Hybridization of Particulate Methane Monooxygenase by Methanobactin-Modified AuNPs.

Particulate methane monooxygenase (pMMO) is a characteristic membrane-bound metalloenzyme of methane-oxidizing bacteria that can catalyze the bioconversion of methane to methanol. However, in order to achieve pMMO-based continuous methane-to-methanol bioconversion, the problems of reducing power in vitro regeneration and pMMO stability need to be overcome. Methanobactin (Mb) is a small copper-chelating molecule that functions not only as electron carrier for pMMO catalysis and pMMO protector against oxygen radicals, but also as an agent for copper acquisition and uptake. In order to improve the activity and stability of pMMO, methanobactin-Cu (Mb-Cu)-modified gold nanoparticle (AuNP)-pMMO nanobiohybrids were straightforwardly synthesized via in situ reduction of HAuCl4 to AuNPs in a membrane fraction before further association with Mb-Cu. Mb-Cu modification can greatly improve the activity and stability of pMMO in the AuNP-pMMO nanobiohybrids. It is shown that the Mb-Cu-modified AuNP-pMMO nanobiohybrids can persistently catalyze the conversion of methane to methanol with hydroquinone as electron donor. The artificial heterogeneous nanobiohybrids exhibited excellent reusability and reproducibility in three cycles of catalysis, and they provide a model for achieving hydroquinone-driven conversion of methane to methanol.

Biosynthesis of (S)-naproxen starch ester by Carica papaya lipase in intermittent opening reaction mode.

Inorder to brought S-naproxen into small intestine, an optically pure (S)-naproxen starch ester was produced by lipase through enantio-selective trans-esterification of racemic naproxen methyl ester with pretreatment starch in solvent system. With carefully selection of the reaction medium (isooctane), lipase (Carica Papaya Lipase, CPL) and the reaction mode (intermittent opening), a high conversion rate (48.6%) and enantiomeric excess of product (99.6%) was obtained. The slow release macromolecular (S)-Naproxen had been synthesized to improve the efficacy of racemic naproxen and overcome its side effects. The enanitomeric ratio of CPL (E=52.5) was higher than CRL (E=22) and greatly influenced by the byproduct methyl alcohol. The intermittent opening reaction mode was the effective way to remove the inhibition of methyl alcohol and to improve the enantio-selectivity of CPL. S-naproxen starch was confirmed by HPLC and 1H NMR. This method may also apply to preparation the other optically pure 2-phenylpropionic acid derivatives. S-naproxen starch was a new optically pure derivatives possessing emulsifying and slow release properties would be widely applied to the food, pharmaceutical and biomedical industries.

Methanobactin-mediated synthesis of gold nanoparticles supported over Al2O3 toward an efficient catalyst for glucose oxidation.

Methanobactin (Mb) is a copper-binding peptide that appears to function as an agent for copper sequestration and uptake in methanotrophs. Mb can also bind and reduce Au(III) to Au(0). In this paper, Au/Al2O3 catalysts prepared by a novel incipient wetness-Mb-mediated bioreduction method were used for glucose oxidation. The catalysts were characterized, and the analysis revealed that very small gold nanoparticles with a particle size <4 nm were prepared by the incipient wetness-Mb-mediated bioreduction method, even at 1.0% Au loading (w/w). The influence of Au loading, calcination temperature and calcination time on the specific activity of Au/Al2O3 catalysts was systematically investigated. Experimental results showed that decomposing the Mb molecules properly by calcinations can enhance the specific activity of Au/Al2O3 catalysts, though they acted as reductant and protective agents during the catalyst preparation. Au/Al2O3 catalysts synthesized by the method exhibited optimum specific activity under operational synthesis conditions of Au loading of 1.0 wt % and calcined at 450 °C for 2 h. The catalysts were reused eight times, without a significant decrease in specific activity. To our knowledge, this is the first attempt at the preparation of Au/Al2O3 catalysts by Mb-mediated in situ synthesis of gold nanoparticles.

Methanobactin-mediated one-step synthesis of gold nanoparticles.

