Lise Kvittingen

Use of salt hydrates to buffer optimal water level during lipase catalysed in synthesis in organic media: a practical procedure for organic chemists

Enzyme catalyzed reactions in mainly organic media depend very much on the amount of water in the system. We have shown that addition of appropriate solid salt hydrates to the reaction mixture is a simple and convenient method to obtain optimal water level conditions throughout the reaction. As a model reaction the esterification of butanoic acid with butanol catalysed by lipase from Candida rugosa was chosen. Variations in the amount of enzyme, in the solvent and in the concentration of reactants were made.

Alginate as immobilization matrix and stabilizing agent in a two-phase liquid system: Application in lipase-catalysed reactions

Alginate was evaluated as an immobilization matrix for enzyme-catalyzed reactions in organic solvents. In contrast to most hydrogels, calcium alginate was found to be stable in a range of organic solvents and to retain the enzyme inside the gel matrix. In hydrophobic solvents, the alginate gel (greater than 95% water) thus provided a stable, two-phase liquid system. The lipase from Candida cylindracea, after immobilization in alginate beads, catalysed esterification and transesterification in n-hexane under both batch and continuous-flow conditions. The operational stability of the lipase was markedly enhanced by alginate entrapment. In the esterification of butanoic acid with n-butanol, better results were obtained in the typical hydrophilic calcium alginate beads than in less hydrophilic matrices. The effects of substrate concentration, matrix area, and polarity of the substrate alcohols and of the organic solvent on the esterification activity were examined. The transesterification of octyl 2-bromopropanoate with ethanol was less efficient than that of ethyl 2-bromopropanoate with octanol. By using the hydrophilic alginate gel as an immobilization matrix in combination with a mobile hydrophobic phase, a two-phase liquid system was achieved with definite advantages for a continuous, enzyme-catalysed process.

Regioselective lipase-catalyzed transesterification of tributyrin. Influence of salt hydrates on acyl migration

The influence of the nature of solid salt hydrates on the rate of hexanoic acid catalyzed acyl migration in 1,2-dibutyrin has been examined in hexane. The results show that the rate of acyl migration is faster when hydrogen phosphate salts are included compared to sulfate salts. The rate is essentially the same in the presence of sulfate salts and without salts. In regioselective lipase-catalyzed acidolysis of tributyrin with hexanoic acid, the use of hydrogen phosphate salts to control water activity leads to a higher rate of migration than the use of sulfate salts. Minor differences are observed in interesterification with ethyl hexanoate.

Alginate as Immobilization Material for Biocatalysts in Organic Solventsa

Entrapment in Ca-alginate gels can be accomplished under very mild conditions and is widely used for immobilization of whole Due to the high porosity of the alginate gel, free enzyme will normally leak out. However, in typical hydrophobic solvents, the enzyme will be retained inside the hydrogel bead. In these systems, the alginate gel beads will retain their volume. To establish the operational ability of alginate in continuous processes, some basic studies have been carried out and the results are summarized in this work. The volume stability (shrinkage) of calcium alginate gel beads made from both “high-G” (Laminaria hyperborea stipes, LF 10/60, provided by Protan A/S, Drammen, Norway) and “low-G” (Macrocystis pyriferu obtained from the Kelco Division of Merck, San Diego, California) alginates was studied for a range of organic solvents. The leakage of enzyme from “high-G” gel beads was also studied. The alginate beads were applied for lipase-catalyzed reactions in nonaqueous solvents. Lipase-catalyzed esterification and transesterification reactions in organic solvents are by now well established.6.’ These reactions have also been carried out in immobilized systems using different matrices such as hydrophobic and hydrophilic photo-cross-linkable resin prepolymers and urethane prepolymer~,’.~ as well as silica” and celite.’ The use of calcium alginate+ntrapped lipase for hydrolysis of triglycerides G-blocks.

Mixing conditions for enzyme catalysis in organic solvents

Abstract When performing enzyme catalysed reactions in organic solvents the agitation method may have an influence on the result. It has been observed that stirring with magnet bars may cause a considerable decrease in the reaction rate, probably due to damage to the enzyme. This applies to both free and immobilised enzyme. However, use of a shaker or an over-head stirrer does not inactivate the enzyme.

Appreciated Abroad, Depreciated at Home

Ellen Gleditsch (1879–1968) became Norway’s first authority on radioactivity and the country’s second female full professor. From her many years abroad—in Marie Curie’s laboratory in Paris and at Yale University in New Haven with Bertram Boltram—she became internationally acknowledged and developed an extensive personal and scientific network. In the Norwegian scientific community she was, however, less appreciated, and her appointment as a professor in 1929 caused controversy. Despite the recommendation of the expert committee, her predecessor and his allies spread the view that Gleditsch was a diligent but outdated researcher with little scientific promise—a view that apparently persists in the Norwegian chemical community today. In addition to her scientific work, Gleditsch acquired political influence by joining the International Federation of University Women in 1920; she later became the president of both the Norwegian section and the worldwide organization. She worked in particular to establish scholarships enabling women to go abroad. I once worked with a learned man who was reputed to hate women. On one occasion he stated that the new collaborator was a rare exception. When he was asked why he said, “She does not scream.” I heard this several months later and have kept it as a great compliment; yes—the biggest in my scientific career. —Ellen Gleditsch I once worked with a learned man who was reputed to hate women. On one occasion he stated that the new collaborator was a rare exception. When he was asked why he said, “She does not scream.” I heard this several months later and have kept it as a great compliment; yes—the biggest in my scientific career. —Ellen Gleditsch

