Recombinant production of serine hydroxymethyl transferase from Streptococcus thermophilus and its preliminary evaluation as a biocatalyst.

The glyA gene encoding a serine hydroxymethyl transferase (SHMT) with threonine aldolase activity was isolated from Streptococcus thermophilus YKA-184 chromosomal DNA. This aldolase is a pyridoxal 5'-phosphate-dependent enzyme that stereospecifically catalyzes the interconversion of L-threonine to glycine and acetaldehyde. The enzyme was overexpressed in Escherichia coli M15 as a recombinant protein of 45 kDa with a His6-tag at its N-terminus. The recombinant enzyme was purified to homogeneity by a single chromatographic step using Ni-nitrilotriacetic acid affinity, obtaining a high activity-recovery yield (83%). Lyophilized and precipitated enzymes were stable at least for 10 weeks when stored at -20 degrees C and 4 degrees C. It was observed that the Km for L-allo-threonine was 38-fold higher than that for L-threonine, suggesting this enzyme can be classified as a specific L-allo-threonine aldolase. The optimum pH range of threonine aldolase activity for the recombinant SHMT was pH 6-7. When tested for aldol addition reactions with non-natural aldehydes, such as benzyloxyacetaldehyde and (R)-N-Cbz-alaninal, two possible beta-hydroxy-alpha-amino acid diastereoisomers were produced, but with moderate stereospecificity. The enzyme showed potential as a biocatalyst for the stereoselective synthesis of beta-hydroxy-alpha-amino acids.

The effect of molecular shape on the thermotropic liquid crystal behavior of monolauroylated amino acid glyceride conjugates.

Monoacylglycerol amino acid conjugates constitute a novel class of specific biocompatible surfactants that can be considered analogues to partial glycerides and lysophospholipids. They consist of one aliphatic chain and one polar head, i.e., the amino acid, linked through a glycerol moiety. In a previous work, we synthesized monolauroylated amino acid glyceride conjugates, 1-O-lauroyl-rac-glycero-3-O-(N(alpha)-acetyl-L-amino acid), changing the amino acid headgroup systematically: arginine (compound 2), aspartic acid (compound 3), glutamic acid (compound 4), asparagine (compound 5), glutamine (compound 6), and tyrosine (compound 7), to elucidate the structure-properties relationship governing the occurrence of their polymorphism. The thermotropism of the new compounds was measured with polarizing light microscopy, differential scanning calorimetry, and X-ray diffraction and compared with the classical monoglyceride rac-1-lauroylglycerol (compound 1). The experiments were performed for a sequence of heating, cooling, and reheating scans. The results showed that compounds 1-6 exhibit a thermotropic smectic phase. As a consequence, the substitution of the polar head did not engender any curvature into the system, which might lead to the formation of cubic or columnar phases. Interestingly, liquid crystalline phases were not found in the case of compound 7. Small-angle X-ray diffraction data in the gel phase revealed that the substitution of the polar head by the different amino acid structures did not modify significantly the lamellar repeat distance relative to that of the reference one. The observed area per molecule, however, was larger for the new compounds. Consequently, interdigitation was promoted in compounds 2-7. The diffraction patterns were analyzed in terms of electron density profiles, using a modified Caillé theory plus a Gaussian electron density representation (MCG method) on X-ray diffraction data.

Assessment of primary eye and skin irritants by in vitro cytotoxicity and phototoxicity models: an in vitro approach of new arginine-based surfactant-induced irritation.

