The influence of water on protease-catalyzed peptide synthesis in acetonitrile/water mixtures

Protease-catalyzed peptide synthesis in acetonitrile/water mixtures, containing 0-90% water, was investigated. alpha-Chymotrypsin, as well as thermolysin, were deposited on solid supports, prior to exposure to the reaction media. Peptide syntheses were performed using both a kinetically controlled process (chymotrypsin) and an equilibrium-controlled synthesis (thermolysin). The activity of chymotrypsin decreased at low water contents. However, at low water contents (1-10%) hydrolytic side reactions were suppressed and high yields of dipeptides were obtained. Optimal water content for the thermolysin-catalyzed reaction was 4-8%. The dipeptides produced were fully soluble in the reaction mixtures. High operational stability for alpha-chymotrypsin was obtained during 216 h of reaction.     

Native and modified subtilisin 72 as a catalyst of peptide synthesis in media with low water content

The catalytic efficiencies of native subtilisin, its noncovalent complex with polyacrylic acid, and the subtilisin covalently immobilized in a cryogel of polyvinyl alcohol were studied in the reaction of peptide coupling in mixtures of organic solvents with a low water content in dependence on the medium composition, reaction time, and biocatalyst concentration. It was established that, in media with a DMF content > 80%, the synthase activity of modified subtilisins is higher than that of the native subtilisin. The use of N-acylpeptides with a free carboxyl group was found to be possible in organic solvents during the enzymatic synthesis catalyzed by both native and immobilized subtilisin. A series of tetrapeptide p-nitroanilides of the general formula Z-Ala-Ala-Xaa-Yaa-pNA (where Xaa is Leu, or Glu and Yaa is Phe or Asp) was obtained in the presence of immobilized enzyme in yields of 70-98% in DMF-MeCN without any activation of the carboxyl component and without protection of side ionogenic groups of polyfunctional amino acids.     

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.     

Enzymatic synthesis of peptide in acetonitrile/supercritical carbon dioxide

Summary The peptide synthesis from N-acetyl-L-tyrosine ethyl ester (Ac-Tyr-OEt) and amino acid amides was realized using -chymotrypsin (CT) in acetonitrile (MeCN) or acetonitrile/supercritical carbon dioxide (SCCO₂) containing small amounts of water. In both solvent systems there was an optimum water content for peptide synthesis, above which peptide hydrolysis became more important. After an incubation for 5 hours, the yields of the peptide was 64% in MeCN and 91% in MeCN/SCCO₂, respectively.     

Revisiting the effect of water content on enzymatic peptide synthesis in non-aqueous medium

Enzymatic acyl-transfer reaction in organic medium competes with the hydrolytic side reaction depending on the water content. The effect of water content on aminolysis activity of -chymotrypsin for the synthesis of Bz-Tyr-Val-NH₂ in acetonitrile was examined under the conditions which were devoid of the hydrolytic deacylation. Excess H-Val-NH₂ (880 mM) was employed to keep the hydrolysis negligible. The aminolysis rate increased abruptly between 4 and 5% (v/v) water but a further increase in the water content did not affect the reaction rate. This suggests that water added more than 5% (v/v) does not enhance intrinsic enzyme activity but acts only as a nucleophile for the hydrolytic deacylation.     

Mirin-making using long-life koji with low water content

In an attempt to store koji longer than is now possible at room temperature, we investigated ways in which to dry it. Dried koji with a water activity (Aw) of 0.77 containing 0.11 g of water/g of solid was prepared from fresh koji with an Aw of 0.88 containing 0.39 g of water/g of solid. Drying was found to be more efficient above 50°C. The most suitable conditions were at 55°C under 760 mm Hg of pressure for 5–6 h (Method I) when fresh koji was prepared with crushed rice. This dried koji had 84% or more of the original enzyme activity. Other methods, such as drying at 55°C under 20 mm Hg of pressure for 6 h (Method II), and drying by mixing fresh koji with dried steamed rice powder at 23°C for 24 h (Method III) were also examined. For retaining enzyme activity after drying, Method III was theoretically the best. In practice, however, Method I was found to be efficient and inexpensive. For dried koji made using Method I, the α-amylase and neutral protease activities were 80% and over 90%, respectively, of the baseline values after 6 months of storage at 30°C. The number of bacteria in the dried koji was small, but the number of thermotolerant bacteria was almost unchanged. For mirin manufactured using dried koji which was stored for 4 months, the yield, properties and sensory qualities of the mirin were essentially the same as those obtained using fresh koji.     

