Penicillin acylase-catalyzed synthesis of cefazolin in water–solvent mixtures: enhancement effect of ethyl acetate and carbon tetrachloride on the synthetic yield

The effects of organic solvents on the penicillin acylase-catalyzed, kinetically controlled synthesis of cefazolin have been examined in various water–solvent mixtures. In the presence of water-miscible solvents, the initial rate and maximum yield of cefazolin (CEZ) synthesis reaction were found to be reduced. The extent of inhibition was increased with increasing hydrophobicity of the solvent in the reaction mixtures. Enzymatic synthesis of cefazolin was also carried out in the water–solvent biphasic systems. Among the water-immiscible solvents tested, ethyl acetate (EtOAc) and carbon tetrachloride (CCl₄) were found to markedly improve the yield of cefazolin in the two-phase reaction system. Our study showed that the enhancement effect of EtOAc and CCl₄ on the synthetic yield was mainly caused by a reduction of the hydrolysis of acyl donor and product in the two-phase system rather than extraction of the product into the solvent phase.     

Kinetics of enzymatic synthesis of peptides in aqueous/organic biphasic systems. Thermolysin-catalyzed synthesis of N-(benzyloxycarbonyl)-L-phenylalanyl-L-phenylalanine methyl ester

We studied kinetics of thermolysin-catalyzed peptide synthesis in an aqueous/organic biphasic system theoretically and experimentally. As a model reaction producing a condensation product having no dissociating groups, we used the synthesis of N-(benzyloxycarbonyl)-L-phenylalanyl-L-phenylalanine methyl ester (Z-Phe2OMe) from N-(benzyloxycarbonyl)-L-phenylalanine (Z-Phe) and L-phenylalanine methyl ester (PheOMe). Usually, ethyl acetate was used as the organic solvent. First we studied the kinetics of the synthesis of Z-Phe2OMe in a buffer solution saturated with ethyl acetate. Then, factors that may affect the kinetics in the biphasic system were examined. The course of Z-Phe2OMe synthesis in the biphasic system was explained by the rate equations obtained, using the partitions of substrate and product and non-enzymatic decomposition of PheOMe. In the biphasic reaction system, the rate of synthesis was lower for a wide range of pH due to the unfavorable partition of PheOMe in the aqueous phase, but yields were higher than in the buffer solution. The effects of the organic solvents on the rate of synthesis could also be explained by variations in the partition coefficient of PheOMe. Finally, we gave a way to predict the aqueous-phase pH change caused by partitioning of the substrate. The significance of the pH change was shown in connection with the reaction using the immobilized enzyme in an organic solvent.     

Transaminase-catalyzed asymmetric synthesis of <Emphasis Type="SmallCaps">l</Emphasis>-2-aminobutyric acid from achiral reactants

Asymmetric synthesis of an unnatural amino acid was demonstrated by ω-transaminase from Vibrio fluvialis JS17. l-2-Aminobutyric acid was synthesized from 2-oxobutyric acid and benzylamine with an enantiomeric excess higher than 99%. The reaction showed severe product inhibition by benzaldehyde, which was overcome by employing a biphasic reaction system to remove the inhibitory product from the aqueous phase. In a typical biphasic reaction (50 mM 2-oxobutyric acid, 70 mM benzylamine and 2.64 U/ml purified enzyme) using hexane as an extractant, conversion of 2-oxobutyric acid reached 96% in 5 h whereas only 39% conversion was obtained without the product extraction.     

Enzymatic peptide synthesis in organic media: a comparative study of water-miscible and water-immiscible solvent systems.

