Preparation of optically active D-arylglycines for use as side chains for semisynthetic penicillins and cephalosporins using immobilized subtilisins in two-phase systems

Optically active D-arylglycines, which are of interest for preparation of semisynthetic penicillins and cephalosporins, were isolated from the racemic mixtures of their derivatives using immobilized proteolytic enzyme subtilisin (EC No. 3.4.4. 16). The performance of these reactions in two-phase systems, consisting of water and an immiscible organic solvent, improved the yield, purity, and economics of the process by increasing the substrate solubility and reducing the rate of nonenzymatic hydrolysis. The proportion of the organic phase can be as much as 75% of the overall volume without seriously impairing the enzymatic activity. The optically pure D-and L-arylglycines were liberated from their D- and L-derivatives by acid hydrolysis. The substituent influence of the various arylglycine derivatives on the rate of the enzymatic cleavage reaction was investigated.     

Formation of C-C bonds by mandelonitrile lyase in organic solvents

Mandelonitrile lyase (EC 4.1.2.10) catalyzes the formation of D-mandelonitrile from HCN and benzaldehyde. Mandelonitrile lyase was immobilized by adsorption to support materials, for example, Celite. The enzyme preparations were used in diisopropyl ether for production of D-mandelonitrile. In order to obtain optically pure D-mandelonitrile it was necessary to use reaction conditions which favor the enzymatic reaction and suppress the competing spontaneous reaction, which yields a racemic mixture of D, L-mandelonitrile. The effects of substrate concentrations, water content, and support materials on both the spontaneous and enzymatic reactions were studied. The enzymatic reaction was carried out under conditions where the importance of the spontaneous reaction was negligible and high enantiomeric purity of D-mandelonitrile was achieved (at least 98% enantiomeric excess). The operational stability of the enzyme preparations was studied in batch as well as in continuous systems. It was vital to control the water content in the system to maintain an active preparation. In a packed bed reactor the enzyme preparations were shown to be active and stable. The reactors were run for 50 h with only a small decrease in product yield.     

Potential applications of an alcohol-aldehyde/ketone oxidoreductase from thermophilic bacteria

Practical uses of a novel alcohol dehydrogenase from Thermoanaerobium brockii have been examined in crude and purified form. Stoichiometric reduction of NADP (50 mg) was demonstrated with agarose-immobilized enzyme and 0.3 (v/v) 2-propanol solution as reductant. A coenzyme recycle number of 20000 was achieved in enzymatic reactions that employed the alcohol dehydrogenase for NADPH/NADP regeneration. Gram-scale synthesis of chiral R(+) 2-pentanol was shown in a system composed of enzyme, 2-pentanone and 2-propanol as reductant. The effect of temperature, reaction time and substrate concentration on alcohol optical purity was examined. An optical purity of 80% was achieved in the enzymatic synthesis of R(+) 2-pentanol. The enzyme was easily immobilized and stable on an enzyme electrode for analytical detection of alcohols and carbonyls. T. brockii enzyme has potential applications as a commercial alcohol dehydrogenase because of broad substrate specificity and activity at high temperature or high solvent concentration, rare carbonyl si-face stereo-specificity in hydrogen transfer, and high stability and activation of immobilized enzyme.     

Structure-based mutagenesis approaches toward expanding the substrate specificity of D-2-deoxyribose-5-phosphate aldolase

2-Deoxyribose-5-phosphate aldolase (DERA, EC 4.1.2.4) catalyzes the reversible aldol reaction between acetaldehyde and D-glyceraldehyde-3-phosphate to generate D-2-deoxyribose-5-phosphate. It is unique among the aldolases as it catalyzes the reversible asymmetric aldol addition reaction of two aldehydes. In order to expand the substrate scope and stereoselectivity of DERA, structure-based substrate design as well as site-specific mutation has been investigated. Using the 1.05 A crystal structure of DERA in complex with its natural substrate as a guide, five site-directed mutants were designed in order to improve its activity with the unnatural nonphosphorylated substrate, D-2-deoxyribose. Of these, the S238D variant exhibited a 2.5-fold improvement over the wild-type enzyme in the retroaldol reaction of 2-deoxyribose. Interestingly, this S238D mutant enzyme was shown to accept 3-azidopropinaldehyde as a substrate in a sequential asymmetric aldol reaction to form a deoxy-azidoethyl pyranose, which is a precursor to the corresponding lactone and the cholesterol-lowering agent Lipitor. This azidoaldehyde is not a substrate for the wild-type enzyme. Another structure-based design of new nonphosphorylated substrates was focused on the aldol reaction with inversion in enantioselectivity using the wild type or the S238D variant as the catalyst and 2-methyl-substituted aldehydes as substrates. An example was demonstrated in the asymmetric synthesis of a deoxypyranose as a new effective synthon for the total synthesis of epothilones. In addition, to facilitate the discovery of new enzymatic reactions, the engineered E. coli strain SELECT (Deltaace, adhC, DE3) was developed to be used in the future for selection of DERA variants with novel nonphosphorylated acceptor specificity.     