Engineering biocatalytic systems in organic media with low water content

The use of organic media in biocatalysis stems from the fact that in many cases biocatalytic processes can hardly be conducted (if at all) in aqueous solutions because of extremely low solubilities of substrates and/or unfavorable shift of the reaction equilibrium in water. The growing interest in this biotechnological area that has sprung up over the past few years has resulted in various approaches to enzyme stabilization against organic solvents. Thus, the main goal of the present review is to formulate a comprehensive classification of numerous successful nonaqueous biocatalytic systems based on a few fundamental principles. Typical examples are considered, along with the advantages and drawbacks inherent in each of the approaches discussed.     

Modeling the performance of immobilized α-chymotrypsin catalyzed peptide synthesis in acetonitrile medium

A model was developed which describes simultaneous reaction and internal diffusion for kinetically controlled, immobilized α-chymotrypsin-catalyzed, oligopeptide synthesis in acetonitrile medium. The model combines the equations that describe the intrinsic kinetics of four different reactions and the physical characteristics of three different support materials, as determined experimentally, to predict the apparent initial activity and nucleophile selectivity of the immobilized biocatalyst. The model is able to predict reasonably well the experimentally observed initial rate and nucleophile selectivity vs. enzyme loading profiles. The reduction in observed initial rate with enzyme loading when fast reactions are carried out with α-chymotrypsin immobilized on celite, and the larger influence of mass transfer limitations on the initial reaction rates than on nucleophile selectivities are correctly predicted by the numerical calculations. The model is general in terms of its application to other systems — enzymes, reactions, support materials and/or kinetic schemes — as long as the intrinsic kinetics and the characteristics of the enzyme and support material are known.     

Stability and catalytic properties of penicillin acylase in systems with low water content

Stability and catalytic properties of native and immobilized penicillin acylase were studied in systems with low water content. Preparations of both native and immobilized penicillin acylase demonstrated the catalytic activity even in solid-phase systems which contained 3-5 wt. % of water. The stability and catalytic activity of penicillin acylase at low water content depended on the thermodynamic water activity (aw) in the system.     

Estimating glutathione synthesis with deuterated water: a model for peptide biosynthesis

Glutathione (GSH), an intracellular tripeptide that combats oxidative stress, must be continually replaced due to loss through conjugation and destruction. Previous methods, estimating the synthesis of GSH in vivo, used constant infusions of labeled amino acid precursors. We developed a new method based on incorporation of ²H from orally supplied ²H₂O into stable C-H bonds on the tripeptide. The incorporation of ²H₂O into GSH was studied in rabbits over a 2-week period. The method estimated N, the maximum number of C-H bonds in GSH that equilibrate with ²H₂O as amino acids. GSH was analyzed by liquid chromatography/mass spectrometry after derivatization to yield GSH-N-ethylmaleimide (GSNEM). A model, which simulated the expected abundance at each mass isotopomer for the GSNEM ion at various values for N, was used to find the best fit to the data. The plateau labeling fit best a model with N = 6 of a possible 10 C-H bonds. Thus, the amino acid precursors do not completely equilibrate with ²H₂O prior to GSH synthesis. Advantages of this new method include replacing costly amino acid infusions with the oral administration of ²H₂O and a statistical basis for estimating N.     

Solid-phase peptide synthesis using nanoparticulate amino acids in water.

Solid-phase peptide synthesis has many advantages compared with solution peptide synthesis. However, this procedure requires a large amount of organic solvents. Since safe organic solvent waste disposal is an important environmental problem, a technology based on coupling reaction of suspended nanoparticle reactants in water was studied. Fmoc-amino acids are used widely, but most of them show low solubility in water. We prepared well-dispersible Fmoc-amino acid nanoparticles in water by pulverization using a planetary ball mill in the presence of poly(ethylene glycol). Leu-enkephalin amide was prepared successfully using the nanoparticulate Fmoc-amino acid on a poly(ethylene glycol)-grafted Rink amide resin in water.     

