Stirred Tank Two-Liquid Phase Biocatalytic Reactor Studies: Kinetics, Evaluation and Modelling of Substrate Mass Transfer

Kinetic measurements of the benzyl acetate hydrolysis by pig liver esterase in a two-liquid phase stirred tank reactor were made at a variety of aqueous phase enzyme concentrations and stirrer speeds. All experiments were performed in an inverted liquid-liquid system at a high phase ratio. The results were explained in terms of the aqueous phase bulk reaction model developed from previous Lewis cell studies. An algorithm is presented for the indirect measurement of the substrate mass transfer coefficient and consequently a model was developed to predict reaction rates. While the model describes the kinetics effectively, and could therefore be used to predict reactor behaviour, no difference was observed between kinetic measurements made at a stirrer speed of 750 and 1000 rpm.     

Asymmetric reduction of a ketone by wet and lyophilized cells of Geotrichum candidum in organic solvents

Sixteen organic co-solvents were screened for stereoselective reduction of 1-acetonapthone in aqueous media by whole cells of Geotrichum candidum. Benzyl alcohol was found to be a good co-solvent as it afforded a high coversion and reduced deactivation of the cells. Half-lives of the wet and lyophilized whole cell biocatalysts in pure benzyl alcohol were 23.07 and 11.21 hours, respectively. The initial reaction rates at 30°C were 13.1 and 11.0μmol/min, respectively, for the wet and lyophilized cells. With optimized conditions in a reaction medium containing phosphate buffer and benzyl alcohol (1:1 by vol) with 230mM 1-acetonapthone, more than 98% and 81% conversion (ee >99%) was achieved in 5 hours with the wet and lyophilized cells, respectively. Both the cell preparations showed maximum conversion at 30°C. A thermodynamic characterization revealed that the wet cells were more thermostable than the lyophilized cells. The calculated half-life of the wet cells at pH 7 was 93 hours, whereas that of the lyophilized cells was 71 hours at the same condition.     

Gas chromatographic–mass spectrometric identification and quantitation of benzyl alcohol in serum after derivatization with perfluorooctanoyl chloride: a new derivative

Benzyl alcohol is commonly used as an antibacterial agent in a variety of pharmaceutical formulations. Several fatalities in neonates have been linked to benzyl alcohol poisoning. Most methods for measuring benzyl alcohol concentrations in serum utilize direct extraction followed by high-performance liquid chromatography. We describe here a novel derivatization of benzyl alcohol using perfluorooctanoyl chloride after extraction from human serum for analysis by gas chromatography–mass spectrometry (GC–MS). The derivative was eluted at a significantly higher temperature respective to underivatized molecule and the method was free from interferences from more volatile components in serum and hemolyzed specimens. Another advantage of this derivatization technique is the conversion of low-molecular-mass benzyl alcohol (M_r 108) to a high-molecular-mass derivative (M_r 504). The positive identification of benzyl alcohol can be achieved by observing a distinct molecular ion at m/z 504 as well as the base peak at m/z 91. Quantitation of benzyl alcohol in human serum can easily be achieved by using 3,4-dimethylphenol as an internal standard. The within run and between run precisions (using serum standard of benzyl alcohol: 25 mg/l) were 2.7% (mean=24.1, S.D.=0.66 mg/l, n=8) and 4.2% (mean=24.3, S.D.=1.03 mg/l, n=8), respectively. The assay was linear for the serum benzyl alcohol concentrations of 2 mg/l to 200 mg/l and the detection limit was 0.1 mg/l. We observed no carry-over (memory effect) problem in our assay as when 2 μl ethyl acetate was injected into the GC–MS system after analyzing serum specimens containing 200 mg/l of benzyl alcohol, we observed no peak for either benzyl alcohol or the internal standard in the total ion chromatogram.     

