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.     

Kinetic analysis and modeling of the liquid-liquid conversion of emulsified di-rhamnolipids by Naringinase from Penicillium decumbens

The enzymatic conversion of an aggregate-forming substrate was kinetically analyzed and a model was applied for the prediction of reaction-time courses. An L-rhamnose molecule from a di-rhamnolipid is cleaved by Naringinase from Penicillium decumbens leading to a mono-rhamnolipid. Optimal reaction rates were found when both, substrate and product build large co-aggregates in a slightly acidic aqueous phase. On the other hand, reaction rates were independent of initial di-rhamnolipid concentration and this was interpreted by assuming that the reaction occurs in the aqueous phase according to Michaelis-Menten kinetics in combination with competitive L-rhamnose inhibition. Rhamnolipids were therefore assumed to be highly concentrated in aggregates, a second liquid phase, whereas diffusive rhamnolipid transport from and to the aqueous phase occurs due to the enzymatic reaction. Furthermore, ideal surfactant mixing between di- and mono-rhamnolipid was assumed for interpretation of the negative effect of the last on the reaction rate. A model was created that describes the system accordingly. The comparison of the experimental data, were in excellent agreement with the predicted values. The findings of this study may beneficially be adapted for any bioconversion involving aggregate-forming substrate and/or product being catalyzed by hydrophilic enzymes.     

Microbial Oxidation of Tetradecanols and Related Substances in Organic Solvents

Microbial oxidations of n-tetradecane, tetradecanols and tetradecanoic acid were investigated by using intact cells of Corynebacterium equi, a hydrocarbon-assimilating bacterium, in an aqueous phase and organic solvents. The bacterial cells were hydrophobic and could be well dispersed in all organic solvents employed to give homogeneous reaction mixtures, and among them, isooctane was found to be the best for the reaction. n-Tetradecane and tetradecanoic acid were completely oxidized in the aqueous phase, but not in isooctane. In contrast, 1-tetradecanol was oxidized much more readily in isooctane than in the aqueous phase, and an oxidation product identified as myristyl myristate was accumulated in isooctane at the conversion rate of 80%. 2-Tetradecanol was also readily oxidized in isooctane, and 2-tetradecanone was obtained at the conversion rate of nearly 100%. Similar results were obtained when toluene and n-hexane were used as the solvent in place of isooctane, while no reaction was observed when chloroform was employed.     

Oxygen transfer enhancement in aqueous/perfluorocarbon fermentation systems: I. experimental observations

A new fiber-optic dissolved oxygen sensing technique was applied to the study of two-phase aqueous/perfluorocarbon (pfc) dispersions. These dispersions were examined for their oxygen transfer enhancement capability in the absence and presence of an oxygen-consuming reaction. For the pfc-in-water dispersions, oxygen uptake rate (OUR) enhancements were equal both with and without oxygen-consuming cells present in the aqueous phase. In contrast, for water-in-pfc dispersions, OUR enhancements inthe presence of reaction were limited by oxygen diffusion across the aqueous phase droplets. Nevertheless, enhancement factors of 5-10 on an aqueous phase volume basis were obtained in a 75% pfc dispersion.These oxygen transfer enhancements were directly translatable into enhancements in overall fermenter productivity for actual microbial cultivation systems.     

Lipidic sponge phase crystallization of membrane proteins

Bicontinuous lipidic cubic phases can be used as a host for growing crystals of membrane proteins. Since the cubic phase is stiff, handling is difficult and time-consuming. Moreover, the conventional cubic phase may interfere with the hydrophilic domains of membrane proteins due to the limited size of the aqueous pores. Here, we introduce a new crystallization method that makes use of a liquid analogue of the cubic phase, the sponge phase. This phase facilitates a considerable increase in the allowed size of aqueous domains of membrane proteins, and is easily generalised to a conventional vapour diffusion crystallisation experiment, including the use of nanoliter drop crystallization robots. The appearance of the sponge phase was confirmed by visual inspection, small-angle X-ray scattering and NMR spectroscopy. Crystals of the reaction centre from Rhodobacter sphaeroides were obtained by a conventional hanging-drop experiment, were harvested directly without the addition of lipase or cryoprotectant, and the structure was refined to 2.2 Angstroms resolution. In contrast to our earlier lipidic cubic phase reaction centre structure, the mobile ubiquinone could be built and refined. The practical advantages of the sponge phase make it a potent tool for crystallization of membrane proteins.     

