Cascade enzymatic synthesis of a statin side chain precursor - the role of reaction engineering in process optimization.

Statins are an important class of drugs used to lower blood cholesterol levels and are often used to combat cardiovascular disease. In view of the importance of safe and reliable supply and production of statins in modern medicine and the global need for sustainable processes, various biocatalytic strategies for their synthesis have been investigated. In this work, a novel biocatalytic route to a statin side chain precursor was investigated in a one-pot cascade reaction starting from the protected alcohol N-(3-hydroxypropyl)-2-phenylacetamide, which is oxidized to the corresponding aldehyde in the first reaction step, and then reacts with two equivalents of acetaldehyde to form the final product N-(2-((2S,4S,6S)-4,6-dihydroxytetrahydro-2H-pyran-2-yl)ethyl)-2-phenylacetamide (phenylacetamide-lactol). To study this complex reaction, an enzyme reaction engineering approach was used, i.e. the kinetics of all reactions occurring in the cascade (including side reactions) were determined. The obtained kinetic model together with the simulations gave an insight into the system and indicated the best reactor mode for the studied reaction, which was fed-batch with acetaldehyde feed to minimize its negative effect on the enzyme activity during the reaction. The mathematical model of the process was developed and used to simulate different scenarios and to find the reaction conditions (enzyme and coenzyme concentration, substrate feed concentration and flow rate) at which the highest yield of phenylacetamide-lactol (75%) can be obtained. In the end, our goal was to show that this novel cascade route is an interesting alternative for the synthesis of the statin side chain precursor and that is why we also calculated an initial estimate of the potential value addition.

Thermostability Engineering of a Class II Pyruvate Aldolase from Escherichia coli by in Vivo Folding Interference.

The use of enzymes in industrial processes is often limited by the unavailability of biocatalysts with prolonged stability. Thermostable enzymes allow increased process temperature and thus higher substrate and product solubility, reuse of expensive biocatalysts, resistance against organic solvents, and better "evolvability" of enzymes. In this work, we have used an activity-independent method for the selection of thermostable variants of any protein in Thermus thermophilus through folding interference at high temperature of a thermostable antibiotic reporter protein at the C-terminus of a fusion protein. To generate a monomeric folding reporter, we have increased the thermostability of the moderately thermostable Hph5 variant of the hygromycin B phosphotransferase from Escherichia coli to meet the method requirements. The final Hph17 variant showed 1.5 °C higher melting temperature (T m) and 3-fold longer half-life at 65 °C compared to parental Hph5, with no changes in the steady-state kinetic parameters. Additionally, we demonstrate the validity of the reporter by stabilizing the 2-keto-3-deoxy-l-rhamnonate aldolase from E. coli (YfaU). The most thermostable multiple-mutated variants thus obtained, YfaU99 and YfaU103, showed increases of 2 and 2.9 °C in T m compared to the wild-type enzyme but severely lower retro-aldol activities (150- and 120-fold, respectively). After segregation of the mutations, the most thermostable single variant, Q107R, showed a T m 8.9 °C higher, a 16-fold improvement in half-life at 60 °C and higher operational stability than the wild-type, without substantial modification of the kinetic parameters.

A cascade reaction for the synthesis of d-fagomine precursor revisited: Kinetic insight and understanding of the system.