Extensive efforts have been made, recently, to find surfactants with lower irritation potential than those presently commercially available, for use in pharmaceutical and cosmetic preparations. Cytotoxic and phototoxic effects of a novel family of dicationic arginine-diglyceride surfactant compounds, 1,2-diacyl,3-O-(l-arginyl)-rac-glycerol with alkyl chain lengths in the range from 8 to 14 carbon atoms, were compared to three commercial surfactants. The end-points used to assess toxicity were the red blood cell lysis assay and uptake of the vital dye neutral red 24h after dosing (NRU), respectively. Two immortalized cell lines, murine fibroblast cell line, 3T3, and one human keratinocyte cell line, HaCaT, were used as in vitro models to predict the potential phototoxicity which could result in irritation, determined by resazurin reduction to resorufin and neutral red uptake (NRU). All tested surfactants had cytotoxicity effects as demonstrated by and decrease of NR uptake, which showed a clear concentration-response relationship. Concentrations resulting in 50% inhibition of NR uptake (IC(50)) range from 1 microM(-1) (hexadecyl trimethyl ammonium bromide) to 565 microM(-1) (12,12-l-arginine). Erythrocyte haemolysis also showed a clear concentration-response relationship, the 50% of haemolysis ranged from 37 microM(-1) (10,10-l-arginine) to 151 microM(-1) (sodium lauryl sulphate). Phototoxicity was performed with 12,12-l-acetyl-arginine, the most stable chemical structure. The validated 3T3 NRU photoxicity assay was used and revealed a phototoxic potential.

Purification of non-toxic, biodegradable arginine-based gemini surfactants, bis(Args), by ion exchange chromatography.

This paper presents an environmentally improved procedure for the preparative purification of a series of arginine-based gemini surfactants. The technique used was cation-exchange chromatography. Mixtures of boric-borate buffer, co-solvent (ethanol), and sodium chloride were tested as eluents. The influence of the buffer pH and the amount of co-solvent on the chromatographic process was studied for the model compound bis(Nalpha-lauroyl-L-arginine) 1,3-propanediamide dihydrochloride, C3(LA)2, and purification conditions were established. The method was scaled-up to the multigram level for C3(LA)2 and the rest of the series. The proposed preparative procedure involves simple equipment, low cost materials, and minimal amounts of solvent (water/ethanol), with low toxicity.

Chemo-enzymatic synthesis of arginine-based gemini surfactants.

A novel chemo-enzymatic synthesis of arginine-based gemini cationic surfactants bis(Args) is reported. These compounds consist of two single N(alpha)-acyl-arginine structures connected through the alfa-carboxylic groups of the arginine residues by a alpha, omega-diaminoalkane spacer chain. N(alpha)-Acyl-L-arginine alkyl ester derivatives were the starting building blocks for the synthesis. The best strategy found consisted of two steps. First, the quantitative acylation of one amino group of the spacer by the carboxylic ester of the N(alpha)-acyl-arginine took place spontaneously, at the melting point of the alpha,omega-diaminoalkane, in a solvent-free system. The second step was the papain-catalyzed reaction between another N(alpha)-acyl-arginine alkyl ester and the free aliphatic amino group of the derivative formed in the first step. Reactions were carried out in solid-to-solid and solution systems using low-toxic potential solvents. Changes in reaction performance and product yield were studied for the following variables: organic solvent, support for enzyme deposition and substrate concentration. The best yields (70%) were achieved in solid-to-solid systems and in ethanol at a(w) = 0.07. Bis(Args) analogs of 8, 10 and 12 carbon atoms using 1,3-diaminopropane and 1, 3-diamino-2-hydroxy-propane as hydrocarbon spacers were prepared at the 6-7 gram level employing the methodology developed. The overall yields which include reaction and purification varied from 51% to 65% of pure (97-98% by HPLC) product.

Qualitative and quantitative analysis of new alkyl amide arginine surfactants by high-performance liquid chromatography and capillary electrophoresis.

A protocol for the qualitative and quantitative analysis of novel arginine-based cationic surfactants using HPLC and CE was studied and compared. The optimization of the analytical conditions was carried out through a systematic variation of the experimental parameters such as mobile phase, eluent conditions, ion pairing and amount of sample for HPLC, and type of buffer, ion strength, type and amount of organic solvent, sample injection time, applied voltage and column washing and conditioning for CE.

Enzymatic synthesis of arginine-based cationic surfactants.