Influence of solvent and water activity on kinetically controlled peptide synthesis.

alpha-Chymotrypsin deposited on Celite was used to catalyse peptide synthesis reactions between N-protected amino acid esters and leucine amide in organic media with low water content. The influence of the solvent and the thermodynamic water activity on the reaction kinetics was studied. The substrate specificity in the reactions was shown to be a combination of the substrate specificity of the enzyme in aqueous media and the influence of the solvents. The magnitude of the solvent effects differed greatly depending on the substrates used. In hydrophobic solvents high reaction rates were observed and the competing hydrolysis of the ester substrate occurred to only a minor extent. Reactions occurred at water activities as low as 0.11, but the rate constants increased with increasing water activity and were about two orders of magnitude higher at the highest water activity tested (0.97).     

Asymmetric enzymatic oxidoreductions in organic solvents

It is now beyond doubt that enzymes can vigorously work even in neat organic solvents containing little or no water. Switching the enzymatic reaction medium from aqueous to nonaqueous can make previously problematic processes feasible through, for example, increased substrate solubility or diminished side reactions. Moreover, when placed in this highly unnatural milieu, enzymes exhibit new and potentially valuable properties, including greater stability, markedly altered selectivity that can be readily controlled by the solvent, and molecular memory. Consequently, novel synthetic and biotechnological opportunities ensue, as illustrated herein by those based on enzymatic oxidoreductions such as the asymmetric peroxidase-catalyzed sulfoxidation of organic sulfides.     

Production of glucose syrups in highly concentrated systems

We have investigated the hydrolysis of maltodextrins in a high concentration (up to 70%), by means of enzymatic and acid catalysis. The study revealed that the equilibrium compositions of the catalyzed reactions were kinetically determined by the selectivity of the catalyst, the substrate concentration and the reaction time. A model comprising a set of two kinetic equations was used to describe the hydrolysis and condensation reactions of glucoamylase-catalyzed reactions, even to highly concentrated systems. Increased substrate concentration resulted in the formation of more condensation products. The enzyme inhibition was low and was found to be independent of the substrate concentration.     

Thermodynamic activity‐based intrinsic enzyme kinetic sheds light on enzyme–solvent interactions

The reaction medium has major impact on biocatalytic reaction systems and on their economic significance. To allow for tailored medium engineering, thermodynamic phenomena, intrinsic enzyme kinetics, and enzyme–solvent interactions have to be discriminated. To this end, enzyme reaction kinetic modeling was coupled with thermodynamic calculations based on investigations of the alcohol dehydrogenase from Lactobacillus brevis (LbADH) in monophasic water/methyl tert‐butyl ether (MTBE) mixtures as a model solvent. Substrate concentrations and substrate thermodynamic activities were varied separately to identify the individual thermodynamic and kinetic effects on the enzyme activity. Microkinetic parameters based on concentration and thermodynamic activity were derived to successfully identify a positive effect of MTBE on the availability of the substrate to the enzyme, but a negative effect on the enzyme performance. In conclusion, thermodynamic activity‐based kinetic modeling might be a suitable tool to initially curtail the type of enzyme–solvent interactions and thus, a powerful first step to potentially understand the phenomena that occur in nonconventional media in more detail. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 33:96–103, 2017     

Chemoenzymatic synthesis of new derivatives of glycyrrhetinic acid with antiviral activity. Molecular docking study

We present an efficient approach to the synthesis of a series of glycyrrhetinic acid derivatives. Six derivatives, five of them new compounds, were obtained through chemoenzymatic reactions in very good to excellent yield. In order to find the optimal reaction conditions, the influence of various parameters such as enzyme source, nucleophile:substrate ratio, enzyme:substrate ratio, solvent and temperature was studied. The excellent results obtained by lipase catalysis made the procedure very efficient considering their advantages such as mild reaction conditions and low environmental impact. Moreover, in order to explain the reactivity of glycyrrhetinic acid and the acetylated derivative to different nucleophiles in the enzymatic reactions, molecular docking studies were carried out. In addition, one of the synthesized compounds exhibited remarkable antiviral activity against TK + and TK- strains of Herpes simplex virus type 1 (HSV-1), sensitive and resistant to acyclovir (ACV) treatment.     