Reductive biotransformation by wild type and mutant strains of Saccharomyces cerevisiae in aqueous-organic solvent biphasic systems

Whole cells of Saccharomyces cerevisiae analyzed the conversion of benzaldehyde to benzyl alcohol in aqueous-organic biphasic media. Reaction rate increased dramatically as moisture content of the solvent was increased in the range 0% to 2%. The highest biotransformation rates were observed when hexane was used as organic solvent. Benzaldehyde was also converted to benzyl alcohol by a cell-free crude extract in biphasic systems containing hexane, although the rate of product formation was much lower. Mutant strains of S. cerevisiae lacking some or all of the ADH isoenzymes, ADH I, II, and III, manifested similar rates for bioconversion of benzaldehyde to benzyl alcohol in both aqueous and two-phase systems. In general, conversion rates observed in aqueous media were 2 to 3 times higher than those observed in hexane containing 2% moisture.     

Lewis cell studies to determine reactor design data for two-liquid-phase bacterial and enzymic reactions

Substrate transfer rates from organic to aqueous phases were measured in the presence and absence of biocatalyst in the reaction medium, using modified Lewis cells. These measurements, in combination with intrinsic aqueous phase biocatalytic reaction kinetics, were used to confirm that benzyl acetate hydrolysis by pig liver esterase and toluene oxidation by a strain of Pseudomonas putida occur uniformly throughout the bulk of the aqueous phase. Such data may be used to provide a basis for two-liquid-phase biocatalytic reactor design.     

大孔树脂固定化脂肪酶及在微水相中催化合成生物柴油的研究

以不同大孔树脂吸附法固定化假丝酵母99_125脂肪酶,在微水有机相中的应用表明非极性树脂NKA是最佳的固定化载体。分别以正庚烷及磷酸盐缓冲液作为固定化介质,发现在正庚烷介质中树脂NKA的固定化效率能够达到98.98%,与采用磷酸盐缓冲液作为介质相比,固定化酶的水解活力和表观酶活回收率分别提高了4.07和3.43倍。考察了在微水相中固定化酶催化合成生物柴油的催化性能,结果表明,在给酶量为1.92∶1(初始酶粉与树脂的质量比),pH值为7.4,体系水含量为15%(水与油的质量比),反应温度为40℃条件下,固定化酶具有最佳的催化能力;以正庚烷为介质固定化脂肪酶催化合成生物柴油,采用三次流加甲醇的方式,单批转化率最高达到97.3%,连续反应19批以后转化率仍保持为70.2%。     

Oxidation of benzyl alcohol by whole cells of Pichia pastoris and by alcohol oxidase in aqueous and nonaqueous reaction media

The low substrate specificity of alcohol oxidase from Pichia pastoris makes this enzyme system of potential biotechnological interest. Whole cells of Pichia pastoris are able to oxidize benzyl alcohol to benzaldehyde in aqueous reaction media. The low water solubility of the reactant and product of this bioconversion, combined with the ability of both to strongly inhibit the reaction, favor the use of nonaqueous reaction fluids. Purified alcohol oxidase was shown to function in a number of 2-phase reaction systems of varied aqueous to organic phase ratios (0.01-0.05 v/v). The apparent V(max) and K(m) were 5.26 g/Lh and 7.41 g/L respectively, for the oxidation of benzyl alcohol to benzaldehyde in hexane containing 3% aqueous phase. The volume of the aqueous phase had a strong effect on the reaction, with an aqueous: organic ratio of 3-5% found to be optimum. The enzyme could be firmly immobilized on DEAE-Biogel (Biorad) to enhance stability and biocatalyst recovery.     

Highly Selective Oxidation of Benzyl Alcohol Using Engineered <Emphasis Type="Italic">Gluconobacter Oxydans</Emphasis> in Biphasic System

The Gluconobacter oxydans M5 with disruption of the pyrroloquinoline quinine-dependent membrane-bound aldehyde dehydrogenase (ALDH) was used for the oxidation of benzyl alcohol. The selectivity toward benzaldehyde showed an obvious increase for the engineered strain, which reached the 67.3%, while the wild strain had only 2.8%. Meantime, the aqueous/isooctane (1:1) biphasic system was used for the further improvement of selectivity. By these methods, nearly 100% selectivity and conversion rate could be obtained within 1 h at the optimum initial benzyl alcohol concentration of 5.0 g/l.     