A novel enzymatic approach for the synthesis of arginine N-alkyl amide and ester derivatives is reported. Papain deposited onto solid support materials was used as catalyst for the amide and ester bond formation between Z-Arg-OMe and various long-chain alkyl amines and alcohols (H2N-Cn2, HO-Cn; n = 8-16) in organic media. Changes in enzymatic activity and product yield were studied for the following variables: organic solvent, aqueous buffer content, support for the enzyme deposition, presence of additives, enzyme loading, substrate concentration, and reaction temperature. The best yields (81-89%) of arginine N-alkyl amide derivatives were obtained at 25 degrees C in acetonitrile with an aqueous buffer content ranging from 0 to 1% (v/v) depending on the substrate concentration. The synthesis of arginine alkyl ester derivatives was carried out in solvent-free systems at 50 or 65 degrees C depending on the fatty alcohol chain length. In this case, product yields ranging from 86 to 89% were obtained with a molar ratio Z-Arg-OMe/fatty alcohol of 0.01. Papain deposited onto polyamide gave, in all cases, both the highest enzymatic activities and yields. Under the best reaction conditions the syntheses were scaled up to the production of 2 g of final product. The overall yields, which include reaction, Nalpha-benzyloxycarbonyl group (Z) deprotection and purification, varied from 53 to 77% of pure (99.9% by HPLC) product.

Reaction medium engineering in enzymatic peptide fragment condensation: synthesis of eledoisin and LH-RH.

The influence of different reaction systems on alpha-chymotrypsin-catalyzed synthesis of eledoisin and LH-RH peptides from (7 + 4) and (5 + 5) fragments was investigated. The peptide yield was determined in the following systems: buffered aqueous media, frozen solutions, organic media, and cosolvent mixtures. The experimental set up was tailored to allow the screening of an array of conditions with minimum consumption of peptide fragments (2.1 and 2.5 mM). The best yields (22% yield for eledoisin and 68% yield for LH-RH) were obtained in buffered aqueous solutions. It was found that the choice of buffer had a strong influence on the peptide yield; boric-borate and ammonium acetate buffers at pH 9, gave the best results. In buffered aqueous systems, both syntheses were scaled up by using a 10-fold increase in fragment concentration (21 and 25 mM). Under these conditions the yields rose to 57% and 80% of eledoisin and LH-RH, respectively. Moreover, during the synthesis of eledoisin and in the presence of boric-borate buffer pH 9, the peptide precipitated from the reaction medium preventing a secondary hydrolysis and facilitating the in situ product purification.

Neoglycopeptide synthesis and purification in multi-gram scale: preparation of O-(2,3,4,6-tetra-O-acetyl-beta-D-galactopyranosyl)-N alpha-fluoren-9-yl-methoxycarbonyl-hydroxyproline and its use in the pilot-scale synthesis of the potent analgesic glycopeptide O1.5-beta-D-galactopyranosyl [DMet2, Hyp5]enkephalinamide.

The preparation of a beta-galactosylated hydroxyproline derivative and its use in the multi-gram solid-phase synthesis of the potent analgesic neoglycopeptide O1.5-beta-D-galactopyranosyl [D-Met2, Hyp5]enkephalinamide is described in this paper. The most closely related impurities have been identified, isolated and characterized. Significant aspects of the synthesis and purification affecting yields and purity of both the building block and the target neoglycopeptide are discussed.

Enzymatic condensation of cholecystokinin CCK-8 (4-6) and CCK-8 (7-8) peptide fragments in organic media.