Peptide synthesis was carried out in a variety of organic solvents with low contents of water. The enzyme was deposited on the support material, celite, from an aqueous buffer solution. After evaporation of the water the biocatalyst was suspended in the reaction mixtures. The chymotrypsin-catalyzed reaction between Z-Phe-OMe and Leu-NH2 was used as a model reaction. Under the conditions used ([Z-Phe-OMe]0 less than or equal to 40 mM, [Leu-NH2]0/([Z-Phe-OMe]0 = 1.5) the reaction was first order with respect to Z-Phe-OMe. Tris buffer, pH 7.8, was the best buffer to use in the preparation of the biocatalyst. In water-miscible solvents the reaction rate increased with increasing water content, but the final yield of peptide decreased due to the competing hydrolysis of Z-Phe-OMe. Among the water-miscible solvents, acetonitrile was the most suitable, giving 91% yield with 4% (by vol.) water. In water-immiscible solvents the reaction rate and the product distribution were little affected by water additions in the range between 0% and 2% (vol. %) in excess of water saturation. The reaction rates correlated well with the log P values of the solvent. The highest yield (93%) was obtained in ethyl acetate; in this solvent the reaction was also fast. Under most reaction conditions used the reaction product was stable; secondary hydrolysis of the peptide formed was normally negligible. The method presented is a combination of kinetically controlled peptide synthesis (giving high reaction rates) and thermodynamically controlled peptide synthesis (giving stable reaction products).     

Kinetics and equilibrium of enzymatic synthesis of peptides in aqueous/organic biphasic systems. Thermolysin-catalyzed synthesis of N-(benzyloxycarbonyl)-L-aspartyl-L-phenylalanine methyl ester

We studied kinetics and the equilibrium relationship for the thermolysin-catalyzed synthesis of N-(benzyloxycarbonyl)-L-aspartyl-L-phenylalanine methyl ester (Z-Asp-PheOMe) from N-(benzyloxycarbonyl)-L-aspartic acid (Z-Asp) and L-phenylalanine methyl ester (PheOMe) in an aqueous-organic biphasic system. This is a model reaction giving a condensation product with dissociating groups. The kinetics for the synthesis of Z-Asp-PheOMe in aqueous solution saturated with ethyl acetate was expressed by a rate equation for the rapid-equilibrium random bireactant mechanism, and the reverse hydrolysis reaction was zero-order with respect to Z-Asp-PheOMe concentration. The courses of synthesis of Z-Asp-PheOMe in the biphasic system were well explained, by the rate equations obtained for the aqueous solution and by the partition of substrate and condensation product between the both phases. The rate of synthesis in the biphasic system was much lower than in aqueous solution due to the unfavorable partition of PheOMe in the aqueous phase. The equation for the equilibrium yield of Z-Asp-PheOMe in the biphasic system was derived assuming that only the non-ionized forms of the substrate and condensation product exist in the organic phase. It was found theoretically and experimentally that the yield of Z-Asp-PheOMe is maximum at the aqueous-phase pH of around 5, lower than for synthesis in aqueous solution. The effect of the organic solvent on the rate and equilibrium for the synthesis of Z-Asp-PheOMe could be explained by the variation in the partition coefficient. The effect of the partitioning of substrate on the aqueous-phase pH change was also shown.     

Enzymatic production of 2-amino-2,3-dimethylbutyramide by cyanide-resistant nitrile hydratase

A novel enzymatic route for the synthesis of 2-amino-2,3-dimethylbutyramide (ADBA), important intermediate of highly potent and broad-spectrum imidazolinone herbicides, from 2-amino-2,3-dimethylbutyronitrile (ADBN) was developed. Strain Rhodococcus boritolerans CCTCC M 208108 harboring nitrile hydratase (NHase) towards ADBN was screened through a sophisticated colorimetric screening method and was found to be resistant to cyanide (5 mM). Resting cells of R. boritolerans CCTCC M 208108 also proved to be tolerant against high product concentration (40 g l⁻¹) and alkaline pH (pH 9.3). A preparative scale process for continuous production of ADBA in both aqueous and biphasic systems was developed and some key parameters of the biocatalytic process were optimized. Inhibition of NHase by cyanide dissociated from ADBN was successfully overcome by temperature control (at 10°C). The product concentration, yield and catalyst productivity were further improved to 50 g l⁻¹, 91% and 6.3 g product/g catalyst using a 30/70 (v/v) n-hexane/water biphasic system. Furthermore, cells of R. boritolerans CCTCC M 208108 could be reused for at lease twice by stopping the continuous reaction before cyanide concentration rose to 2 mM, with the catalyst productivity increasing to 12.3 g product/g catalyst. These results demonstrated that enzymatic synthesis of ADBA using whole cells of R. boritolerans CCTCC M 208108 showed potential for industrial application.     

Enzymatic synthesis of the precursor of Leu-enkephalin in water-immiscible organic solvent systems.

