Jacques C. Baratti

Lipase catalyzed formation of flavour esters

SummaryThirteen commercial lipase preparations were checked for their ability to catalyse the formation of flavour esters (isoamyl or geranyl acetate, propionate and butyrate) by either direct esterification or ester solvolysis in n-heptane. The formation of isoamyl or geranyl butyrates and propionates by direct esterification was catalyzed by the majority of the tested lipases. Acetic acid esters were more difficult to obtain. Transesterification reactions were found to be a good alternative way for ester synthesis.

Hydrolysis of p-nitrophenyl palmitate in n-heptane by the Pseudomonas cepacia lipase: A simple test for the determination of lipase activity in organic media

Abstract The rate of p -nitrophenyl palmitate (pNPP) hydrolysis in n -heptane by a lipase preparation from Pseudomonas cepacia was studied. In organic media, the reaction rate could be easily measured by spectrophotometry after extraction of the liberated p -nitrophenol by an alkaline water phase. Optimal conditions for substrate hydrolysis were selected and proposed for the easy and fast assay of lipase activity in organic media. Agitation and the amount of added water showed little effect on reaction rates. A temperature of 37°C and substrate concentration of 50 m m were selected. The reaction rate in n -heptane was only 0.16% of that in water.

Multi-competitive enzymatic reactions in organic media: a simple test for the determination of lipase fatty acid specificity

Multiple substrate competition was kinetically analyzed to study lipase-catalyzed reactions in organic media. For each substrate, a competitive factor (the ratio of the specificity constants kcat/Km) was measured by reference to the best substrate using a mixture of fatty acid ethyl esters submitted to a solvolysis reaction by n-propanol. A scale of competitive factors was established which quantitatively described the lipase specificity. This principle was applied to the determination of the specificity of four commercial lipase preparations towards fatty acid chain length and degree of unsaturation. The results were not affected by changes in the physicochemical conditions of the reaction (water content, substrate concentration, nature of nucleophile, etc.). The simple test will be a useful tool to characterize lipase specificity.

Microbiological transformations 37. An enantioconvergent synthesis of the β-blocker (R)-Nifenalol® using a combined chemoenzymatic approach

Abstract This work describes the synthesis of (R)- Nif e nalol ® based on an enantioconvergent chemoenzymatic hydrolysis of para-nitrostyrene oxide. A mathematical approach is devised which allowed to optimize the process.

Properties of free and immobilized lipase from Pseudomonas cepacia

The purified lipase from Pseudomonas cepacia (PS, Amano) was immobilized on a commercially available microporous polypropylene support. The enzyme was rapidly and completely adsorbed on the support. Special attention was devoted to the demonstration of the lack of diffusional limitations, either internal or external, when a soluble substrate (p-nitrophenylacetate, pNPA) was used. The activity yield was high (100%) with pNPA and very low (0.4%) with p-nitrophenylpalmitate (pNPP). These values clearly showed that the immobilized enzyme was fully active as soon as activity was assayed on a soluble substrate rather than an insoluble one. With the latter one, the low activity was due mainly to a slow rate of substrate diffusion inside the porous support. The same diffusional phenomenon could explain the complete change of fatty acid specificity of the immobilized lipase. After immobilization, the lipase was mainly specific for short chain fatty acid esters, whereas the free enzyme was mainly specific for long chain esters. The activity-versus-temperature profiles were not greatly affected by immobilization with maximal reaction rates in the range 45 degrees to 50 degrees C for both enzyme preparations. However, immobilization increased enzyme stability mainly by decreasing the sensitivity to temperature of the inactivation reaction. Half-lives at 80 degrees C were 11 and 4 min for the immobilized and free enzymes, respectively. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 56: 181-189, 1997.

Microbiological transformations. Part 45: A green chemistry preparative scale synthesis of enantiopure building blocks of Eliprodil: elaboration of a high substrate concentration epoxide hydrolase-catalyzed hydrolytic kinetic resolution process

Abstract The enantioselective hydrolysis of racemic para-chlorostyrene oxide 2 following a typical ‘green chemistry’ procedure based on the use of two different Epoxide Hydrolases is described. This allows the preparation of both enantiomers of 2 in very high enantiomeric purity. Furthermore, using a ‘one-pot’ sequential bi-enzymatic strategy enabling to overcome the 50% yield limitation intrinsic to any resolution process, rac-2 could be transformed into nearly enantiopure (R)-3 with an overall yield as high as 93%. The methodology developed was based on the use of a biphasic reactor at high substrate concentration, which is highly desirable for any potential industrial process. The obtained chirons are valuable building blocks for the synthesis of various biologically active targets, like (R)-Eliprodil.