Preparation of gold nanoparticles with a narrow size distribution has enormous importance in nanotechnology. Methanobactin (Mb) is a copper-binding small peptide that appears to function as an agent for copper sequestration and uptake in methanotrophs. Mb can also bind and catalytically reduce Au (III) to Au (0). In this study, we demonstrate a facile Mb-mediated one-step synthetic route to prepare monodispersed gold nanoparticles. Continuous reduction of Au (III) by Mb can be achieved by using hydroquinone as the reducing agent. The gold nanoparticles have been characterized by UV-visible spectroscopy. The formation and the surface plasmon resonance properties of the gold nanoparticles are highly dependent on the ratio of Au (III) to Mb in solution. X-ray photoelectron spectroscopy (XPS), fluorescence spectra and Fourier transform-infrared spectroscopy (FT-IR) spectra suggest that Mb molecules catalytically reduce Au (III) to Au (0) with the concomitant production of gold nanoparticles, and then, Mb statically adsorbed onto the surface of gold nanoparticles to form an Mb-gold nanoparticles assembly. This avoids secondary nucleation. The formed gold nanoparticles have been demonstrated to be monodispersed and uniform by transmission electron microscopy (TEM) images. Analysis of these particles shows an average size of 14.9 nm with a standard deviation of 1.1 nm. The gold nanoparticles are extremely stable and can resist aggregation, even after several months.

Biosysthesis of corn starch palmitate by lipase Novozym 435.

Esterification of starch was carried out to expand the usefulness of starch for a myriad of industrial applications. Lipase B from Candida antarctica, immobilized on macroporous acrylic resin (Novozym 435), was used for starch esterification in two reaction systems: micro-solvent system and solvent-free system. The esterification of corn starch with palmitic acid in the solvent-free system and micro-solvent system gave a degree of substitution (DS) of 1.04 and 0.0072 respectively. Esterification of corn starch with palmitic acid was confirmed by UV spectroscopy and IR spectroscopy. The results of emulsifying property analysis showed that the starch palmitate with higher DS contributes to the higher emulsifying property (67.6%) and emulsion stability (79.6%) than the native starch (5.3% and 3.9%). Modified starch obtained by esterification that possesses emulsifying properties and has long chain fatty acids, like palmitic acid, has been widely used in the food, pharmaceutical and biomedical applications industries.

Preparation of Electrode Modified with Methanobactin Functionalized Gold Nanoparticle and Mimic Superoxide Dismutase Activity.

Using a mixed self-assembly method, this research utilized modified Mb-functionalized gold nanoparticles (Mb-AuNPs-MPA) on the gold electrode surface to prepare a biosensor which was applied to detect superoxide anion-free electron mediators. Together with the study on the performance of the sensor, the characteristics of modified nanoclusters were investigated by UV-Vis and FTIR and the electrochemical characteristics of the electrode surface were investigated using electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV), respectively. It was demonstrated that the charge transfer resistance (Rct) of the modified electrode (Mb-MPA-AuNPs/Au) was 7862 Ω, the exchange current density (i0) was 32.7 µA/cm-2. And a pair of reversible and symmetrical redox peaks appeared after the coordination of Cu2+, and the electrical signal response of Cu2+/Mb-AuNPs-MPA/Au reached the highest. The superoxide anion generated in the basic DMSO system was determined by CV using the modified electrode Cu2+/Mb-AuNPs-MPA/Au. It was discovered that the superoxide anion had a strong disproportionation effect.

Environmentally friendly, efficient resolution of racemic secondary alcohols by lipase-catalyzed enantioselective transesterification in ionic liquids in the presence of organic bases.

The lipase-catalyzed enantioselective transesterification of racemic secondary alcohols was studied using vinyl acetate as acyl donor in two imidazolium-based ionic liquids vs. hexane (Scheme), both in the absence and presence of catalytic amounts of organic bases such as triethylamine (Et(3)N) or pyridine. The organic bases generally enhanced both the rate and enantioselectivity of the reaction. Further, the system 1-butyl-3-methyl-1H-imidazolium hexafluorophosphate/Candida antarctica lipase B ([bmim][PF(6)]/CALB) could be readily recycled four times without significant loss in activity or enantioselectivity.