L-Tryptophan synthesis from14C-Anthranilic acid in plants with high and low tryptophan content

The biosynthesis of L-tryptophan (L-trp) from anthranilic acid-¹⁴C (AA-¹⁴C) in. undamaged organs of the seedlings of kohlrabi and pea, with high L-trp content and ma ze plants, with low L-trp content was compared. As for maize the experiments were carried oiut with normal and opaque-2 phenotypes, both with the seedlings and with the ripening kernels. AA-¹⁴C is metabolized in the plants to L-trp pool (i.e. free and bound L-trp, and secondary metabolites) and to glycosyl esters of AA (i.e. to simple glucosyl ester in pea and kohlrabi and more complex glycosides in maize). In maize seedlings L-trp-¹⁴C is synthesized relatively less. (40% in the 1st and 2nd leaf and 33% in the 3rd leaf of the total radioactivity of the incorporated AA-¹⁴C is transferred into the L-trp-¹⁴C pool after 24 h) than in kohlrabi (52% in the hypocotyl and 85% in the cotyledons) and in pea (58% in the 1st and the 2nd internode and 85% in the 3rd and the 4th internode). Thede novo formation of L-trp-¹⁴C is stoped earlier in maize (after 5 h) than in kohlrabi (after 15 h). The level of free L-trp-¹⁴C is relatively low ill maize (15% and 13% of the total radioactivity of the incorporated AA-¹⁴C is converted to free L-trp-¹⁴C and remains in this form after 24 h) in comparison with kohlrabi (31% and 60%) and pea (30% and 49%). In spite of this the formation of L-trp-¹⁴C from AA-¹⁴C is sufficient in maize to incorporate L-trp both into the proteins and into a secondary metabolite that is not yet defined. At the period of seedlings the incorporation in maize of L-trp into the proteins (11% and 10% of the activity of the incorporated AA-¹⁴C) is comparable with that in kohlrabi (11% and 17%), and it is maximum in pea (29% and 36%). Maize, at the stage of germination, thus forms proteins rich in L-trp. The formation of free L-trp is approximately ten times lower in ripening kernels and in the leaves adjacent to the ear and it further decreases in the course of the ripening of the kernels. Although the activity of the biosynthesis of the AA-¹⁴C → L-trp-¹⁴C pathway is relatively lower in maize than in kohlrabi and pea, this pathway is most responsible for the differences in the content of L-trp in these plants. Neither amitrol nor histidine affected the biosynthesis of L-trp in kohlrabi; the interaction of the biosynthetic pathways of L-trp and histidine known in microorganisms is thus not important in a higher plant.     

Enzyme activation by denaturants in organic solvent systems with a low water content.

The effect of urea and guanidine hydrochloride (GdmCl) on the activity of heart lactate dehydrogenase, glycerol-3-phosphate dehydrogenase, hexokinase, inorganic pyrophosphatase, and glyceraldehyde-3-phosphate dehydrogenase was studied in low-water systems. Most of the experiments were made in a system formed with toluene, phospholipids, Triton X-100, and water in a range that varied over 1.0-6.5% (by vol.) [Garza-Ramos, G., Darszon, A., Tuena de Gómez-Puyou, M. & Gómez-Puyou, A. (1990) Biochemistry 29, 751-757]. In such conditions at saturating substrate concentrations, the activity of the enzymes was more than 10 times lower than in all-water media. However the activity of the first four aforementioned enzymes was increased between 4 and 20 times by the denaturants. The most marked activating effect was found with lactate dehydrogenase; with 3.8% (by vol.) water maximal activation was observed with 1.5 M GdmCl (about 20-fold); 4 M urea activated, but to a lower extent. Activation by guanidine thiocyanate was lower than with GdmCl. The activating and inactivating effects of GdmCl on lactate dehydrogenase depended on the amount of water; as the amount of water was increased from 2.0% to 6.0% (by vol.), activation and inactivation took place with progressively lower GdmCl concentrations. When activity was measured as a function of the volume of 1.5 M GdmCl solution, a bell-shaped activation curve was observed. In a low-water system formed with n-octane, hexanol, cetyltrimethylammonium bromide and 3.0% water, a similar activation of lactate dehydrogenase by GdmCl and urea was observed. The water solubility diagrams were modified by GdmCl and urea, and this could reflect on enzyme activity. However, from a comparison of denaturant concentrations on the activity of the enzymes studied, it would seem that, independently of their effect on the characteristics of the low-water systems, denaturants bring about activation through their known mechanism of action on the protein. It is suggested that the effect of denaturants is due to the release of constraints in enzyme catalysis imposed by a low-water environment.     