The precursor of Leu-enkephalin, Z-L-TyrGlyGly-L-Phe-L-LeuOEt, was synthesized from amino acid derivatives with three proteinases without the protection of the side chain of L-Tyr. First, Z-GlyGlyOBut and Z-L-TyrGlyGlyOBut were synthesized in quite a high yield, 83% and 99%, in an aqueous/organic biphasic system by papain and alpha-chymotrypsin, respectively. Then, Z-L-Phe-L-LeuOEt was synthesized by thermolysin from Z-L-Phe and L-LeuOEt either in buffer or in a biphasic system; the yields were 95% and 100%, respectively. The synthesis of Z-L-TyrGlyGly-L-Phe-L-LeuOEt from Z-L-TyrGlyGly and L-Phe-L-LeuOEt was performed effectively by thermolysin immobilized on Amberlite XAD-7 in a buffer and in an aqueous/organic biphasic system, as well as in saturated ethyl acetate, while the yield was low in reactions by free thermolysin. In the reaction with the immobilized enzyme (IME) in saturated ethyl acetate, the maximum yield of the precursor of Leu-enkephalin was 68%. The reasons for effective synthesis with IME are: (1) higher concentration of L-Phe-L-LeuOEt inside support, which resulted in rising the rate of the synthesis reaction and protecting the competitive hydrolysis of Z-L-TyrGlyGly by thermolysin, (2) entrapment of the product inside the support where thermolysin could not act in the case of reaction in buffer, and (3) extraction of the product with the organic solvent in the case of reaction in a biphasic system or in saturated organic solvent.     

Concentration dependent effect of GsMTx4 on mechanosensitive channels of small conductance in <Emphasis Type="Italic">E. coli</Emphasis> spheroplasts

The spider peptide GsMTx4, at saturating concentration of 5 μM, is an effective and specific inhibitor for stretch-activated mechanosensitive (MS) channels found in a variety of eukaryotic cells. Although the structure of the peptide has been solved, the mode of action remains to be determined. Because of its amphipathic structure, the peptide is proposed to interact with lipids at the boundaries of the MS channel proteins. In addition, GsMTx4 has antimicrobial effects, inhibiting growth of several species of bacteria in the range of 5–64 μM. Previous studies on prokaryotic MS channels, which serve as model systems to explore the principle of MS channel gating, have shown that various amphipathic compounds acting at the protein–lipid interface affect MS channel gating. We have therefore analyzed the effect of different concentrations of extracellular GsMTx4 on MS channels of small conductance, MscS and MscK, in the cytoplasmic membrane of wild-type E. coli spheroplasts using the patch-clamp technique. Our study shows that the peptide GsMTx4 exhibits a biphasic response in which peptide concentration determines inhibition or potentiation of activity in prokaryotic MS channels. At low peptide concentrations of 2 and 4 μM the gating of the prokaryotic MS channels was hampered, manifested by a decrease in pressure sensitivity. In contrast, application of peptide at concentrations of 12 and 20 μM facilitated prokaryotic MS channel opening by increasing the pressure sensitivity.     

How to predict product yield in kinetically controlled enzymatic peptide synthesis

The published theory of kinetically controlled enzymatic peptide synthesis needed experimental verification. We carried out the measurement of the peptide yield and estimation of the key parameters alpha and beta for papain-catalyzed synthesis of Mal-L-Phe-L-Ala-LLeuNH(2) from Mal-L-Phe-L-AlaOMe and L-LeuNH(2). The experimental results demonstrate that this theory adequately describes the real process. (c) 1992 John Wiley & Sons, Inc.     