The synthesis of aldol adduct (3S,4R)-6-[(benzyloxycarbonyl)amino]-5,6-dideoxyhex-2-ulose, a precursor of the interesting dietary supplement, iminosugar d-fagomine, was studied in a cascade reaction with three enzymes starting from Cbz-N-3-aminopropanol. This system was studied previously using a statistical optimization method which enabled a 79 % yield of the aldol adduct with a 10 % yield of the undesired amino acid by-product. Here, a kinetic model of the cascade, including enzyme operational stability decay rate and the undesired overoxidation of the intermediate product, was developed. The validated model was instrumental in the optimization of the cascade reaction in the batch reactor. Simulations were carried out to determine the variables with the most significant impact on substrate conversion and product yield. As a result, process conditions were found that provided the aldol adduct in 92 % yield with only 0.7 % yield of the amino acid in a one-pot one-step reaction. Additionally, compared to previous work, this improved process outcome was achieved at lower concentrations of two enzymes used in the reaction. With this study the advantages are demonstrated of a modelling approach in developing complex biocatalytical processes. Mathematical models enable better understanding of the interactions of variables in the investigated system, reduce cost, experimental efforts in the lab and time necessary to obtain results since the simulations are carried out in silico.

Mathematical model of the MenD-catalyzed 1,4-addition (Stetter reaction) of α-ketoglutaric acid to acrylonitrile.

The Stetter reaction, a conjugate umpolung reaction, is well known for cyanide-catalyzed transformations of mostly aromatic aldehydes. Enzymatic Stetter reactions, however, have been largely unexplored, especially with respect to preparative transformations. We have investigated the kinetics of the MenD-catalyzed 1,4-addition of α-ketoglutaric acid to acrylonitrile which has shown that acrylonitrile, while an interesting candidate, is a poor substrate for MenD due to low affinity of the enzyme for this substrate. The kinetic model of the reaction was simplified to double substrate Michaelis-Menten kinetics where the reaction rate linearly depends on acrylonitrile concentration. Experiments at different initial concentrations of acrylonitrile under batch, repetitive batch, and fed-batch reactor conditions were carried out to validate the developed mathematical model. Thiamine diphosphate dependent MenD proved to be quite a robust enzyme; nevertheless, enzyme operational stability decay occurs in the reactor. The spontaneous reactivity of acrylonitrile towards polymerization was also taken into account during mathematical modeling. Almost quantitative conversion of acrylonitrile was achieved in all batch reactor experiments, while the yield of the desired product was dependent on initial acrylonitrile concentration (i.e., the concentration of the stabilizer additive). Using the optimized reactor parameters, it was possible to synthesize the product, 6-cyano-4-oxohexanoic acid, in a concentration of 250 mM. The highest concentration of product was achieved in a repetitive batch reactor experiment. A fed-batch reactor experiment also delivered promising results, especially regarding the short reaction time needed to achieve a 200 mM concentration of product. Hence, the enzymatic Stetter reaction with a highly reactive acceptor substrate can be performed on a preparative scale, which should enable similar transformations with acrylate, methacrylate, and methyl vinyl ketone.

Different strategies for multi-enzyme cascade reaction for chiral vic-1,2-diol production.

The stereoselective three-enzyme cascade for the one-pot synthesis of (1S,2S)-1-phenylpropane-1,2-diol ((1S,2S)-1-PPD) from inexpensive starting substrates, benzaldehyde and acetaldehyde, was explored. By coupling stereoselective carboligation catalyzed by benzoylformate decarboxylase (BFD), L-selective reduction of a carbonyl group with alcohol dehydrogenase from Lactobacillus brevis (ADHLb) as well as the coenzyme regeneration by formate dehydrogenase (FDH), enantiomerically pure diastereoselective 1,2-diol was produced. Two different multi-enzyme system approaches were applied: the sequential two-step one-pot and the simultaneous one-pot cascade. All enzymes were kinetically characterized. The impact of acetaldehyde on the BFD and ADHLb stability was investigated. To overcome the kinetic limitation of acetaldehyde in the carboligation reaction and to reduce its influence on the enzyme stability, experiments were performed in two different excesses of acetaldehyde (100 and 300%). Due to the ADHLb deactivation by acetaldehyde, the simultaneous one-pot cascade proved not to be the first choice for the investigated three-enzyme system. In the sequential cascade with 300% acetaldehyde excess a 100% yield of vic 1,2-diol was reached.