Methanobactin-mediated synthesis of bimetallic Au-Pd/Al2O3 toward an efficient catalyst for glucose oxidation.

A green bioreductive approach with methanobactin was adopted to fabricate bimetallic Au-Pd/Al2O3 catalysts for solvent-free oxidation of glucose to gluconic acid with H2O2 at atmospheric pressure. The catalyst was characterised by diffuse reflectance UV-vis spectroscopy, transmission electron microscopy, X-ray photoelectron spectroscopy, and X-ray diffraction techniques to understand synergistic interactions between Au and Pd. Effects of Au to Pd molar ratio on the catalytic activity of Au-Pd/Al2O3 were investigated. The Au-Pd/Al2O3 catalyst with Au/Pd molar ratio of 0.8:0.2 exhibited excellent catalytic performance. With the catalyst, the oxidation activities of glucose to gluconic acid 2856 mmol min-1 g-1 and selectivity 99.6% were attained at 323 K with H2O2. The results indicated the activity and selectivity was affected by the ratio of Au/Pd on the Al2O3. The formation of Au0.8Pd0.2/Al2O3 was favourable for the catalytic reaction.

Synthesis of L-Ascorbyl Flurbiprofenate by Lipase-Catalyzed Esterification and Transesterification Reactions.

The synthesis of L-ascorbyl flurbiprofenate was achieved by esterification and transesterification in nonaqueous organic medium with Novozym 435 lipase as biocatalyst. The conversion was greatly influenced by the kinds of organic solvents, speed of agitation, catalyst loading amount, reaction time, and molar ratio of acyl donor to L-ascorbic acid. A series of solvents were investigated, and tert-butanol was found to be the most suitable from the standpoint of the substrate solubility and the conversion for both the esterification and transesterification. When flurbiprofen was used as acyl donor, 61.0% of L-ascorbic acid was converted against 46.4% in the presence of flurbiprofen methyl ester. The optimal conversion of L-ascorbic acid was obtained when the initial molar ratio of acyl donor to ascorbic acid was 5 : 1. kinetics parameters were solved by Lineweaver-Burk equation under nonsubstrate inhibition condition. Since transesterification has lower conversion, from the standpoint of productivity and the amount of steps required, esterification is a better method compared to transesterification.

Colorimetric detection of melamine based on methanobactin-mediated synthesis of gold nanoparticles.

A simple and rapid field-portable colorimetric method for the detection of melamine in liquid milk was reported. Methanobactin (Mb) could reduce Au (III) to Au (0) and mediate the synthesis of gold nanoparticles (Au-NPs). Upon the addition of melamine, melamine interacted with oxazolone ring of Mb, which interrupted the formation of Au-NPs. Melamine could also stimulate the aggregation of formed Au-NPs. In this paper, these characteristics have been used to detect melamine in liquid milk by naked eyes observation with a detection limit of 5.56 × 10(-6)M (0.7 mg/kg). Further, the plasmon absorbance of the formed Au-NPs allowed the quantitative detection of melamine by UV-vis spectrometer. A linear correlation was existed between the absorbance and the melamine concentration ranging from 3.90 × 10(-7)M to 3.97 × 10(-6)M with a correlation coefficient of 0.9685. The detection limit (3σ) obtained by UV-vis spectrum was as low as 2.38 × 10(-7)M (i.e., 0.03 mg/kg).

Methanol production from CO(2) by resting cells of the methanotrophic bacterium Methylosinus trichosporium IMV 3011.

Methanol production from carbon dioxide was successfully achieved using resting cells of Methylosinus trichosporium IMV 3011 as biocatalysts. Carbon dioxide was reduced to methanol and extracellular methanol accumulation has been found in the carbon dioxide incubations. However, resting cells of methanotrophs have a finite or intrinsic methanol production capacity due to a limiting supply of intracellular reducing equivalent. It has been found that the catabolism of stored Poly-beta -Hydroxybutyrate (PHB) can provide intracellular reducing equivalents to improve the intrinsic methanol production capacity. The initial nitrogen and copper concentration in the culture medium were studied for the accumulation of PHB by M. trichosporium IMV 3011, to expand its potential uses in methanol production from carbon dioxide reduction. It appeared that the total methanol production capacity was increased with increasing PHB content in cells. Resting cells containing 38.6% PHB exhibited the highest total methanol production capacity. But higher PHB accumulation adversely affected the total methanol production capacity. The effects of methanol production process on the survival and recovery of M. trichosporium IMV 3011 were examined. The results showed that the methanol production from carbon dioxide reduction was not detrimental to the viability of methanotrophs.