Synthesis of dipeptide precursors with an immobilized thermolysin in ethyl acetate

N-(Benzyloxycarbonyl)-L-aspartyl-L-phenylalanine methyl ester (Z-AspPheOMe), a precursor of the aspartame, and N-(benzyloxycarbonyl)-L-phenylalanyl-Lphenylalanine methyl ester (Z-PhePheOMe) were synthesized from the respective amino acid derivatives with an immobilized thermolysin (EC 3.4.24.4) in ethyl acetate. Various factors affecting the synthesis of these dipeptide precursors were clarified. The initial synthetic rate was the highest at the water content of 3.5% for both reactions. The substrate concentration dependencies of the initial synthetic rate of Z-AspkPheOMe and Z-PhePheOMe with the immobilized enzyme in ethyl acetate were different from those in an aqueous buffer solution saturated with ethyl acetate but similar to those in the aqueous/organic biphasic system using the free enzyme. Particularly, the initial synthetic rate of Z-AspPhOMe increased in order higher than first order with respect to the concentration of L-phenylalanine methyl ester (PheOMe), whereas it decreased sharply with the concentration of N-(benzyloxycarbonyl)-L-aspartic acid (Z-Asp). Such kinetic behavior could be explained by regarding the inside of the immobilized enzyme as being a biphasic mode composed from the organic phase and aqueous phase where the enzymatic reaction takes place. The reaction in the aqueous/organic biphasic system using the free enzyme could be simulated by taking into consideration the partition of the substrate and the initial rate of synthesis in the aqueous buffer saturated with ethyl acetate. Based on this analysis, the rate of reaction with the immobilized enzyme in ethyl acetate could also be predicted. Z-AsPheOMe and Z-PhePheOMe were synthesized by the fed-batch method where the acid component of the substrate was intermittently added during the course of reaction and by the batch method. In the synthesis of Z-AspPheOMe, the synthetic rate and maximum yield of reaction as well as the stability of the immobilized enzyme were higher in the fed-batch reaction than those in the batch reaction. In the synthesis of Z-PhePheOMe, the results obtained by both methods were similar. (c) 1994 John Wiley & Sons, Inc.     

Modulating enzyme activity using ionic liquids or surfactants

One of the important strategies for modulating enzyme activity is the use of additives to affect their microenvironment and subsequently make them suitable for use in different industrial processes. Ionic liquids (ILs) have been investigated extensively in recent years as such additives. They are a class of solvents with peculiar properties and a "green" reputation in comparison to classical organic solvents. ILs as co-solvents in aqueous systems have an effect on substrate solubility, enzyme structure and on enzyme–water interactions. These effects can lead to higher reaction yields, improved selectivity, and changes in substrate specificity, and thus there is great potential for IL incorporation in biocatalysis. The use of surfactants, which are usually denaturating agents, as additives in enzymatic reactions is less reviewed in recent years. However, interesting modulations in enzyme activity in their presence have been reported. In the case of surfactants there is a more pronounced effect on the enzyme structure, as can be observed in a number of crystal structures obtained in their presence. For each additive and enzymatic process, a specific optimization process is needed and there is no one-fits-all solution. Combining ILs and surfactants in either mixed micelles or water-in-IL microemulsions for use in enzymatic reaction systems is a promising direction which may further expand the range of enzyme applications in industrial processes. While many reviews exist on the use of ILs in biocatalysis, the present review centers on systems in which ILs or surfactants were able to modulate and improve the natural activity of enzymes in aqueous systems.     