A mathematical model of oxidative deamination of amino acid catalyzed by two D-amino acid oxidases and influence of aeration on enzyme stability.

Two D-amino acid oxidases (DAAO) from different sources (Arthrobacter protophormiae and porcine kidney) were used to oxidatively deaminate D-methionine in the batch reactor. A mathematical model of the process was developed and validated by the experiments carried out without and with oxygen supply by aeration. Kinetic parameters of the model were estimated from the initial reaction rate experiments. Aeration increased the reaction rate in the initial part of the reaction and reduced the time necessary to achieve the final substrate conversion. However, it had a negative influence on the operational stability of enzymes. Operational stability decay rate constants estimated from the experimental data increased with the airflow rate, which indicated lower operational stability of enzymes. It was found that oxygen concentration significantly influenced the stability of DAAO from porcine kidney. Enzyme from microbial source had better operational stability and one order of magnitude lower values of decay rate constants.

Mathematical model for aldol addition catalyzed by two D-fructose-6-phosphate aldolases variants overexpressed in E. coli.

Two D-fructose-6-phosphate aldolase variants namely, single variant FSA A129S and double variant FSA A129S/A165G, were used as catalysts in the aldol addition of dihydroxyacetone (DHA) to N-Cbz-3-aminopropanal. Mathematical model for reaction catalyzed by both enzymes, consisting of kinetic and mass balance equations, was developed. Kinetic parameters were estimated from the experimental data gathered by using the initial reaction rate method. The model was validated in the batch and continuously operated ultrafiltration membrane reactor (UFMR). The same type of kinetic model could be applied for both enzymes. The operational stability of the aldolases was assessed by measuring enzyme activity during the experiments. FSA A129S/A165G had better operational stability in the batch reactor (half-life time 26.7 h) in comparison to FSA A129S (half-life time 5.78 h). Both variants were unstable in the continuously operated UFMR in which half-life times were 1.99 and 3.64 h for FSA A129S and FSA A129S/A165G, respectively.

Aldol addition of dihydroxyacetone to N-Cbz-3-aminopropanal catalyzed by two aldolases variants in microreactors.

Aldol addition of dihydroxyacetone to N-Cbz-3-aminopropanal catalyzed by two d-fructose-6-phosphate aldolase variants, FSA A129S and FSA A129S/A165G, overexpressed in Escherichia coli was studied in microreactors. The presence of organic solvent was necessary due to poor solubility of N-Cbz-3-aminopropanal in water. Hence, three co-solvents were evaluated: ethyl acetate, acetonitrile and dimethylformamide (DMF). The influence of these solvents and their concentration on the enzyme activity was independently tested and it was found that all solvents significantly reduce the activity of FSA depending on their concentration. The reaction was carried out in three different microreactors; two without and one with micromixers. By increasing enzyme concentration, it was possible to achieve higher substrate conversion at lower residence time. Enzyme activity measured at the outlet flow of the microreactor at different residence time revealed that enzymes are more stable at lower residence times due to shorter time of exposure to organic solvent. The reaction in the batch reactor was compared with the results in microreactor with micromixers. Volume productivity was more than three fold higher in microreactor with micromixers than in the batch reactor for both aldolases. It was found to be 0.88Md(-1) and 0.80Md(-1) for FSA A129S and FSA A129S/A165G, respectively.

Complete starch hydrolysis by the synergistic action of amylase and glucoamylase: impact of calcium ions.

Starch hydrolysis was performed by the synergistic action of amylase and glucoamylase. For that purpose glucoamylase (Dextrozyme) and two amylases (Liquozyme and Termamyl) in different combinations were investigated. Experiments were carried out in the repetitive- and fed-batch modes at 65 °C and pH 5.5 with and without the addition of Ca(2+) ions. 100 % conversion of starch to glucose was achieved in batch experiments. Calcium ions significantly enhanced stability of the amylase Termamyl. The intensity of synergism between amylase Termamyl and glucoamylase Dextrozyme was higher than in the experiments carried out with amylase Liquozyme and Dextrozyme. Mathematical model of the complete reaction system was developed. Using the model, a possible explanation of the synergism between the amylase and glucoamylase was provided.