Penicillin G acylase catalyzed acylation of 7-ACA in aqueous two-phase systems using kinetically and thermodynamically controlled strategies: improved enzymatic synthesis of 7-[(1-hydroxy-1-phenyl)-acetamido]-3-acetoxymethyl-Δ3-cephem-4-carboxylic acid

Penicillin G acylase (PGA) catalyzed acylation of 7-aminocephalosporanic acid (7-ACA) with R-mandelic acid and its derivatives gives 7-[(1-hydroxy-1-phenyl)-acetamido]-3-acetoxymethyl-Δ³-cephem-4-carboxylic acid. This compound is a useful intermediate for the synthesis of some 3′-functionalized cephalosporins. However, acylations catalyzed by PGA isolated from Escherichia coli give poor results both considering a kinetical or a thermodynamical approach. In order to improve this enzymatic acylation, polyethylene glycol (PEG 600)-ammonium sulphate aqueous two-phase systems have been studied with the aim to have, during the reaction, a continuous extraction of the acylation product outside of the enzyme environment (the ammonium sulphate phase). This strategy shifts the equilibrium in the thermodynamically controlled synthesis and prevents the hydrolysis of the synthesized antibiotic in the kinetically controlled synthesis. The best results were achieved using PEG 600 (80% in water) equilibrated with 4 M ammonium sulphate. In these conditions, the acylation product was completely partitioned in the PEG phase (K > 200), whereas the substrates maintained a suitable concentration in the enzyme environment. Both in the kinetic (88% yield) and the thermodynamic (75% yield) processes, the results obtained were sensitively improved in comparison with those achieved working in homogeneous solution (phosphate buffer). Using R-mandelic acid methyl ester, the yield increased from 65% (monophasic system) to 88%. The PEG solution, without isolation of the acylation product, was successfully used for the synthesis of Cefamandole and Cefonicid.     

Adaptation of a <Emphasis Type="Italic">Mycobacterium</Emphasis> strain to phenanthrene degradation in a biphasic culture system: influence on interfacial area and droplet size

A phenanthrene-degrading Mycobacterium sp. strain 6PY1 was grown in an aqueous/organic biphasic culture system with phenanthrene as sole carbon source. Its capacity of degradation was studied during sequential inoculum enrichments, reaching complete phenanthrene degradation at a maximim rate of 7 mg l⁻¹ h⁻¹. Water–oil emulsions and biofilm formation were observed in biphasic cultures after four successive enrichments. The factors influencing interfacial area in the emulsions were: the initial phenanthrene concentration, the initial inoculum size, and the silicone oil volume fraction. The results showed that the interfacial area was mainly dependent on the silicone oil/mineral salts medium ratio and the inoculum size.     

Methylosinus trichosporium OB3b whole-cell methane monooxygenase activity in a biphasic matrix

 Reconstituted whole-cell preparations of lyophilized Methylosinus trichosporium OB3b were used to demonstrate soluble methane monooxygenase activity in a two-phase (biphasic) matrix consisting of a buffered aqueous phase and 2,2,4-trimethylpentane (isooctane). The rate of conversion of gaseous propylene to propylene oxide, a non-metabolized liquid, was used as the primary measure of enzyme activity. Appreciable soluble methane monooxygenase activity was detected when the volume of the aqueous phase represented at least 1% of the total volume, although the initial rate of product formation did increase as the volume of the aqueous phase increased. In comparison to the aqueous system, the specific rate and yields in the biphasic system were much less sensitive to increases in the concentrations of formate and protein (the methane monooxygenase). However, there was some evidence that the enzyme system was more stable in the biphasic matrix, since the rate of propylene oxide formation remained linear for an extended period of time. V _(app.) in the biphasic system decreased by a factor of 0.6 relative to the same parameter in the aqueous system. Conversely, K _m(app.) for propylene was 1.6 times greater in the biphasic system. Hence, the apparent catalytic efficiency in the aqueous system was four times that in the biphasic system, as indicated by a decrease in the corresponding ratios of V _(app.) to K _m(app.). Received: 21 July 1995/Received last revision: 1 February 1996/Accepted: 5 December 1996     

Biphasic Modulation by ACTH-Like Peptides of Protein Synthesis in a Cell-Free System from Rat Brain

Brain protein synthesis in a cell-free system was stimulated by 10(-8) M-ACTH1-24. This stimulatory effect was completely inhibited by aurintricarboxylic acid (ATA), an inhibitor of reinitiation of new peptide chains. The N-terminal peptide sequence 4-10 exerted a biphasic modulation of cell-free protein synthesis, i.e., a stimulation at low concentrations (10(-8) and 10(-10) M) and an inhibition at a high concentration (10(-4) M). The D-isomer, ACTH4-10-7-D-phe, also showed a biphasic modulation that, however, was in a direction opposite to that shown by ACTH4-10-7-L-phe at 10(-8) M and 10(-4) M.     