Investigation of behavior of an enzyme in a biphasic system: soybean lipoxygenase-1

Soybean lipoxygenase-1 (EC 1.13.11.12) reaction with linoleic acid as substrate was used to study the biocatalysis in a biphasic system when the reactants have surface-active properties. The poorly water-soluble substrate was initially dissolved in an apolar solvent (octane). The hydroperoxide produced was water soluble and remained in the aqueous phase (borate buffer). The bioreactor was a modified Lewis cell with a well-defined interfacial area between the two phases. Two phenomena were studied separately: the reactant transfer between the two phases and the biocatalyzed reaction in an aqueous medium. This allowed determination of the transfer and the reaction constants. Substrate transfer was found to be affected by the progress of the reaction, because linoleic acid and the hydroperoxy acid have an influence on the interfacial tension. Inactivation of the biocatalyst at the interface was observed in the bioreactor. These results indicate that it is impossible to analyze the system behavior with the method proposed in the literature, which is based on the sequential study of the substrate transfer to the aqueous phase and its biocatalysis by lipoxygenase. The interaction between transfer phenomena and reaction kinetics was studied in the biphasic system. The kinetics were different from those obtained in the aqueous medium. Catalysis and transfer influence each other reciprocally. In this compartmentalized system, cooperativity phenomena were obtained using a nonallosteric enzyme. The evolution of the system was modeled (Runge-Kutta algorithm). The curves obtained were very close to those determined experimentally.     

Computer simulation of damped oscillations during peroxidase-catalyzed oxidation of indole-3-acetic acid

Oscillation patterns in horseradish peroxidase (HRP)-catalyzed oxidation of indole-3-acetic acid (IAA) at neutral pH were studied using computer simulation. Under certain conditions, such as the presence of a reaction promoter and continuous intake of oxygen from the gaseous phase, the simulated system exhibits damped oscillations of the concentrations of oxygen in the aqueous phase, [O(2)](aq), and of all the reaction intermediates. The critical concentration of oxygen in aqueous phase, [O(2)](cr)(aq), was used to describe the nature of the oscillations. The critical concentration is the concentration at which the system abruptly changes its properties. If [O(2)](aq) is higher than [O(2)](cr)(aq) then the reaction develops as an avalanche, otherwise, the reaction stops. The nature of oscillations is accounted for by the interaction of two processes: the consumption/accumulation of oxygen and the accumulation/consumption of reaction intermediates. Oscillations are always damped. Neither HRP or umbelliferone (Umb) deactivation nor IAA consumption can account for the damping. The nature of the damping is determined by the termination reactions of free radical intermediates and ROOH. The three major parameters of oscillations: period of oscillations, initial amplitude of oscillations and the rate of damping were studied as functions of: (i) oxygen concentration in the gaseous phase, (ii) initial oxygen concentration in aqueous phase, (iii) the concentration of IAA and (iv) the initial concentration of HRP.     

Gas phase enantioselective reduction catalyzed by immobilized ketoreductase: Effects of water activity and reaction temperature

The performance (activity, stability, enantioselectivity and productivity) of the commercial ketoreductase immobilized on non-porous glass supports was investigated as functions of the water activity and the reaction temperature in a continuous gas phase reactor. The enantioselective reduction of 2-butanone to (S)-2-butanol with the in situ regeneration of β-nicotinamide adenine dinucleotide phosphate by 2-propanol catalyzed by the immobilized ketoreductase was used as a model reaction. The activity, stability and enantioselectivity were strongly influenced by the water activity and the reaction temperature. The optimal water activity and reaction temperature were obtained at 0.8 and 313–323 K in terms of the productivity, respectively. Successfully, the enantioselectivity for the gas phase system attained the level identical to that for the aqueous phase system.     