Benzyl alcohol penetration into micelles, dielectric constant of the binding site, partition coefficient and high-pressure squeeze-out

The absorbance maximum, lambda max, of a local anesthetic, benzyl alcohol, is shifted to longer wavelengths when solvent polarity is decreased. The shift was approximately a linear function of the dielectric constant of the solvent. This transition in electronic spectra according to the microenvironmental polarity is used to analyze benzyl alcohol binding to surfactant micelles. A facile method is devised to estimate the micelle/water partition coefficient from the dependence of lambda max of benzyl alcohol on surfactant concentrations. The effective dielectric constants of the sodium decyl sulfate, dodecyl sulfate and tetradecyl sulfate micelles were 29, 31 and 33, respectively. The partition coefficient of benzyl alcohol between the micelles and the aqueous phase was 417, 610 and 1089, respectively, in the mole fraction unit. The pressure dependence of the partition coefficient was estimated from the depression of the critical micelle concentration of sodium dodecyl sulfate by benzyl alcohol under high pressure up to 200 MPa. High pressure squeezed out benzyl alcohol molecules from the micelle until about 120 MPa, then started to squeeze in when the pressure was further increased. The volume change of benzyl alcohol by transfer from the aqueous to the micellar phase was calculated from the pressure dependence of the partition coefficient. The volume change, estimated from the thermodynamic argument, was 3.5 +/- 1.1 cm3.mol-1 at 298.15 K, which was in reasonable agreement with the partial molal volume change determined directly from the solution density measurements, 3.1 +/- 0.2 cm3.mol-1. Benzyl alcohol apparently solvates into the micelles close to surface without losing contact with the aqueous phase.     

Kinetic modeling of immobilized-lipase catalyzed transesterification of n-octanol with vinyl acetate in non-aqueous media

Organic esters are employed as solvents, fragrance, flavors, and precursors in a variety of industries. Particularly, aliphatic esters are greatly used in flavor industry, mainly as fixatives and modifiers, and aromatic esters in fragrance compositions. Esters are produced by a variety of methods among which esterification and transesterification with acid catalysts under reflux conditions are prominent. The use of biocatalysts provides an opportunity for carrying out reactions under milder conditions leading to better quality products suitable in fragrance and flavor industry. Transesterification of n-octanol with vinyl acetate was studied at 30 °C as a model reaction by employing different lipases as catalysts such as Psedomonas species lipase immobilized on diatomite, free Candida rugosa lipase. Novozym 435 (lipase B from Candida antarctica; immobilized on macro-porous polyacrylic resin beads) and Lipozyme IM 20 (Mucor miehei lipase immobilized on anionic resin). Novozym 435 was found to be the most active catalyst in heptane as a solvent. A conversion of 82% with 100% selectivity of n-octyl acetate was obtained at 30 °C in 90 min using equimolar quantities of the reactants with 0.833 g l⁻¹ of Novozym 435. Transesterification of other alcohols such as n-decanol, benzyl alcohol, cinnamyl alcohol, 2-ethyl-1-hexanol, 1-phenyl ethyl alcohol, and 2-phenyl ethyl alcohol was also studied with vinyl acetate. The analysis of the initial rate data and progress curve data showed that the reaction obeys the ternary complex bi–bi mechanism with inhibition by n-octanol. The experimental and theoretical values matched very well.

The order of transesterification reactivity of vinyl acetate with various alcohols in presence of Novozym 435 under otherwise identical conditions at 30 °C was found to be as follows:

n-octanol>n-decanol>benzylalcohol>cinnamylalcohol>2-ethyl-1-hexanol>2-phenylethylalcohol>1-phenylethylalcohol.

     

Enzymatic synthesis of benzyl benzoate using different acyl donors: Comparison of solvent-free reaction techniques