Chemically stabilized trypsin used in dipeptide synthesis

Bovine pancreatic trypsin was treated with ethylene glycol bis(succinic acid N-hydroxysuccinimide ester). Approximately 8 of 14 lysines per trypsin molecule were modified. This derivative (EG trypsin) was more stable than native between 30 degrees and 70 degrees C: T50 values were 59 degrees C and 46 degrees C, respective. EG trypsin's half-life of 25 min at 55 degrees C was fivefold greater than native's. EG trypsin had a decreased rate of autolysis and retained more activity in aqueous mixtures of 1,4-dioxan, dimethylformamide, dimethylsulfoxide, and acetonitrile. EG trypsin had lower Km values for both amide and ester substrates; its kcat values for two amides (benzoyl-L-arginine p-nitroanilide and benzyloxycarbonyl glycyl-glycyl-arginyl-7-amino-4-methyl coumarin) increased, whereas its kcat value for an ester (thiobenzoyl benzoyloxycarbonyl-L-lysinate) decreased slightly. The specific activity (kcat/Km) of EG trypsin was increased for both amide and ester substrates. EG trypsin gave higher yields and reaction rates than native in kinetically controlled synthesis of benzoyl argininyl-leucinamide in acetonitrile and in t-butanol. Highest peptide yields occurred with EG trypsin in 95% acetonitrile, where 90% of the substrate was converted to product. No peptide synthesis occurred in 95% DMF with either form of trypsin.     

Convolution analysis of transcription by yeast DNA-dependent ribonucleic acid polymerase A. A mathematical method for studying ribonucleic acid chain elongation.

The rate of initiation of RNA synthesis catalysed by yeast RNA polymerase A on native calf thymus DNA decayed exponentially with a half-life of about 4.3 min. The rate constant for initiation was unaffected by preincubating the enzyme with DNA, or by decreasing the concentration of GTP 4-fold. The rate of RNA synthesis was constant for 15--20 min and then decreased. Each enzyme molecule made no more than one RNA molecule. In this situation, initiation, elongation and total RNA synthesis are related by a convolution integral. Solution of the convolution integral revealed that the rate of elongation was apparently biphasic. Analysis of the size of the RNA product showed that this biphasic profile arose because most but not all of the enzyme stopped RNA synthesis soon after initiation.     

Enzymatic synthesis of alkyl beta-D-xylosides by transxylosylation and reverse hydrolysis

The Trichoderma reesei beta-xylosidase (EC 3.2.1.37) is used to catalyze the production of alkyl beta-D-xyloside. Two general methods of production are tested and compared using the same enzyme: transglycosylation and reverse hydrolysis. Using both methods, primary, secondary, and tertiary alcohols are studied as acceptors. In kinetically controlled process (transglycosylation), the chosen donor is methyl beta-D-xyloside and primary, secondary, and tertiary alkyl alcohols are accepted. In the equilibrium-controlled synthesis, the donor is xylose whereas acceptors are only primary and secondary alcohols. The influence of the donor concentration is investigated in both processes. The yields of the kinetically controlled reactions are higher compared with those of the equilibrium-controlled synthesis. The specificity of the beta linkage is confirmed by proton nuclear magnetic resonance ((1)H NMR) analysis. (c) 1994 John Wiley & Sons, Inc.     

Enzyme peptide synthesis and semisynthesis: Kinetic and thermodynamic aspects

The hydrolysis/synthesis equilibrium of the peptide bond is governed by the relative magnitudes of the corresponding Gibbs' energies of hydrolysis to non-ionized products and of their ionization. The positive energy change in peptide hydrolysis to non-ionized products is the thermodynamic basis for the acyl and leaving group specificity of proteinases. With a proteinase of suitable specificity, some peptide bonds can be synthesized by a thermodynamically controlled enzyme aminolysis of specific acylamino or peptide acids; any peptide bond can be formed by a kinetically controlled enzyme aminolysis of the corresponding acylamino or peptide esters.     