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.     

Enzymatic hydrolysis of soluble starch in a polyethylene glycol-dextran aqueous two-phase system

Hydrolysis of soluble starch by glucoamylase and β-amylase was investigated as a model reaction in an aqueous two-phase system consisting of polyethylene glycol (PEG) and dextran (DEX). Changes in glucose concentration observed in the batch reaction experiments with glucoamylase were almost identical for the aqueous two-phase and pure water systems, showing that the enzymic reactions investigated were not influenced by the presence of PEG and DEX. The partition of β-amylase into the DEX phase was insufficient compared to that of glucoamylase. Hence, the former enzyme was crosslinked with glutaraldehyde to increase its apparent molecular weight and, as a consequence, the partition coefficient, defined as the concentration ratio of the component partitioned into the PEG phase to that into the DEX phase, was decreased to 17% of that of the original enzyme. In the operation in which the enzyme and substrate are partitioned selectively into the DEX phase and allowed to react there while the product, thus transferring to the PEG phase, is recovered, the aqueous two-phase system with a smaller partition coefficient provided longer operational stability.     

Kinetics of the extraction of succinic acid with tri-n-octylamine in 1-octanol solution

Kinetic studies for the extraction of succinic acid from aqueous solution with 1-octanol solutions of tri-n-octylamine (TOA) were carried out using a stirred cell with a microporous hydrophobic membrane. The interfacial concentrations of species were correlated and thus the intrinsic kinetics was obtained. The overall extraction process was controlled by the chemical reaction at or near the interface between the aqueous and organic phases. The formation reaction of succinic acid-TOA complex was found to be first order with respect to the concentration of succinic acid in the aqueous phase and the order of 0.5 with respect to that of TOA in the organic phase with a rate constant of (3.14 +/- 0.6) x 10(-8) m(2.5) x mol(-0.5) x s(-1). The dissociation reaction of succinic acid-TOA complex was found to be the second-order with respect to that of succinic acid-TOA complex in the organic phase and the order of -2 with respect to that of TOA in the organic phase with a rate constant of (1.44 +/- 1.4) x 10(-4) mol x m(-2) x s(-1).     

Continuous enzymatic production of peptide precursor in aqueous/organic biphasic medium

N-(benzyloxycarbonyl)-L-aspartic acid (Z-L-Asp) has generally been used as a carboxyl substrate for the enzymatic synthesis of a precursor of aspartame (synthetic sweetener); however, alternative inexpensive protection groups have been in demand for lowering the total cost of its industrial-scale production. A formyl group (F-) was found to be a more desirable protecting group for the N-terminus of amino acid derivatives due to its low cost of preparation, introduction, and removal. The yield of F-AspPheOMe (N-formyl-L-aspartyl-L-phe- nylalanine methylester), however, was found to be <10% in a conventional aqueous medium. We found that F-L-Asp and L-PheOMe were partitioned mainly to the aqueous phase in an aqueous/organic biphasic medium, whereas F-AspPheOMe partitioned to the organic phase, especially when some extracting agents were added. In this study, simultaneous operation of an enzymatic reaction and a product separation by liquid-liquid extraction was thus applied to the F-AspPheOMe synthesis. We succeeded in synthesizing F-AspPheOMe continuously in an aqueous/tributylphosphate (TBP) biphasic medium with >95% yield, which was about tenfold higher than that in an aqueous monophasic medium.     

Use of aqueous two-phase systems for in situ extraction of water soluble antibiotics during their synthesis by enzymes immobilized on porous supports

