviously, the shift of the IR absorption peak and/or peak splitting of nitrile group on BSA are correlated with the change of BSA conformation in AOT reverse micelle. So we conclude that the nitrile infrared probe can be used to study protein conformation in a reverse micelle.

A bioelectrochemical method for the quantitative description of the Hofmeister effect of ionic liquids in aqueous solution.

It is imperative to establish a simple, efficient, and practical method to investigate the Hofmeister effect of ionic liquids (ILs) on the behavior of proteins (enzymes). In this study, the effects of the cations and anions of different ILs in aqueous media on the structural stability of horseradish peroxidase (HRP), a model oxidoreductase, were systematically investigated using electrochemical methods. It is found that without ILs no direct electron transfer current signals of HRP appear at bare glassy carbon electrode (GCE) in phosphate buffer (pH 7.0) even after incubation and accumulation at a negative potential. In the presence of ILs, however, a current signal occurs at GCE, depending on the structure of the IL and its concentration. A linear relationship between the peak currents and the scan rates demonstrates that the direct electron transfer is a surface-confined thin-layer electrochemical process. The redox signal at GCE is from the heme of HRP. An IL has a perturbing effect on the HRP structure. The anodic peak current of HRP at GCE, the catalytic activity of HRP, and the secondary structure of HRP are well correlated. Different cations or anions at varied concentrations have different effects on the structural stability of HRP, resulting in different current signals at GCE. Thus, the anodic peak current of HRP at GCE can be used as an indicator to quantitatively characterize the effect of ILs on the structural stability of HRP. The present Hofmeister series for cations and anions is in good agreement with that reported elsewhere. To our knowledge, this is a first attempt to establish a simple and practical electrochemical method to correlate Hofmeister effects with characteristics of ions and solvents. The present investigation not only deepens our understanding of the complex electrochemical behavior of proteins in ILs media but also offers a practical guidance to designing "green" and biocompatible ILs for protein (enzyme) separation, purification, and enzymatic catalysis and conversion.

Design of nanoporous metals with bimodal pore size distributions for enhanced biosensing.

Nanoporous gold (np-Au) has shown great potential in catalysis, plasmonics, sensing, etc. In this work, by two-step dealloying a well-designed AuAgAl ternary precursor alloy, np-Au with bimodal ligament/pore size distributions is successfully fabricated. The first dealloying in HCl solution removes Al and generates a nanoporous AuAg alloy which would be mildly annealed at 200 °C for 30 min to homogenize the alloy ligament and enlarge the ligament/pore size. Next, the nanoporous AuAg alloy is further dealloyed in a HNO(3) solution to etch Ag and fabricate np-Au with a hierarchical microstructure. This novel bimodal np-Au is demonstrated to exhibit enhanced electrocatalytic activity towards H(2)O(2) reduction and be a better support for the fabrication of an oxidase-based biosensor compared with normal np-Au, with a uniform pore/ligament size of 30-40 nm. In a proof-of-concept study, a sensitive glucose biosensor with a linear range up to 21 mM is fabricated by immobilization of glucose oxidase on the bimodal np-Au.

The catalytic efficiency of lipase in a novel water-in-[Bmim][PF6] microemulsion stabilized by both AOT and Triton X-100.

In the water-in-[Bmim][PF(6)] microemulsion stabilized by both AOT and Triton X-100, the lipase-catalyzed hydrolysis of 4-nitrophenyl butyrate (p-NPB) was investigated to evaluate the catalytic efficiency of lipase in this novel microemulsion. The structural parameters of the microemulsion and the conditions of the enzymatic reaction affect the catalytic activity of lipase, especially the concentration of Tris-HCl buffer. Under optimum conditions, the catalytic activity of lipase in the present microemulsion is much higher than that in H(2)O saturated [Bmim][PF(6)]. When the partitioning of the substrate in the microemulsion is taken into account, the catalytic efficiency of lipase in this novel microemulsion is 14.3 times that in H(2)O saturated [Bmim][PF(6)] due to the significant decrease of the Michaelis constant in the microemulsion. Due to the large interface, high water activity, and probably the activating effect of the imidazolium cation in the water pool, the present microemulsion is demonstrated to be a promising medium for the lipase-catalyzed hydrolytic reaction. To demonstrate an important biocatalytic application in the IL-based microemulsion, the lipase-catalyzed synthesis of the flavoring agent benzyl acetate via transesterification of vinyl acetate with benzyl alcohol was also studied in the medium. Due to the high dispersion of lipase, large interface and removal of the byproduct, a maximum yield of 94% was obtained, indicating that the novel microemulsion is really important and useful.

Effect of the structure of imidazolium cations in [BF4](-)-type ionic liquids on direct electrochemistry and electrocatalysis of horseradish peroxidase in Nafion films.

The direct electrochemistry and bioelectrocatalysis of horseradish peroxidase (HRP) in Nafion films at glassy carbon electrode (GCE) was investigated in three [BF(4)](-)-type room-temperature ionic liquids (ILs) to understand the structural effect of imidazolium cations. The three ILs are 1-ethyl-3-methylimidazolium tetrafluoroborate ([Emim][BF(4)]), 1-butyl-3-methylimidazolium tetrafluoroborate ([Bmim][BF(4)]) and 1-hexyl-3-methylimidazolium tetrafluoroborate ([Hmim][BF(4)]). A small amount of water in the three ILs is indispensable for maintaining the electrochemical activity of HRP in Nafion films, and the optimum water contents decrease with the increase of alkyl chain length on imidazole ring. Analysis shows that the optimum water contents are primarily determined by the hydrophilicity of ILs used. In contrast to aqueous medium, ILs media facilitate the direct electron transfer of HRP, and the electrochemical parameters obtained in different ILs are obviously related to the nature of ILs. The direct electron transfer between HRP and GCE is a surface-confined quasi-reversible single electron transfer process. The apparent heterogeneous electron transfer rate constant decreases gradually with the increase of alkyl chain length on imidazole ring, but the changing extent is relatively small. The electrocatalytic reduction current of H(2)O(2) at the present electrode decreases obviously with the increase of alkyl chain length, and the mass transfer of H(2)O(2) via diffusion in ILs should be responsible for the change. In addition, the modified electrode has good stability and reproducibility; the ability to tolerate high levels of F(-) has been greatly enhanced due to the use of Nafion film. When an appropriate mediator is included in the sensing layer, a sensitive nonaqueous biosensor could be fabricated.