In this study, benzyl benzoate was successfully synthesized via enzymatic acylation using three immobilized enzymes as biocatalysts. Different acyl donors (benzoic acid and benzoic anhydride), operation regimes (batch, fed-batch), mixing modes (conventional mechanical stirring and ultrasound), process parameters (temperature, substrate molar ratio of acyl donor to acyl acceptor), presence or absence of solvents, enzyme amount and type were evaluated. Benzoic acid is a solid that is difficult to solubilize and, thus, was not efficient as acyl donor for the synthesis of benzyl benzoate. On the other hand, benzoic anhydride was very effective for the acylation of benzyl benzoate, and the presence of an excess of benzyl alcohol was essential to ensure the solute-solvent intermolecular attractions and good substrate solubilization, allowing the ester synthesis to be performed in the absence of organic solvents. The ultrasound was effective in increasing increase the initial reaction rate and the final conversion (88 %). However, the Lipozyme TL-IM and RM-IM supports were damaged, and the reuse was unfeasible. The batch and fed-batch approaches in conventional stirring ensured high conversions of 92 and 90 %, respectively, for batch (anhydride: alcohol 1:6) and fed-batch (1:3) using the Lipozyme TL-IM as biocatalyst. The controlled addition of the anhydride (fed-batch) allowed the reduction of alcohol molar ratio but decreased the reaction rates, and the maximum conversions were reached only after 24 h, while the batch approach had 92 % of conversion after 6 h. The yield of benzyl benzoate was high at 6 wt.% of enzyme, low temperature (50 °C), and simple reactor operation (batch). Results show the feasibility of the synthesis of benzyl benzoate via acylation using a green process that may be an alternative route to the chemical synthesis.     

Lipase catalyzed synthesis of benzyl acetate in solvent-free medium using vinyl acetate as acyl donor

Use of vinyl acetate as acyl donor in transesterification of benzyl alcohol catalyzed by a commercially available lipase (Lipozyme RM IM) gave 100% conversion in 10 min. The excess acyl donor and the enzyme could be recovered and reused. Unlike the chemical catalytic processes, it produced no undesirable side product.     

Modelling and experimental studies on lipase-catalyzed isoamyl acetate synthesis in a microreactor

A lipase-catalyzed synthesis of isoamyl acetate was studied in a continuously operated pressure-driven microreactor. The esterification of isoamyl alcohol and acetic acid occurred at the interface between n-hexane and an aqueous phase with dissolved lipase B from Candida antarctica. By adjusting flow rates of both phases, a parallel laminar flow with liquid–liquid boundary in the middle of the microchannel could be reestablished and a separation of phases was achieved at the y-shaped exit of the microreactor. Since product remained in the organic phase, this also enabled its continuous separation from the aqueous phase with the enzyme. A three-dimensional mathematical model was developed, considering the velocity profile developed for steady-state conditions between two immiscible fluids. The model contained convection, diffusion, and enzyme reaction terms, where esterification rate was described with a Ping-Pong Bi-Bi mechanism and inhibition by both substrates. Experimental data, which were in good agreement with model simulations, have demonstrated 35% conversion at residence time 36.5 s at 45 °C and at 0.5 M acetic acid and isoamyl alcohol inlet concentrations, which is much faster as in any literature reported so far. According to model simulations, obtained by non-equidistant finite differences numerical solutions of complex non-linear equations system, further microreactor design and process optimization are feasible.     

Lactose hydrolysis by immobilized beta-galactosidase in capillary bed reactor

The hydrolysis of lactose by immobilized beta-galactosidase was studied in a continuous-flow capillary bed reactor operating at 30 degrees C. Solutions containing 50, 100, and 150 g lactose and 0.5 g sodium acetate/L were fed to the reactor. Lactose conversions ranging from 24% to greater than 99% were achieved at reactor space times ranging from 0.06 to 6.3 min. These conversion data were successfully modeled in terms of a plug flow reactor model and a form of Michaelis-Menten kinetics which included competitive inhibition by both the alpha and beta forms of galactose.     

Production of geranyl acetate and other acetates by direct esterification catalyzed by mycelium of Rhizopus delemar in organic solvent

Dry mycelium of Rhizopus delemar MIM catalyzed the formation of geranyl acetate using 110 mM geraniol and acetic acid at 55°C in heptane to give 11.9 g/l (55% molar conversion). Geranyl acetate was produced at 72.5-75 g/l after 10 days by semi-continuous addition of the substrates. Rhizopus delemar also catalyzed the direct acetylation of different primary alcohols with molar conversions ranging from 65 to 98%.     

