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Methods used for the regeneration of cofactors in organic media are reviewed. Substrate-driven regeneration methods include the use of a second substrate of the same enzyme and the use of a second enzyme and its substrate. The use of mediators in oxidoreductions is described and examples of photochemical and electrochemical regeneration methods are presented. General problems and possibilities of
Synthesis of partial glycerides in a solvent-free system has been investigated with various acyl donors and glycerol as substrates and a 1,3-specific immobilized lipase to catalyze the reaction. Capric acid was the most efficient acyl donor, compared with ethyl caprate and tricaprin. However, to obtain a high yield of dicaprin and a low amount of tricaprin, ethyl caprate was the acyl donor of choi
α-Chymotrpysin (EC 3.4 21.1) was immobilized by deposition on celite and subsequent cross-linking with glutaraldehyde. The effects of different mixtures of aqueous buffer and acetonitrile on the immobilized preparation were evaluated using a dipeptide synthesis as model reaction. The initial reaction rate at 6-95% of water increased with increasing water content. The maximum yield of peptide had t
A depeptide synthesis was drastically influenced by the reaction temperature, in the range from −30° to 25°C. This article shows the kinetic reasons of this effect. α‐Chymotrypsin was immobilized on celite and used in four different water‐miscible solvents containing small amounts of water‐miscible solvents containing small amounts of water. The reaction studied was the aminolysis of N‐acetyl‐L‐ph
The effect of the addition of sorbitol on the activity and stability of enzymes was examined by monitoring transesterification reactions performed in organic media at various water activities (aw = 0.08 to 0.97). Lipases from Chromobacterium viscosum and Candida rugosa immobilized on celite, and chymotrypsin, free or immobilized on celite, were used. When the sorbitol‐containing enzymes were emplo
A novel total enzymatic synthesis of [Leu]- and [Met]-enkephalin derivatives was accomplished in low-water content systems at a preparative scale. α-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 rea
Lipase-catalyzed transesterification of high-oleic-acid rapeseed oil with stearic acid or methyl stearate was investigated. High yields of 1,3-distearoyl-2-monooleyl glycerol (36%) and 1(3)-2-dioleyl-1(3)-monostearoyl glycerol (27%) with small incorporation of stearic acid in the 2-position were obtained by using lipase from Rhizopus arrhizus immobilized on polypropylene powder and ethyl stearate
Noncovalent complexes were formed by lyophilization of aqueous solutions containing horse liver alcohol dehydrogenase, NAD+ and a polymer [ethyl cellulose or poly(vinyl butyral)]. The complexes expressed higher specific catalytic activity in organic solvents as compared to a corresponding amount of enzyme deposited on to Celite or lyophilized enzyme powder. The noncovalent complexes were soluble i
In order to evaluate whether there are differences with regard to functionality in starch gel modification between 1‐and 2‐monoglycerides, the gelatinisation of potato starch in the presence of monolaurin, monopalmitin and monoolein has been examined. When the temperature was increased by 1°C/min from 20°C to 80°C there was a significant difference in gel volume between samples containing 1‐monola
Scanning calorimetry measurements of different amounts of chymotrypsin in water alone gave a temperature of denaturation (Td) value of 54°C. However, when high‐molecular‐weight poly(ethylene glycol) was added to aqueous solutions of chymotrypsin, the thermostability of the enzyme was enhanced. For example, the addition of 20% (w/w) of poly(ethylene glycol) of molecular weight of 100,000 increased
Rhizopus arrhizus lipase immobilized on celite was used to prepare isomerically pure 2-monoglycerides by alcoholysis of triglycerides in organic media. Reaction parameters such as choice of solvent, choice of alcohol, and alcohol concentration were studied. When 12.5 mM tripalmitin was used as substrate, methyl-tert-butyl ether was the best solvent for alcoholysis at water activity 0.11. Ethanol g
A technique of continuous water activity control was used to examine the effects of water activity on enzyme catalysis in organic media. Esterification catalyzed by Rhizopus arrhizus lipase was preferably carried out at a water activity of 0.33, which resulted in both maximal initial reaction rate and a high yield. When Pseudomonas lipase was used as catalyst it was beneficial to start the reactio
A recent method for exposing enzymes to organic solvents is reviewed. By complex formation between the enzyme and polymers that per se are soluble in organic solvents it is possible to disperse the enzyme in the organic medium in such a way that an optically transparent (in the visible region) solution is obtained. After reaction, the separation of the enzyme from the organic medium can be obtaine
Mixing solutions of polymers dissolved in chloroform resulted in turbid solutions that parted into two separate phases upon standing. Each phase consisted primarily of one of the two polymers and contained only small amounts of the other. An enzyme (α‐chymotrypsin) added to the two‐phase system partitioned preferentially to one of the phases; this was observed with native enzyme and with enzyme as
The progress of enzymatic peptide synthesis catalyzed by α-chymotrypsin and subtilisin from Bacillus subtilis strain 72 (subtilisin 72) in low-water systems was studied. The initial reaction mixture consisted of the solvent, the acyl-group donor (MalAlaAlaPheOMe or ZAlaAlaPheOMe, Mal, maleyl, Z, benzyloxycarbonyl), the nucleophile XaaNH2 (Xaa = Phe, Leu or Ala), and the enzyme adsorbed on porous s
The use of β-glucosidase for synthesizing octyl-β-glucoside was studied. Almond β-glucosidase immobilized on XAD-4, glucose, and octanol were mixed at controlled water activity. A water activity of at least 0.67 was needed for synthesis of octyl-β-glucoside, and the reaction rate increased with increasing water activity. The final yield decreased with increasing water activity. The best results we
Octanoylglucose was synthesized from insoluble glucose and octanoic acid in acetonitrile, using lipase from Candida antarctica as catalyst. The initial concentration of octanoic acid was optimized to give complete conversion of glucose to monoester and avoid diester formation. The acylation occurred exclusively at the primary hydroxyl group on the glucose ring.
Pepsin‐catalyzed synthesis of protected peptides was studied in two‐phase systems containing up to 50% (by volume) of aqueous phase. A methodological study was carried out to determine the optimum conditions for the synthesis of the model protected peptide Z‐Phe‐Phe‐OMe. Several parameters such as concentrations of carboxylic and amino components, pH of the aqueous phase, ratio of organic to aqueo
Lipase from Candida rugosa immobilized on Celite was employed as the biocatalyst in order to examine the effect of the reaction medium upon enzymic activity and selectivity. As the model reaction, transesterification between tributyrin and pentan‐2‐ol in iso‐octane (2,2,4‐trimethylpentane) was chosen. A small amount of water (0.05%, v/v) was added to the reaction medium. Enhanced transesterificati
