Enzyme reaction method and a method for enzymatically producing an optically active cyanohydrin

Abstract

The present invention relates to an enzyme reaction method which comprises performing an enzyme reaction, using an immobilized enzyme having a water content of 10% by weight or more as an enzyme and using an organic solvent substantially immiscible with water as a reaction solvent, under such conditions that a liquid phase forms a homogeneous system without phase separation although it is saturated with water or an aqueous buffer; a method for performing an enzyme reaction using an aldehyde compound as a substrate, which comprises removing a carboxylic acid compound contained in an aldehyde compound by subjecting the aldehyde compound to an alkaline treatment before starting the enzyme reaction; a method for performing an enzyme reaction using an aldehyde compound as a substrate, which comprises reducing a carboxylic acid compound content in the aldehyde compound to 0.1 wt % or less by subjecting the aldehyde compound to an alkaline treatment before starting the enzyme reaction; a method for enzymatically producing an optically active cyanohydrin from a carbonyl compound and prussic acid containing an acidic substance as a stabilizer, which comprises subjecting the prussic acid to a treatment for reducing inhibitory effect of the stabilizer on an enzyme, and performing an enzyme reaction to synthesize the optically active cyanohydrin using the treated prussic acid; a method for enzymatically producing an optically active cyanohydrin, which comprises dissolving prussic acid in an organic solvent substantially immiscible with water to give an organic solution of prussic acid, adding a buffer to this solution in a saturation amount or more, mixing, collecting the organic phase, and performing an enzyme reaction to synthesize the optically active cyanohydrin using the organic phase as prussic acid; as well as a method for enzymatically producing an optically active cyanohydrin, which comprises performing distillation of a reaction solution after completion of an enzyme reaction to separate and collect unreacted prussic acid and organic solvent therefrom, and repeatedly using the collected prussic acid and organic solvent at least once.

Description

BACKGROUND OF THE INVENTION [0001] The present invention relates to an enzyme reaction method and a method for enzymatically producing an optically active cyanohydrin. [0002] An enzyme reaction advantageously uses an organic solvent as a reaction solvent so as to increase the concentration of a substrate or product that is hard to dissolve in water. Such an enzyme reaction in an organic solvent has therefore been used as a reaction system for various enzymes. However, in contrast to an aqueous environment in which an enzyme is stable and active, an enzyme is often denatured and thus generally unstable in an organic solvent system. For this reason, in cases where enzyme reactions are applied to industrial syntheses of useful substances, the construction of reaction systems depends on the nature of enzymes to be used. A choice between high concentration reaction conditions and the stability of enzyme is therefore made according to individual circumstances. Particularly, in a method wh...

AI Technical Summary

Benefits of technology

The present invention provides an improved method for synthesizing optically active cyanohydrins using an immobilized enzyme and an aldehyde compound as a substrate. The method allows for a high concentration and high yield of the desired product, with the use of a substrate-containing organic solvent saturated with water or an aqueous buffer. The method also includes the use of an alkaline treatment to remove carboxylic acid compounds that may inhibit the enzyme reaction. The invention also provides a simple and effective separation and recovery of the enzyme from the reaction mixture. Overall, the invention provides a more efficient and effective method for synthesizing optically active cyanohydrins.

Problems solved by technology

However, in contrast to an aqueous environment in which an enzyme is stable and active, an enzyme is often denatured and thus generally unstable in an organic solvent system.

Accordingly, such a conventionally known reaction using hydroxynitrile lyase in an organic solvent system involves a problem of extended reaction time.

In addition, an aromatic carbonyl compound used as a substrate for this enzyme has low solubility in water, so that it is not practical to carry out such a reaction in an aqueous system due to low substrate and product concentrations.

An aqueous reaction system involves a problem of insufficient efficiency attributed to low substrate and product concentrations because a carbonyl substrate, such as aldehyde or ketone, generally has low solubility in water.

A mixed solvent system comprising a mixture of a polar solvent and water also involves a problem of insufficient efficiency although it provides some increases in substrate concentration, as compared with a simple aqueous system.

This system involves an additional problem that a polar solvent is likely to affect the stability of the enzyme.

The use of an organic solvent saturated with water or an aqueous buffer achieves higher substrate and product concentrations, but on the other hand, it provides a low reaction rate because the water content is too low.

In practice, however, an enzyme comes into direct contact with an organic solvent in this system, so that the enzyme and contaminant proteins are likely to be denatured by the organic solvent.

The two-phase system therefore involves difficulties of the separation of a reaction mixture into two phases.

However, there is no knowledge regarding acceptable concentration and effective removal of impurities such as benzoic acid, which act as inhibitors against enzyme reactions.

However, distillation is not easily applied to industrial processes because it requires a distillation plant, it cannot completely separate some impurities such as benzoic acid due to their sublimation properties, and it may accelerate the production of carboxylic acids due to the application of heat.

However, none of these reports has mention an industrially produced prussic acid containing a stabilizer which seriously affects the activity of hydroxynitrile lyase.

First, the previous reports took little notice of the above fact because prussic acid used therein was prepared for laboratory use in a very small amount, but not industrially produced, so that it contained no stabilizer.

However, recycling of the distillate was previously unknown although collection of an optically active cyanohydrin through distillation of a reaction product solution is a known process.

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