Enzymatic esterification between n-alcohol homologs and n-caprylic acid in non-aqueous medium under microwave irradiation

The enzymatic esterification between n-alcohol homologs and n-caprylic acid catalyzed by lipozyme RM IM (LRI) in microwave field was investigated. Some interesting findings were obtained. The optimum reaction temperature slightly shifted from that in enzymatic esterification by conventional heating. n-Alcohol homologs used in this experiment showed substrate specificity in terms of the odd and even carbon numbers. THF expressed abnormal solvent effect. Whereas in the contrastive enzymatic esterification by conventional heating, the above mentioned substrate specificity and solvent effect were not observed. All the above phenomena could be explained by both thermal and non-thermal effect of microwave on enzyme and substrates. Further investigation revealed that microwave irradiation reduced the apparent activation energy of the enzymatic reaction according to Arrhenius equation, which is considered as one of the causes increasing initial reaction rate.     

Low reaction temperature increases the selectivity in an enzymatic reaction due to substrate solvation effects

Immobilized a-chymotrypsin was used as catalyst for studying temperature effects on acyl transfer reactions (acyl-donor: Bz-TyrOEt) in a water-immiscible organic solvent. The solubility of the two nucleophiles, Phe-NH and water, decreased with decreasing temperature. The relative decrease for the amide was larger (2.4-fold) than for water. Therefore the thermodynamic activity (estimated by the relative saturation) increased more for this substrate and hence the selectivity in the reaction was increased.     

Stabilization of Candida antarctica lipase B in hydrophilic organic solvent by rational design of hydrogen bond

Enzymatic reactions conducted in organic solvents have many advantages. However, organic solvent molecules may replace water molecules at the protein surface and penetrate into the enzyme, which could lead to the denaturation of the enzyme or changes in its reaction kinetics and substrate specificity. Thus, it is important to enhance the stability of enzymes in organic solvents. To date, there has been no efficient rational approach developed to enhance enzyme stability in hydrophilic solvents. We developed a rational approach to enzyme design. The design rules were established by investigating stable mutants from previous studies of directed evolution. Candida antarctica lipase B (CalB) was used as a target enzyme due to its versatile applications in organic solvents. The N97Q, N264Q, and D265E mutants of CalB showed higher organic solvent stability than the wild type.     

High-Yielding Enzymatic α-Glucosylation of Pyridoxine by Marine α-Glucosidase from Aplysia fasciata

We recently succeeded in the identification and purification of an interesting marine exo-α-glucosidase (EC 3.2.1.20) from the anaspidean mollusc Aplysia fasciata. The enzyme was characterized by good transglycosylation activity toward different acceptors using maltose as donor. High-yielding enzymatic α-glycosylation of pyridoxine using this marine enzyme is reported here; the reaction has been optimized, reaching 80% molar yield of products (pyridoxine monoglucosides 24 g/l; pyridoxine isomaltoside 35 g/l). High selectivity toward the 5′ position is observed for both monoglucoside and disaccharide formation. This is the first report describing the enzymatic production of pyridoxine isomaltoside.     

New insights on nucleoside 2′-deoxyribosyltransferases: a versatile Biocatalyst for one-pot one-step synthesis of nucleoside analogs

In recent years, glycosiltransferases have arisen as standard biocatalysts for the enzymatic synthesis of a wide variety of natural and non-natural nucleosides. Such enzymatic synthesis of nucleoside analogs catalyzed by nucleoside phosphorylases and 2′-deoxyribosyltransferases (NDTs) has demonstrated to be an efficient alternative to the traditional multistep chemical methods, since chemical glycosylation reactions include several protection–deprotection steps. This minireview exhaustively covers literature reports on this topic with the final aim of presenting NDTs as an efficient option to nucleoside phosphorylases for the synthesis of natural and non-natural nucleosides. Detailed comments about structure and catalytic mechanism of described NDTs, as well as their possible biological role, substrate specificity, and advances in detection of new enzyme specificities towards different non-natural nucleoside synthesis are included. In addition, optimization of enzymatic transglycosylation reactions and their application in the synthesis of natural and non-natural nucleosides have been described. Finally, immobilization of NDTs is shown as a practical procedure which leads to the preparation of very interesting biocatalysts applicable to industrial nucleoside synthesis.     