Effect of different variables on the efficiency of the Baker's yeast cell disruption process to obtain alcohol dehydrogenase activity.

Cell disruption process of dry baker's yeast was studied in this work to obtain maximum activity of alcohol dehydrogenase (ADH). Disruption by ultrasonication, glass beads, and combination of these two methods was compared. A 1.8-fold increase of ADH activity can be achieved by combining glass beads with ultrasonication in comparison to ultrasonication. To achieve maximum volume activity of ADH, the effect of different variables on the cell disruption process was investigated (time, glass bead diameter, mass of glass beads, and ultrasound amplitude). Using the Design-Expert© software, 24 factorial experimental design was performed. Two ultrasound probes were tested: MS 73 and KE 76. Optimal conditions (process variables) for cell disruption process were obtained. Optimal ADH activities after cell disruption with MS 73 and KE 76 probes were 1,890.9 and 1,531.7 U cm⁻³, respectively. Necessary ultrasonication time and ultrasound amplitude should be at the maximum values in the investigated variable range (30 min and 62 %). Bead size should be at maximum (4 mm) when using MS 73 probe and at minimum (0.3 mm) when using KE 76 probe. Partial purification of the enzyme was carried out and it was kinetically characterized using several oxidation and reduction systems.

Mathematical modeling of maize starch liquefaction catalyzed by α-amylases from Bacillus licheniformis: effect of calcium, pH and temperature.

The first step of starch hydrolysis, i.e. liquefaction has been studied in this work. Two commercial α-amylases from Bacilllus licheniformis, known as Termamyl and Liquozyme have been used for this purpose. Using starch as the substrate, kinetics of both enzymes has been determined at optimal pH and temperature (pH 7, T = 80 °C) and at 65 °C and pH 5.5. Michaelis-Menten model with uncompetitive product inhibition was used to describe enzyme kinetics. Mathematical models were developed and validated in the repetitive batch and fed-batch reactor. Enzyme inactivation was described by the two-step inactivation model. All experiments were performed with and without calcium ions. The activities of both tested amylases are approximately one hundred times higher at 80 °C than at 65 °C. Lower inactivation rates of enzymes were noticed in the experiments performed at 65 °C without the addition of calcium than in the experiments at 80 °C. Calcium ions in the reaction medium significantly enhance amylase stability at 80 °C and pH 7. At other process conditions (65 °C and pH 5.5) a weaker calcium stabilizing effect was detected.

Coenzyme regeneration in hexanol oxidation catalyzed by alcohol dehydrogenase.

The enzymatic ways of coenzyme regeneration include the addition of a second enzyme to the system or the addition of the co-substrate. In the present study, both methods of enzymatic coenzyme (NAD(+)) regeneration were studied and compared in the reaction of hexanol oxidation catalyzed by alcohol dehydrogenase (ADH). As a source of ADH, commercial isolated enzyme and the whole baker's yeast cells were used. First, coenzyme regeneration was employed in the reaction of acetaldehyde reduction catalyzed by the same enzyme that catalyzed the main reaction, and then NAD(+) regeneration was applied in the reaction of pyruvate reduction catalyzed by L-lactate dehydrogenase (L-LDH). Hexanal was obtained as the product of hexanol oxidation catalyzed by isolated ADH while hexaonic acid was detected as a product of the same reaction catalyzed by baker's yeast cells. All of the used biocatalysts were kinetically characterized. The mass reactions were described by the mathematical models. All models were validated in the batch reactor. One hundred percent hexanol conversion was obtained using permeabilized yeast cells using both methods of cofactor regeneration. By using isolated enzyme ADH, the higher conversion was achieved in a system with cofactor regeneration catalyzed by L-LDH.