Enantioselective Hydrolysis of p-Nitrostyrene Oxide by an Epoxide Hydrolase Preparation from Aspergillus niger

The epoxide hydrolase activity of Aspergillus niger was synthesized during growth of the fungus and was shown to be associated with the soluble cell fraction. An enzyme preparation was worked out which could be used in place of the whole mycelium as biocatalyst for the hydrolysis of epoxides. The effect of four different cosolvents on enzyme activity was investigated. Consequently, dimethylsulfoxide (DMSO) was selected for epoxide solubilization. The effect of temperature on both reaction rate and enzyme stability was studied in the presence of DMSO (0.2 volume ratio). A temperature of 25°C was selected for the reaction of bioconversion. With a substrate concentration of 4.5 mM a batch reactor showed that the enzyme preparation hydrolyzed para‐nitrostyrene oxide with very high enantioselectivity. The (S) enantiomer of the epoxide remained in the reaction mixture and showed an enantiomeric excess higher than 99%. The substrate concentration could be increased to 20 mM without affecting the enantiomeric excess and degree of conversion. Therefore, the method is potentially useful for the preparative resolution of epoxides. Application are in the field of chiral synthons which are important building blocks in organic synthesis.

Asymmetric hydrolysis of racemic para-nitrostyrene oxide using an epoxide hydrolase preparation from Aspergillus niger

Abstract A lyophilized epoxide hydrolase preparation was isolated from the fungus Aspergillus niger . The preparation could be used in place of the whole mycelium as biocatalyst for the enantioselective hydrolysis of racemic para-nitrostyrene oxide. The cosolvent used for substrate dissolution showed slightly different effects on enzyme activity and stability. Dimethylsulfoxide (DMSO) was selected as the less inhibitory cosolvent among those tested. The enzyme preparation was first proved efficient by running a batch reactor at a low substrate concentration of 4 m m . The hydrolysis of para-nitrostyrene oxide was fast (around 5 h) and with high enantioselectivity ( E = 41). The (S) enantiomer of the epoxide remained in the reaction mixture with an enantiomeric excess (ee) higher than 97% for a conversion of 47%. The substrate concentration had been optimized. It could be increased to 330 m m (54 g l −1 ) without affecting the ee; therefore, the method is potentially useful for the preparative resolution of epoxides. Applications are in the field of chiral synthons which are important building blocks in organic synthesis.

Lipase-catalyzed reactions in organic media: competition and applications

Lipases (triacylglycerol acylhydrolase, EC 3.1.1.3) have been used in organic media for the catalysis of reactions such as hydrolysis, esterification and transesterification. In these conditions it was confirmed that all reactions proceed through an acyl enzyme intermediate in two successive steps: acyl enzyme formation and solvolysis. The competition between two acyl acceptors (acyl donors) for reaction with a donor (acceptor) is described for the first time. A kinetic model is proposed using a competitive factor which is in good accordance with experimental results. The model was used successfully for the prediction of alcohol (acid) separations and resolutions by lipases.

Microbiological transformations. Part 39: Determination of the regioselectivity occurring during oxirane ring opening by epoxide hydrolases: a theoretical analysis and a new method for its determination

Abstract In the course of this work we have devised new equations as well as a new method allowing for the total determination of the regioselectivity occurring during biohydrolysis of a racemic epoxide by an epoxide hydrolase. This determination is achievable by simply studying the racemic epoxide as a substrate. The results showed that, depending on the enantioselectivity (E value) and the regioselectivity involved, the absolute configuration as well as the enantiopurity of the residual epoxide and of the formed diol appear to be highly variable. For a specific enzyme/substrate couple, the yield and enantiopurity of the less reactive (remaining) epoxide—and thus the possibility to prepare it in enantiopure form—exclusively depend upon the enzyme enantioselectivity. On the other hand, the ee of the formed diol (eep) depends upon the enantioselectivity and on the regioselectivity of the oxirane ring opening. A theoretical analysis based on the material balance, as well as several practical examples, are provided to illustrate the various possibilities of such biohydrolyses.