Yields of kinetically controlled synthesis of antibiotics catalyzed by penicillin G acylase from Escherichia coli (PGA) have been greatly increased by continuous extraction of water soluble products (cephalexin) away from the surroundings of the enzyme. In this way its very rapid enzymatic hydrolysis has been avoided. Enzymes covalently immobilized inside porous supports acting in aqueous two-phase systems have been used to achieve such improvements of synthetic yields. Before the reaction is started, the porous structure of the biocatalyst can be washed and filled with one selected phase. In this way, when the pre-equilibrated biocatalyst is mixed with the second phase (where the reaction product will be extracted), the immobilized enzyme remains in the first selected phase in spite of its possibly different natural trend. Partition coefficients (K) of cephalexin in very different aqueous two-phase systems were firstly evaluated. High K values were obtained under drastic conditions. The best K value for cephalexin (23) was found in 100% PEG 600-3 M ammonium sulfate where cephalexin was extracted to the PEG phase. Pre-incubation of immobilized PGA derivatives in ammonium sulfate and further suspension with 100% PEG 600 allowed us to obtain a 90% synthetic yield of cephalexin from 150 mM phenylglycine methyl ester and 100 mM 7-amino desacetoxicephalosporanic acid (7-ADCA). In this reaction system, the immobilized enzyme remains in the ammonium sulfate phase and hydrolysis of the antibiotic becomes suppressed because of its continuous extraction to the PEG phase. On the contrary, synthetic yields of a similar process carried out in monophasic systems were much lower (55%) because of a rapid enzymatic hydrolysis of cephalexin.     

A novel approach to biotransformations in aqueous-organic two-phase systems: enzymatic synthesis of alkyl beta-[D]-glucosides using microencapsulated beta-glucosidase

A novel approach to enzymatic biotransformations in aqueous-organic two-phase systems was developed where the aqueous phase was contained within permeable polymeric capsules suspended in organic solvent. Microencapsulated beta-glucosidase, used as a model enzyme, was shown to retain its catalytic activity for a considerable time and was repeatedly used in batch experiments after recharging the microcapsules with solid glucose. The reaction conditions for the synthesis of hexyl beta-[D]-glucopyranoside were optimized with regard to the polymer composition of the microcapsules, pH, and the volume ratio of aqueous to organic phases. The potential for further improvement in the efficiency of the system was demonstrated by designing a bioreactor which incorporated units for product recovery and recycling of the organic solvent. Other advantages of the proposed methodology include facile control over the size and composition of the microcapsules, and mild reaction conditions during their preparation.     

Enhancement of hydrolytic activity of sphingolipid ceramide N-deacylase in the aqueous-organic biphasic system

Lysoglycosphingolipids were produced from glycosphingolipids by using sphingolipid ceramide N-deacylase, which cleaves the N-acyl linkage between fatty acids and sphingosine bases in various glycosphingolipids. The enzyme reaction was done in a biphasic media prepared with water;-immiscible organic solvent and aqueous buffer solution containing the enzyme. We investigated the effects of organic solvents and detergents on lysoglycosphingolipid production in the biphasic system. Among the organic solvents tested, n-butylbenzene, cumene, cyclodecane, cyclohexane, n-decane, diisopropylether, n-heptadecane, and methylcyclohexane promoted hydrolysis of GM1, whereas benzene, chloroform, ethyl acetate, and toluene inhibited GM1 hydrolysis. Hydrolysis of asialo GM1, GD1a, GalCer, and sulfatide was also enhanced by the addition of n-decane. The hydrolytic activity of the enzyme was enhanced by the addition of 0.8% sodium taurodeoxycholate or sodium cholate to the aqueous phase. The most effective hydrolysis of various glycosphingolipids by the enzyme was thus obtained in the aqueous-n-decane biphasic system containing 0.8% sodium taurodeoxycholate. Under this condition, the fatty acids released from GM1 by the action of the enzyme were trapped and diffused into the organic phase, while lysoGM1 remained in the aqueous phase.Thus the almost complete hydrolysis of GM1 was achieved using the biphasic system, while at most 70% of hydrolysis was obtained using normal aqueous media possibly due to the inhibition of hydrolysis reaction by accumulation of fatty acids in the reaction mixture.     