The influence of age and task specialization on the production and perception of honey bee pheromones

The production and perception of honey bee pheromones were studied in workers of different ages and task specializations. Electroantennogram responses increased to a maximum by days 6–12 and no differences were seen between bees performing different tasks. Levels of production of isopentyl acetate, octyl acetate, (E)-2-octenyl acetate, butyl acetate, and hexyl acetate increased with age to maximal levels at days 30–40. Production of 2-nonanol, 2-nonyl acetate, benzyl acetate, and benzyl alcohol differed from the above compounds, with most workers producing very low levels and a few producing very high levels. When examined by task specialization, foragers produced higher levels of benzyl acetate, benzyl alcohol, and 2-nonanol than guards, fanners, or comb bees respectively.     

Production of immobilized penicillin acylase using aqueous polymer systems for enzyme purification and in situ immobilization

A novel approach for the isolation and purification of penicillin acylase (PA), which couples aqueous two-phase partitioning and enzyme immobilization has been investigated.A PA yield of 90% was achieved by treating E. coli cells with 4% butyl acetate, freeze-thawing step, and pressure homogenization. PA purification (93% recovery) was achieved by (1) removing cell debris via precipitation with polyethylene glycol (PEG 2000); (2) aqueous two-phase partitioning using a PEG 2000 + phosphate system (87% recovery).An in situ enzyme immobilization approach, using oxirane acrylic or aldehyde-agarose beads dispersed in the PEG-rich phase, was explored for the conversion of penicillin G to 6-aminopenicillanic acid. An appropriate immobilization reaction time was found. The catalytic performance of the enzyme, when immobilized, was found not to be affected by recycling of the phase-forming components.     

Generation of Phenylpropanoid Pathway-Derived Volatiles in Transgenic Plants: Rose Alcohol Acetyltransferase Produces Phenylethyl Acetate and Benzyl Acetate in Petunia Flowers

Esters are important contributors to the aroma of numerous flowers and fruits. Acetate esters such as geranyl acetate, phenylethyl acetate and benzyl acetate are generated as a result of the action of alcohol acetyltransferases (AATs). Numerous homologous AATs from various plants have been characterized using in-vitro assays. To study the function of rose alcohol acetyltransferase (RhAAT) in planta, we generated transgenic petunia plants expressing the rose gene under the control of a CaMV-35S promoter. Although the preferred substrate of RhAAT in vitro is geraniol, in transgenic petunia flowers, it used phenylethyl alcohol and benzyl alcohol to produce the corresponding acetate esters, not generated by control flowers. The level of benzyl alcohol emitted by the flowers of different transgenic lines was ca. three times higher than that of phenylethyl alcohol, which corresponded to the ratio between the respective products, i.e. ca. three times more benzyl acetate than phenylethyl acetate. Feeding of transgenic petunia tissues with geraniol or octanol led to the production of their respective acetates, suggesting the dependence of volatile production on substrate availability.     

Enhanced synthesis of isoamyl acetate using liquid-gas biphasic system by the transesterification reaction of isoamyl alcohol obtained from fusel oil

In this work, the transesterification reaction of isoamyl alcohol obtained from fusel oil and leading to the synthesis of isoamyl acetate was conducted simultaneously with in situ ethanol removal, which allows to shift the reaction equilibrium toward ester synthesis. The extracellular Aspergillus oryzae lipase was immobilized into calcium alginate. Effects of immobilization conditions on the loading efficiency and on the specific activity of entrapped lipase were investigated. The kinetic transfer of volatile reactants from the reactor was investigated using an experimentally first order kinetic model, in order to approve the feasibility of the liquid-gas system with continuous ethanol removal in the ester synthesis. The effects of the most influent parameters affecting the reaction have been also investigated using a Doehlert matrix design. The better operating conditions for isoamyl acetate synthesis were: a temperature of 68.5°C and a respective isoamyl alcohol and A. oryzae lipase concentration of 0.72 M and 2.39 g/L. At these conditions, the resulting reaction conversion and ethanol extraction yields were of 89.55 and 69.60%, respectively. The use of the fluidized bed reactor with continuous ethanol removal has allowed to improve the reaction conversion which was two times than the conversion higher obtained in batch reactor. Furthermore, under the optimized conditions in the fluidized bed reactor, the reaction conversion and the ethanol extraction yields were increased by 44.8 and 36.2%, respectively.