[Methane monooxygenases hydroxylase from a type II methanotroph: purification and physical-chemical properties].

Methanotrophs can catalyze hydroxylate of methane and some hydrocarbon. Which play an important role in mitigating global warming and have also potential significance for industrial applications or bioremediation. A high activity of hydroxylase, a crucial component in sMMO, from Methylosinus trichosporium IMV 3011 has been purified to homologues by using chromatographic techniques. The molecular weight of the hydroxylase determined by gel filtration is 201.3 kD, and SDS-PAGE showed that hydroxylase consists of three subunits(alpha beta gamma) with molecular weights of 58kD, 36kD and 23kD respectively, drawing a comparison both methods indicated that the hydroxylase is a homodimer with an (alpha beta gamma)2 configuration. Purified hydroxylase has a pI at 5.2 judged by thin layer isoelectric focusing. The purified hydroxylase contains 3.02 mol of iron per mol of protein. The stability pH for the hydroxylase in solution is 5.8-8.0 and the stability temperature is below 35 degrees C. The cells form show a long, bent, and rod-shaped with even surface observed by scanning electron microscopy.

[Continuous biosynthesis of epoxypropane in a methanotrophic attached-films reactor].

Using a fluidized bed as immobilization system, mixed culture methanotrophic attached-films were developed on diatomite particles. The Methane Monooxygenase (MMO) activity was found to increase obviously as soon as the lag phase ended. Greater than 90% of the MMO activity in the bed was attached. Biofilm concentration of 3.3-3.7 mg dry weight cell/g DS was observed. Batch experiments were performed to explore the possibility of producing epoxypropane by a cooxidation process. The effect of methane on the oxidation of propene to epoxypropane and the effect of propene on the growth of methanotroph were also studied. In continuous experiments, optimum mixed gaseous substrates (methane: 35%; propene: 20%; oxygen: 45%) were continuously circulated through the fluidized bed reactor to remove product. Initial epoxypropane productivity was 110-150 mumol/d. The bioreactor operated continuously for 25 d without obvious loss of epoxypropane productivity.

Fine separation and characterization of Candida rugosa lipase isoenzymes.

Commercial Candida rugosa lipase has been separated into two distinct fractions (CRLA and CRLB) by anion-exchange chromatography. As analyzed on SDS-polyacrylamide gel electrophoresis, CRLA and CRLB are homogenous. At high ionic strength, CRLA and CRLB have similar hydrophobicity and UV spectra, suggesting that the open extent of the large hydrophobic pockets of CRLA and CRLB may be similar. At low ionic strength, using "hydrophobic interfacial affinity chromatography", both CRLA and CRLB have been separated into four isofractions. They have different hydrophobicity and UV spectra, suggesting that the open extent of the large hydrophobic pocket of the four forms may be different. Further, the conversion of CRL isoenzymes in the process of organic solvent treatment and ester hydrolysis were examined. The results clearly showed not only that CRLB had been converted to CRLA, but also that CRLA sub-fractions with different open extent of large hydrophobic pocket had been converted

Particulate methane monooxygenase from Methylosinus trichosporium is a copper-containing enzyme.

Particulate methane monooxygenase (pMMO) has been exfoliated and isolated from membranes of the Methylosinus trichosporium IMV 3011. It appears that the stability of pMMO in the exfoliation process is increased with increasing copper concentration in the growth medium, but extensive intracytoplasmic membrane formed under higher copper concentration may inhibit the exfoliation of active pMMO from membrane. The highest total activity of purified pMMO is obtained with an initial concentration of 6 microM Cu in the growth medium. The purified MMO contains only copper and does not utilize NADH as electron donor. Treatment of purified pMMO with EDTA resulted in little change in copper level, suggesting that the copper in the pMMO is tightly bound with pMMO.