Amino-benzosuberone: a novel warhead for selective inhibition of human aminopeptidase-N/CD13

This paper describes the design and synthesis of compounds belonging to a novel class of highly selective mammalian CD13 inhibitors. Racemic homologues of 3-amino-2-tetralone 1 were synthesised and evaluated for their ability to selectively inhibit the membrane-bound, zinc-dependent aminopeptidase-N/CD13 (EC 3.4.11.2). Some of these novel non-peptidic compounds are potent, competitive inhibitors of the mammalian enzyme, with K(i) values in the low micromolar range in spite of their minimal size (MW <200 Da). Moreover, they show an interesting selectivity profile against representative members of the aminopeptidase family, that is leucine aminopeptidase (EC 3.4.11.1), Aeromonas proteolytica aminopeptidase (EC 3.4.11.10) and the aminopeptidase activity of leukotriene A4 hydrolase (EC 3.3.2.6). The amino-benzosuberone derivative 4 is the most promising compound in terms of potency, stability and selectivity. A hypothetical binding mode of 4 to the catalytic zinc and several conserved active site residues is proposed, based on the observed structure-activity relationships, structural insights from aminopeptidase-N homologues of known three-dimensional structure.     

Kinetics of coupled enzyme reactions

A theory has been developed for the kinetics of coupled enzyme reactions. This theory does not assume that the first reaction is irreversible. The validity of this theory is confirmed by a model system consisting of enoyl-CoA hydratase (EC 4.2.1.17) and 3-hydroxyacyl-CoA dehydrogenase (EC 1.1.1.35) with 2,4-decadienoyl coenzyme A (CoA) as a substrate. This theory, in contrast to the conventional theory, proves to be indispensible for dealing with coupled enzyme systems where the equilibrium constant of the first reaction is small and/or the concentration of the coupling enzyme is higher than that of the intermediate. Equations derived on the basis of this theory can be used to calculate steady-state velocities of coupled enzyme reactions and to predict the time course of coupled enzyme reactions during the pre steady state.     

A comparison of lipase-catalysed ester and lactone synthesis in low-water systems: analysis of optimum water activity.

We investigated the effects of the lyophilisation medium (enzyme plus buffer salt and additives) and of water activity (a(w)) on the catalytic properties of lipase from Chromobacterium viscosum (lipase CV) in organic solvents; catalysis of ester and lactone synthesis were compared and, despite the similarities of the reactive groups involved in these reactions, some interesting differences were observed. Including 2-[N-morpholino]ethanesulfonic acid (MES) buffer in the lyophilisation medium of lipase CV increased its catalytic activity in transesterification and lactonisation, although the buffer salt requirement for maximal activity differed between the two reactions. Sorbitol, glucose, lactose, 18-crown-6 (crown ether 18-C-6), beta-cyclodextrin and bovine serum albumin were employed as alternative additives in the transesterification reaction, but were not as effective as MES buffer. Salt hydrates were used to investigate the effect of a(w) on esterification and lactonisation reactions catalysed by lipase CV. The maximum rate of hexadecanolide synthesis in toluene occurred at a(w) = 0.48. The optimum a(w) for the transesterification reaction in heptane/alcohol mixtures depended on the alcohol substrate employed (1-heptanol, 2-heptanol, or 3-methyl-3-hexanol) but not on the acyl donor (p-NP acetate or caprylate). The optimum a(w) values for both reactions were unchanged when a common solvent system (toluene/1-heptanol) was employed, indicating that the dependence of enzyme activity on a(w) is an intrinsic property of the enzyme-catalysed reaction and not a function of the solvent or other additives.     

Designing enzymatic kyotorphin synthesis in organic media with low water content

Design of enzymatic kyotorphin synthesis in low water media has been carried out as a function of enzyme nature, the immobilization support material and the reaction medium, by using N-benzoyl-L-tyrosine ethyl ester and L-argininamide as substrates. Native and chemically-glycated alpha-chymotrypsin deposited on supports with different degrees of aquaphilicity (celite, polypropylene PP, and polyamide PA6) were used as catalysts. Binary organic solvent systems of ethanol and different water-immiscible organic cosolvents (ethylacetate, tert-butanol, chloroform, toluene, n-hexane, and n-octane) were studied as reaction media at constant water content (3% v/v). The greater the water binding affinity of the support the lower the synthetic activity of deposited enzymes: the activity of the celite derivative was 4x greater than the polyamide derivative. The enzyme glycation process hardly modified the catalytic ability of the celite derivative, but resulted in a moderate increase in operational stability. The presence of hydrophobic organic cosolvents in the water/ethanol reaction medium significantly increased enzyme activity, whereas the selectivity of the reaction remained high. Hexane was shown to be the best cosolvent, the synthetic activity of the celite derivative in hexane-ethanol (77 : 20%, v/v) being 130x greater than that in 97% (v/v) ethanol.