Optimization of butanediol dimethacrylate crosslinked polystyrene: A novel polymeric support for solid phase peptide synthesis

Summary Studies leading to optimization of butanediol dimethacrylate-crosslinked polystyrene supports (BDDMA-PS) for solid phase peptide synthesis are delineated. BDDMA-PS copolymers with different crosslink densities were prepared and functionalised with chloromethyl groups. The reactivity of the Lys(2-Cl−Z)−OH residue bound to these polymers through a benzyl ester linkage was investigated by following the kinetics of acylation by the HOBt active ester of Boc-Alanine. From the results it was observed that the rate of peptide bond formation was maximum for a 2% BDDMA crosslinked resin. This resin was compared with a 2% DVB-crosslinked polystyrene resin (DVB-PS). Synthesis of an extremely insoluble, hydrophobic, antiparallel β-sheeted difficult sequence peptide LMVGGVVIA (β 34–42), C-terminal fragment of β-amyloid protein, β (1–42), was carried out on both 2% DVB-PS and 2% BDDMA-crosslinked polystyrene supports. The synthesis of the peptide was carried out using Boc amino acid strategy. Greater extent of swelling of the resino peptide, increased coupling efficiency during the assembly of amino acids and relatively high purity of synthesised peptide were observed in the case of 2% BDDMA-PS polymer.     

Peptide synthesis with a proteinase from the extremely thermophilic organism Thermus Rt41A

A proteinase isolated from Thermus RT41a was immobilized to controlled pore glass beads and was used in the free and immobilized forms for peptide synthesis. The observed maximum yield was the same in both cases. a number of dipeptides were produced from amino acid esters and amides. The best acyl components, from those tested, were found to be Ac-Phe-OEt and Bz-Ala-OMe. Tur-NH(2), Trp-NH(2), Leu-pNA, and Val-pNA were all reactive nucleophiles.The kinetically controlled synthesis of Bz-ala-Tyr-NH(2) was optimized by studying the effect of pH, temperature, solvent concentration, ionic strength, and nucleophile and acyl donor concentration, ionic strength, and nucleophile and acyl donor concentration on the maximum yield. The initial conditions used were 25 mM Bz-ala-OMe, 25 mM Tyr-NH(2), 70 degrees C, pH 8.0, and 10% v/v dimethylformamide (DMF). The optimum conditions were 90% v/v DMF using 80 mM bz-Ala-OMe and 615 mM Tyr-NH(2) at 40 degrees C and pH 10. These conditions increased the maximum conversion from 0.75% to 26% (of the original ester concentration). In a number of other cosolvents, the best peptide yields were observed with acetonitrile and ethyl acetate. In 90% acetonitrile similar yields were observed to those in 90% DMF under optimized conditions except that the acyl donor and nucleophile concentrations could be reduced to 25 mM and 100mM, respectively. The effect of the blocking group on the nucleophile was also investigated; -betaNA and -pNA as blocking groups improved the yields markedly. The blocking and leaving groups of the acyldonor had no effect on the dipeptide yield. (c) 1994 John Wiley & Sons, Inc.     

Kinetic Hysteresis in Collagen Folding

The triple helix of collagen shows a steep unfolding transition upon heating, whereas less steep and more gradual refolding is observed upon cooling. The shape of the hysteresis loop depends on the rate of temperature change as well as the peptide concentration. Experimental heating and cooling rates are usually much faster than rates of unfolding and refolding. In this work, collagen model peptides were used to study hysteresis quantitatively. Their unfolding and refolding profiles were recorded at different heating and cooling rates, and at different peptide concentrations. Data were fitted assuming kinetic mechanisms in which three chains combine to a helix with or without an intermediate that acts as a nucleus. A quantitative fit was achieved with the same kinetic model for the forward and backward reactions. Transitions of exogenously trimerized collagen models were also analyzed with a simplified kinetic mechanism. It follows that true equilibrium transitions can only be measured at high concentrations of polypeptide chains with slow scanning rates, for example, 0.1°C/h at 0.25 mM peptide concentration of (Gly-Pro-Pro)₁₀. (Gly-Pro-4(R)Hyp)₁₀ folds ∼2000 times faster than (Gly-Pro-Pro)₁₀. This was explained by a more stable nucleus, whereas the rate of propagation was almost equal. The analysis presented here can be used to derive kinetic and thermodynamic data for collagenous and other systems with kinetically controlled hysteresis.