The kinetically controlled condensation reaction of Z-Gly-Trp-Met-OR(1) (R(1): Et, Al, Cam) and H-Asp-(OR(2))-Phe-NH(2) (R(2): H, Bu(t)) catalyzed by alpha-chymotrypsin deposited onto polyamide in organic media was studied. The effect of the drying process of the enzyme-support preparation, substrate concentrations, reaction medium, acyl donor, and nucleophile structure on both enzymatic activity and pentapeptide yield was investigated. The immobilized preparation directly equilibrated at a(w) = 0.113, gave higher enzymatic activities than dried with vacuum first, and then equilibrated at a(w) = 0.113. The addition of triethylamine to the reaction medium increased dramatically the enzymatic activity. However, the pentapeptide yield was affected neither by the drying procedure nor by the addition of triethylamine. The donor ester Z-Gly-Trp-Met-OAl gave initial reaction rates 2.6 times higher than the conventional ethyl ester derivative but rendered similar yields. The best results were obtained using Z-Gly-Trp-Met-OCam as acyl-donor ester; 80% yield and initial reaction rates 4 times higher than the ethyl ester derivative. In all cases, acetonitrile containing Tris-HCl 50 mM pH 9 buffer (0.5% v/v) and triethylamine (0.5% v/v) was found to be the best reaction system. Under these conditions, it was possible to use the nucleophile H-Asp-Phe-NH(2) with beta-unprotected aspartic acid residue. In this case, 50% yield was obtained, but economic considerations could lead to select it as nucleophile. Finally, the fragment condensation reaction was carried out at gram scale, obtaining a 39% yield which included the reaction, removal of protecting groups and purification steps. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 56: 456-463, 1997.

Enzymatic synthesis of a CCK-8 tripeptide fragment in organic media.

The enzymatic synthesis of the tripeptide derivative Z-Gly-Trp-Met-OEt is reported. This tripeptide is a fragment of the cholecystokinin C-terminal octapeptide CCK-8. Studies on the alpha-chymotrypsin catalyzed coupling reaction between Z-Gly-Trp-R(1) and Met-R(2) have focused on low water content media, using deposited enzyme on inert supports such as Celite and polyamide. The effect of additives (polar organic solvents), the acyl-donor ester structure, the C-alpha protecting group of the nucleophile, enzyme loading, and substrate concentration were tested. The best reaction medium found was acetonitrile containing buffer (0.5%, v/v) and triethylamine (0.5%, v/v) using the enzyme deposited on Celite as catalyst (8 mg of alpha-chymotrypsin/g of Celite). A reaction yield of 81% was obtained with Z-Gly-Trp-OCam as acyl donor, at an initial concentration of 80 mM. The tripeptide synthesis was scaled up to the production of 2 g of pure tripeptide with an overall yield of 71%, including reaction and purification steps.

Enzymatic peptide synthesis in low water content systems: preparative enzymatic synthesis of [Leu]- and [Met]-enkephalin derivatives.

A novel total enzymatic synthesis of [Leu]- and [Met]-enkephalin derivatives was accomplished in low-water content systems at a preparative scale. alpha-Chymotrypsin, papain, thermolysin and bromelain adsorbed on Celite were used as catalysts. Organic solvents such as acetonitrile and ethyl acetate with small amounts of buffer added or at specific water activity were used as reaction media. Simple readily available amino acid ester derivatives were used as starting building blocks. This feature allowed the possibility of using the products in one step directly as acyl-donor ester, without any chemical or enzymatic modification, in the next enzymatic coupling. The optimal strategy for the synthesis of the enkephalin derivatives was different depending on the carboxy terminal group. The preparation of the carboxy-terminal amide derivatives (R-Tyr-Gly-Gly-Phe-Leu[Met]-NH2) was achieved via 4 + 1 fragment condensation catalyzed by alpha-chymotrypsin. The carboxy-terminal ethyl ester derivatives (R-Tyr-Gly-Gly-Phe-Leu[Met]-OEt) were obtained via 2 + 3 condensation catalyzed by bromelain, a quite unusual protease for peptide synthesis but more effective than papain in this coupling. Both syntheses were carried out in four enzymatic steps and one or two chemical deprotection steps routinely used in peptide synthesis. The overall yields of pentapeptide derivatives were between 40-54% of pure product.

Enzymatic synthesis of X-Phe-Leu-NH2 in low water content systems: influence of the N-alpha protecting group and the reaction medium composition.