Evaluation of factors influencing the enantioselective enzymatic esterification of lactic acid in ionic liquid.

In this paper esterification of ethanol and lactic acid catalyzed by Candida antarctica B (Novozyme 435) in ionic liquid (Cyphos 104) was studied. The influence of different variables on lipase enantioselectivity and lactic acid conversion was investigated. The variables investigated were ionic liquid mass/lipase mass ratio, water content, alcohol excess and temperature. Using the Design Expert software 2(3) factorial experimental plan (two levels, three factors) was performed to ascertain the effect of selected variables and their interactions on the ethyl lactate enantiomeric excess and lactic acid conversion. The results of the experiments and statistical processing suggest that temperature and alcohol excess have the highest effect on the ethyl lactate enantiomeric excess, while temperature and water content have the highest influence on the lactic acid conversion. The statistical mathematical model developed on the basis of the experimental data showed that the highest enantiomeric excess achieved in the investigated variable range is 34.3%, and the highest conversion is 63.8% at the initial conditions of water content at 8%; 11-fold molar excess of alcohol and temperature at 30 °C.

Optimization of laccase production by Trametes versicolor cultivated on industrial waste.

Laccases are very interesting biocatalysts for several industrial applications. Its production by different white-rot fungi can be stimulated by a variety of inducing substrates, and the use of lignocellulosic wastes or industrial by-products is one of the possible approaches to reduce production costs. In this work, various industrial wastes were tested for laccase production by Trametes versicolor MZKI G-99. Solid waste from chemomechanical treatment facility of a paper manufacturing plant showed the highest potential for laccase production. Enzyme production during submerged cultivation of T. versicolor on the chosen industrial waste has been further improved by medium optimization using genetic algorithm. Concentrations of five components in the medium were optimized within 60 shake-flasks experiments, where the highest laccase activity of 2,378 U dm(-3) was achieved. Waste from the paper industry containing microparticles of CaCO(3) was found to stimulate the formation of freely dispersed mycelium and laccase production during submerged cultivation of T. versicolor. It was proven to be a safe and inexpensive substrate for commercial production of laccase and might be more widely applicable for metabolite production by filamentous fungi.

Modelling as a tool of enzyme reaction engineering for enzyme reactor development.

Strategy of the development of model for enzyme reactor at laboratory scale with respect to the modelling of kinetics is presented. The recent literature on the mathematic modelling on enzyme reaction rate is emphasized.

Guidelines for reporting of biocatalytic reactions.

Enzymes and whole cells are being increasingly applied in research and industry, but the adoption of biocatalysis relies strongly on useful scientific literature. Unfortunately, too many published papers lack essential information needed to reproduce and understand the results. Here, members of the scientific committee of the European Federation of Biotechnology Section on Applied Biocatalysis (ESAB) provide practical guidelines for reporting experiments. The document embraces the recommendations of the STRENDA initiative (Standards for Reporting Enzymology Data) in the context of pure enzymology and provides further guidelines and explanations on topics of crucial relevance for biocatalysis. In particular, guidelines are given on issues such as the selectivity, specificity, productivity and stability of biocatalysts, as well as on methodological problems related to reactions in multiphase systems. We believe that adoption and use of these guidelines could greatly increase the value and impact of published work in biocatalysis, and hence promote the further growth of applications.

Mathematical model for Trametes versicolor growth in submerged cultivation.