Lipase-catalyzed synthesis of 6-O-vinylacetyl glucose in acetonitrile

6-O-Vinylacetyl glucose was synthesized with an immobilized lipase using vinylacetic acid and glucose in water-miscible organic solvents, of which acetonitrile gave the highest conversion of 35% at 50 mM glucose and 600 mM vinylacetic acid. The addition of 3Å molecular sieves at 60 mg ml^–1 increased the conversion to 74%. The product, 6-O-vinylacetyl glucose, was polymerized to yield a water-soluble polymer with a molecular mass of about 5000.     

Effect of water activity on thermolysin-catalyzed peptide synthesis in organic solvents

Summary The effect of water activity on the rate of thermolysin-catalyzed synthesis of an aspartame precursor has been investigated in water-miscible and water-immiscible solvents. In both cases, the enzyme reaction rate at a given water activity was found to be significantly different depending on the nature of the solvent. The reaction rates in water-immiscible solvents, where the water activities were close to 1.0, were found to be significantly dependent on the volume ratio of water to organic media and the hydrophobicity of the solvent. These data suggest that the enzyme reaction in the solvent is influenced appreciably by other factors in addition to the water activity.     

Solid phase peptide synthesis in water VI: evaluation of water-soluble coupling reagents for solid phase peptide synthesis in aqueous media

Solid phase peptide synthesis requires large amounts of organic solvents, the safe disposal of which is an important environmental issue. Peptide synthesis, if performed in water and using less or nontoxic reagents, circumvents the disposal problem. Our ultimate aim is to develop an "environment-friendly" solid phase peptide synthesis (SPPS) methodology. Previously, we showed that SPPS in water is feasible. To perform SPPS in water, the coupling reagent must be water-soluble and maintain its reactivity in water. For this report, we tested the efficacy of the water-soluble coupling reagents, 2-(5-norbornene-2,3-dicarboximido)-1,1,3,3-tetramethyluronium tetrafluoroborate (TNTU) and 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMT-MM), towards SPPS in water. We successfully synthesized Leu-enkephalin amide on a solid support suspended in aqueous 50% EtOH using DMT-MM and 2-(4-sulfophenylsulfonyl)ethoxycarbonylamino acids.     

Hydration-induced conformational and flexibility changes in lysozyme at low water content

Using laser Raman spectroscopy, we are able to study conformational changes that occur as previously-dried hen egg-white lysozyme is sequentially rehydrated. Parallel n.m.r. exchangeability studies enable us to monitor flexibility changes also during this rehdyration. The results are consistent with a general loosening up of the protein at a water content of ～0.08 g water/g protein, followed by (probably small) local conformational changes. The enzyme regains its activity only after both these processes have gone to completion; thus these solvent-related changes may be necessary before activity can recommence.     

Salt hydrates buffer water activity during chymotrypsin-catalysed peptide synthesis.

Chymotrypsin (EC 3.4.21.1) powder suspended in hexane in the presence of Na2CO3.10H2O is a good catalyst for peptide synthesis. The salt hydrate releases water to fix the thermodynamic water activity of the system in accord with its dissociation pressure. Salt hydrates can be useful to buffer water activity in mainly organic enzyme reaction mixtures at a value permitting activity of the catalyst while minimising hydrolytic side reactions.     

Augmented water binding and low cellular water content in erythrocytes of camel and camelids.

We investigated a link between hemoglobin primary structure, hemoglobin hydrophobicity-hydrophilicity, and erythrocyte water content in various mammalian species. Some hemoglobin molecules, particularly those of the camel and camelids, contain more charged amino acid residues and are more hydrophilic than the hemoglobins of human and a number of other mammalian species. To test the in vivo significance of these alterations of hemoglobin primary structure, we determined the osmotically unresponsive erythrocyte water fractions in mannit solutions of various osmolarities at 4 degreesC. Among the species investigated, the size of the osmotically unresponsive erythrocyte water fraction relates in a positive linear way to hemoglobin hydrophilicity. The extreme low total erythrocyte water content of camel erythrocytes (1.1-1.3 g water/g dry mass) may be explained by a comparatively high osmotically unresponsive erythrocyte water fraction. It is proposed that alterations of hemoglobin sequences of camel and camelids may be the part of a natural selection process aimed at protecting these animals against osmotic dehydration in arid environments.