Response from Kvittingen

Professor Klibanov has endeavoured to answer the question ‘Why did biocatalysis in organic media not take off in the 1930s?’, which Peter Halling and I recently formulated in this journal1xWhy did biocatalysis in organic media not take off in the 1930s?. Halling, P. and Kvittingen, L. Trends Biotechnol. 1999; 17: 343–344Abstract | Full Text | Full Text PDF | PubMed | Scopus (13)See all References1. Our question was based on the work of the Polish biochemist Ernest Aleksander Sym who, in the 1930s, published papers on enzyme catalysis in organic solvents, and from whom we had included two quotes2xUber die esterasewirkung III. Sym, E.A. Biochem. Z. 1933; 258: 304–324See all References, 3xEine methode der enzymatischen estersynthesen. Sym, E.A. Enzymologia. 1936; 1: 156–160See all References.In Klibanov’s summary, he proposes the following two reasons in answer to the title’s question: (1) ‘the enabling scientific advance of the mid-1980s that typical, ordinary enzymes can function in organic media was not made. That lipases can do so must have been seen as a mere extension of their natural abnormal reaction habitat (lipid–water interfaces)’; and (2) ‘at that time there was no demand for enantiopure compounds’.I would like to comment on the first proposed reason. First, at the time of the papers we quoted, the distinction between lipases and esterases on the basis of interfacial behaviour was not an established fact, although it was under discussion. This can be illustrated by another quote from one of Sym’s papers: ‘When the reaction components of an enzyme catalysis are long-chain fatty acids and glycerin and the combination of these, the enzymes that catalyse the hydrolysis and the esterification are called lipases. When short-chain fatty acids are included in the reaction components, the enzyme catalysis is considered, in a narrow sense, to be an action of an esterase. According to this classification, we should assume that an active pancreas preparation consists of two enzymes: a lipase and an esterase. However, these ferments seem to be identical’4xUber die esterasewirkung IV. Sym, E.A. Biochem. Z. 1933; 262: 406–425See all References4.Second, Sym did not limit his use of enzymes in organic media to lipases, he also used pig-liver esterase4xUber die esterasewirkung IV. Sym, E.A. Biochem. Z. 1933; 262: 406–425See all References4. Moreover, Bourquelot and Bridel5xSynthese des glucosides d’alcools a l’aide de l’emulsine et reversibilite des actions fermentaires. Bourquelot, E. and Bridel, M. Ann. Chim. Phys. 1913; 29: 145–218See all References5 had investigated nonlipases (e.g. emulsions of almonds) in organic media more than 20 years before Sym started his work. Sym cited his precedents, and seemed well aware on whose shoulder he stood, so there was therefore no reason for him to limit his choice of enzyme, when used in organic solvents, to lipases.From the above, it seems likely that Professor Klibanov’s argument that ‘Sym’s findings would have been seen as stemming from the unique nature of lipases’ is not supported by historical evidence.

Regioselective hydrolysis of diesters of (Z)- and (E)-2-methyl-butenedioic acids by PLE

Abstract Dimethyl, diethyl and dipropyl esters of (Z)- and (E)-2-methyl-butenedioic acids (citraconic and mesaconic acid) were hydrolysed by pig live esterase, PLE. For the diethyl esters the enzyme showed low regioselectivity for both the (Z)- and the (E)-isomers. However, the hydrolysis of dimethyl and dipropyl (Z)-2-methyl-butenedioic acid gave a ratio of the monoesters of 95:5 and 85:15.

Control of Water Activity by Using Salt Hydrates in Enzyme Catalysed Esterifications in Organic Media

Enzymes used as catalysts in organic media are known to need some water to function. We have used salt hydrates to control the water content in the reaction mixture and studied the effect when the composition in a model reaction was changed. As a model reaction was chosen the esterification of butanoic acid with butanol catalysed by lipase from Candida rugosa. Some other lipases were tested to determine the water content for optimal activity.

Highly regioselective hydrolysis of tricarballylates (retro-fats) and citrates by subtilisin

Trimethyl and triethyl esters of tricarballylic acid and citric acid were hydrolysed with porcine liver esterase(PLE) to the isomeric diesters. In all cases the hydrolysis took place with poor regioselectivity (maximum 50% excess). However, the hydrolysis of trimethyl and triethyl esters of tricarballylic acid and of the triethyl ester of citric acid with subtilisin was absolutely regioselective and the symmetric 1,5-diester was obtained.