固定化青霉素酰化酶在光-pH敏感可回用两水相中裂解青霉素G为6-APA

两水相体系在发展中存在的关键问题是相体系回收困难.由于生产成本及降低污染的原因, 用过的相体系需要回收和重复使用.用环境敏感型溶解可逆聚合物形成可回用两水相体系是当前是为可行的回收方法。本文在光敏感可回用高聚物PNBC与pH敏感型可回用高聚物PADB形成的两水相体系中进行固定化青霉素酰化酶的相转移催化青霉素G产生6-APA的反应。在这个两水相体系中,通过优化,在1% NaCl 存在下,６－ＡＰＡ的分配系数可达5.78。催化动力学显示,达平衡的时间近7h,反应最高得率约85.3%（pH 7.8, 20℃）。较相近条件下的单水相反应得率提高近20%。在反应过程中,通过底物及产物的分配系数检测,发现底物分配系数变化不大,而产物6-APA及苯乙酸的分配系数发生很大变化,从而引起产物的得率变化。在两水相中,底物及产物主要分配在上相,固定化酶分配在下相,底物青霉素G进入下相经酶催化产生的6-APA及苯乙酸又转入上相,从而解除了青霉素酰化酶催化反应的底物及产物抑制作用,达到提高产物得率的效果。此外,采用固定化酶较固定化细胞效率高,占用下相体积小,较游离酶稳定性高,且完全单侧分配在下相。因此,在两水相中进行固定化酶的催化反应具有明显的优越性。形成两水相的高聚物PNBC通过488 nm 的激光照射或经滤光的450nm 光源照射得到回收;pH敏感型成相聚合物PADB可通等电点 4.1沉淀可实现循环利用,高聚物的回收率在95%-98%之间,按此回收率计算,聚合物可使用60次以上。     

Flow injection analysis-based methodology for automatic on-line monitoring and quality control for biodiesel production

An automated on-line approach based on determination of free and bound glycerol was here proposed to monitor biodiesel production. The method was based on liquid-liquid extraction of glycerol from the biodiesel to an aqueous ethanolic phase in which glycerol is oxidized to formaldehyde with meta periodate with subsequent reaction with acetylacetone. The reaction product was photometrically measured at 410 nm. Free and bound glycerol were differentiated by glycerides hydrolysis with potassium ethylate. The experimental set-up consisted of a flow-injection manifold for liquid-liquid extraction without phase separation and iterative change of the flow direction that enabled: (a) filling the flow manifold with a meta periodate-acetylacetone acceptor phase; (b) sampling of small amounts (microl) from the reactor; (c) determination of free glycerol by extraction from biodiesel to the aqueous phase with simultaneous oxidation-reaction with acetylacetone in the acceptor phase; (d) continuous monitoring of the aqueous phase by passage through a photometric detector; (e) filling the flow manifold with a potassium ethylate-meta periodate-acetylacetone new acceptor phase; (d) repetition of steps b-to-d to determine total glycerol after saponification of the bound glycerol by potassium ethylate; and (f) determination of bound glycerol by difference between the second and first analyses. The results showed that the proposed automated on-line method is a suitable option in routine analysis during biodiesel production.     

Enzymatic esterification in organic media: role of water and organic solvent in kinetics and yield of butyl butyrate synthesis

Summary The respective roles of organic solvent and of water in butyl butyrate synthesis from n-butanol and n-butyric acid in n-hexane by Mucor miehei lipase have been investigated by analysis of the kinetics and the reaction balances. Esterificaton was found to take place in both low water systems containing solid enzyme in hexane and in biphasic aqueous enzyme solution/hexane systems. In the solid enzyme system, the enzyme adsorbed the water produced, thus delaying the appearance of a discrete aqueous phase. As expected, the presence of some water was indispensable for this system, as its removal or exclusion by various means (adsorption, distillation) affected enzyme activity. However, water removal had little effect on the final yield of esterification. Reaction velocities were quite similar for the solid enzyme/hexane system and for the biphasic aqueous enzyme solution/hexane system. In the latter case, the butyl butyrate formed was almost exclusively found in the organic phase. Ethyl butyrate, a more polar compound, was synthesized with a lower yield. These results allow the conclusion that the reaction took place in a phase consisting of either solid hydrated enzyme with no discrete aqueous phase or of an aqueous enzyme solution by basically similar mechanisms according to the amount of water available to the system, the esterification being driven to completion by transfer of the ester into the organic phase because of a favourable partition coefficient. Offprint requests to: F. Monot