The influence of eight different N-terminal protecting groups (For, Ac, Boc, Fmoc, Mal, Pheac, Aloc and Z) on the alpha-chymotrypsin-catalyzed synthesis of the dipeptide derivative X-Phe-Leu-NH2 in organic media was studied. Groups such as Ac, For, Boc, Z, Mal, Pheac and Alloc always rendered good peptide yields (92% to 99%) either in acetonitrile or in ethyl acetate. Good correlations were found between molecular and physico-chemical characteristics of the N-alpha moiety such as the hydrophobicity (log P), ovality and dipole moment and the global reaction rate parameter k'. High k' values were obtained with the less hydrophobic groups, Ac, For and Mal, that have ovality values close to one and the highest dipole moments. Furthermore, it was found that the relative rate of hydrolysis and aminolysis of the acyl-enzyme intermediate expressed as the partition parameter p is affected by the N-alpha moiety of the acyl donor. Correlations between this parameter and the dipole moment of the protecting group were observed.

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).

Racemization free coupling of peptide segments. Synthesis of an insect neuropeptide.

The total synthesis of the insect neuropeptide derivative Z-Gly-Gly-Ser-Leu-Tyr-Ser-Phe-Gly-Leu-NH2 has been carried out by a convergent solid phase strategy. For the coupling of the N-terminal pentapeptide to the C-terminal tetrapeptide, three different methods were assayed. Racemization of the acyl activated amino acid during the fragment condensation reaction was monitored by HPLC. Best results were obtained by enzymatic coupling in a low water containing media using adsorbed alpha-chymotrypsin. An optically pure product was obtained in 82% yield after 1 h of reaction. Chemical methods such as DIC/HOBt and BOP/HOBt/NMM always rendered highly optically impure products containing 10-20% of the D-epimer.

Application of empirical design methodologies to the study of the influence of reaction conditions and N-alpha-protecting group structure on the enzymatic X-Phe-Leu-NH(2) dipeptide synthesis in buffer/dimethylformamide solvents systems.

The influence of five different N-terminal protecting groups (For, Ac, Boc, Z, and Fmoc) and reaction conditions (temperature and dimethylformamide content) on the alpha-chymotrypsin-catalyzed synthesis of the dipeptide derivative X-Phe-Leu-NH(2) was studied. Groups such as For, Ac, Boc, and Z always rendered good peptide yields (82% to 85%) at low reaction temperatures and DMF concentrations, which depended on the N-alpha protection choice. Boc and Z were the most reactive N-alpha groups and, in addition, the most suitable for peptide synthesis. On the other hand, the use of empirical design methodologies allowed, with minimal experimentation and by multiple regression, to deduce an equation, which correlates the logarithm of the first order kinetic constant (log k') with reaction temperature, DMF concentration, and hydrophobicity (log P values) of the different protecting groups. The predictive value of the equation was tested by comparing the performance of another protective group, such as Aloc, with the performance predicted by said equation. Experimental and calculated k' values were found to be in good agreement.

Substrate specificity of alpha-chymotrypsin-catalyzed esterification in organic media.

11 amino acid derivatives were tested as alpha-chymotrypsin substrates in the esterification reaction with methanol in organic media. The reactions were carried out in water-saturated ethyl acetate and in acetonitrile containing 4% water. alpha-Chymotrypsin adsorbed on Celite was used as a catalyst. From initial reaction rate measurements, the Michaelis-Menten parameters Vmax and KM were determined. All the amino acid derivatives tested were esterified, and the highest values of kcat/KM were obtained with the N-acylated aromatic amino acids. Correlations between Michaelis-Menten parameters and physical properties of the substrates such as molar refractivity (MR) and log P were deduced. The results show that the specificity of the alpha-chymotrypsin towards the side chain of the amino acids in organic media is the same as that in aqueous media. However, the specificity towards the N-protecting group is opposite to that in water, so the reaction medium affects the interaction of this part of the molecule with the enzyme to a large extent.

Catalytic properties of alpha-chymotrypsin in organic media.

alpha-Chymotrypsin was deposited on Celite and the resulting immobilized preparations were used to carry out peptide synthesis reactions in organic media with only small amounts of water present. The influence of different parts of the donor ester and acceptor nucleophile substrate molecules on the kinetics of the enzymatic reactions was studied. The specificity of alpha-chymotrypsin in organic media was a combination of its substrate specificity in aqueous media and solvent effects. The kinetics of peptide synthesis can thus be modulated by using suitable solvents and protecting groups.

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).