Trametes versicolor is a white-rot fungus known as a producer of extracellular enzymes such as laccase, manganese-peroxidase, and lignin-peroxidase. The production of these enzymes requires detailed knowledge of the growth characteristics and physiology of the fungus. Submerged cultivations of T. versicolor on glucose, fructose, and sucrose as sole carbon sources were performed in shake flasks. Sucrose hydrolysis catalyzed by the whole cells of T. versicolor was considered as one-step enzymatic reaction described with Michaelis-Menten kinetics. Kinetic parameters of invertase-catalyzed sucrose hydrolysis were estimated (K (m) = 7.99 g dm(-3) and V (m) = 0.304 h(-1)). Monod model was used for description of kinetics of T. versicolor growth on glucose and fructose as sole carbon sources. Growth associated model parameters were estimated from the experimental results obtained by independent experiments (mu(G)(max) = 0.14 h(-1), K(G)(S) = 8.06 g dm(-3), mu(F)(max) = 0.37 h(-1) and K(F)(S) = 54.8 g dm(-3)). Developed mathematical model is in good agreement with the experimental results.

Oxidation of Coniferyl Alcohol Catalyzed by Laccases from Trametes versicolor.

Oxidation of coniferyl alcohol catalyzed by commercial laccase and crude laccase produced during the submerged cultivation of Trametes versicolor MZKI G-99 in a medium containing the waste from paper industry was investigated. pH of 6.6 and temperature of 35 °C was found to be optimal for coniferyl alcohol oxidation catalyzed by commercial laccase. Based on the initial reaction rate measurements, apparent Michaelis-Menten kinetic parameters for commercial laccase were determined in an aqueous media (Vm = 4.387 U mg-1, Km = 0.025 mmol dm-3), as well as in 1:1 (v/v) methanol: phosphate buffer mixture (Vm = 0.979 U mg-1, Km = 0.019 mmol dm-3). Inhibition of substrate was found for crude laccase and the following apparent kinetic parameters Vm = 9.272 U mg-1, Km = 0.045 mmol dm-3 and Ki = 0.002 mmol dm-3 were estimated. Mathematical model of batch process, which includes double-substrate Michaelis-Menten kinetics with oxygen as the second substrate and mass balances, has been developed and validated in experiments with or without additional aeration. 100 % conversions of up to 0.8 mmol dm-3 of coniferyl alcohol in batch experiment due to the high operational stability of enzymes was realized with both laccases.

Mathematical modeling of maltose hydrolysis in different types of reactor.

A commercial enzyme Dextrozyme was tested as catalyst for maltose hydrolysis at two different temperatures: 40 and 65 degrees C at pH 5.5. Its operational stability was studied in different reactor types: batch, repetitive batch, fed-batch and continuously operated enzyme membrane reactor. Dextrozyme was more active at 65 degrees C, but operational stability decay was observed during the prolonged use in the reactor at this temperature. The reactor efficiencies were compared according to the volumetric productivity, biocatalyst productivity and enzyme consumption. The best reactor type according to the volumetric productivity for maltose hydrolysis is batch and the best reactor type according to the biocatalyst productivity and enzyme consumption is continuously operated enzyme membrane reactor. The mathematical model developed for the maltose hydrolysis in the different reactors was validated by the experiments at both temperatures. The Michaelis-Menten kinetics describing maltose hydrolysis was used.

Mathematical modelling of NADH oxidation catalyzed by new NADH oxidase from Lactobacillus brevis in continuously operated enzyme membrane reactor.

NADH oxidase from Lactobacillus brevis was kinetically characterized in two different buffers: Tris-HCl and glycine-sodium pyrophosphate (pH 9.0). Reaction kinetics was described using the Michaelis-Menten model with product (NAD(+)) inhibition. It was found that this type of inhibition is uncompetitive. Experiments in the continuously operated enzyme membrane reactor revealed a strong enzyme deactivation at two different residence times: 12 and 60 min. A stronger deactivation was observed at the lower residence time in the glycine-sodium pyrophosphate buffer. Enzyme deactivation was assumed to be of the first order. The developed mathematical model for the continuously operated enzyme membrane reactor described these experiments very well. The mathematical model simulations revealed that a high enzyme concentration (up to 30 g cm(-3)) is necessary to obtain and maintain the stationary NADH conversion near 100% for a longer period of time.