U.S. patent application number 10/990053 was filed with the patent office on 2005-04-28 for enzyme reaction method and a method for enzymatically producing an optically active cyanohydrin.
This patent application is currently assigned to NIPPON SHOKUBAI CO., LTD.. Invention is credited to Dobashi, Yukio, Semba, Hisashi.
Application Number | 20050089977 10/990053 |
Document ID | / |
Family ID | 27343612 |
Filed Date | 2005-04-28 |
United States Patent
Application |
20050089977 |
Kind Code |
A1 |
Semba, Hisashi ; et
al. |
April 28, 2005 |
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.
Inventors: |
Semba, Hisashi; (Ibaraki,
JP) ; Dobashi, Yukio; (Ibaraki, JP) |
Correspondence
Address: |
FINNEGAN, HENDERSON, FARABOW, GARRETT & DUNNER
LLP
901 NEW YORK AVENUE, NW
WASHINGTON
DC
20001-4413
US
|
Assignee: |
NIPPON SHOKUBAI CO., LTD.
|
Family ID: |
27343612 |
Appl. No.: |
10/990053 |
Filed: |
November 15, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
10990053 |
Nov 15, 2004 |
|
|
|
09870821 |
Jun 1, 2001 |
|
|
|
Current U.S.
Class: |
435/128 |
Current CPC
Class: |
C12P 13/004 20130101;
C12N 9/88 20130101 |
Class at
Publication: |
435/128 |
International
Class: |
C12P 013/00 |
Foreign Application Data
Date |
Code |
Application Number |
Jun 2, 2000 |
JP |
166578/2000 |
Jun 2, 2000 |
JP |
166579/2000 |
Jul 7, 2000 |
JP |
206130/2000 |
Claims
1-10. (canceled)
11. A method for enzymatically producing an optically active
cyanohydrin from a carbonyl compound and prussic acid containing an
acidic substance as a stabilizer, said prussic acid providing an
aqueous phase with pH 5 or less when dissolved at a concentration
of 1.5 M in an organic solvent substantially immiscible with water,
mixed with pure water at such a ratio that the mixture separates
into organic and aqueous phases, and then allowed to stand, wherein
said method comprises: subjecting said 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.
12. A method for enzymatically producing an optically active
cyanohydrin from prussic acid and carbonyl compound, 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.
13. The method according to claim 12, wherein the buffer has
buffering ability in a range of pH 4 to pH 7.
14. The method according to claim 11, wherein the enzyme reaction
is catalyzed by hydroxynitrile lyase.
15. The method according to claim 12, wherein the enzyme reaction
is catalyzed by hydroxynitrile lyase.
16. A method for enzymatically producing an optically active
cyanohydrin from prussic acid and carbonyl compound, 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.
17. The method according to claim 16, wherein the reaction solution
after completion of an enzyme reaction is obtained from the method
according to claim 1.
18. The method according to claim 16, wherein the reaction solution
after completion of an enzyme reaction is obtained from the method
according to claim 5.
19. The method according to claim 16, wherein the reaction solution
after completion of an enzyme reaction is obtained from the method
according to claim 6.
20. The method according to claim 16, wherein the reaction solution
after completion of an enzyme reaction is obtained from the method
according to claim 11.
21. The method according to claim 16, wherein the reaction solution
after completion of an enzyme reaction is obtained from the method
according to claim 12.
22. A method of performing an enzymatic reaction, comprising:
treating a solution comprising an aldehyde compound with an
alkaline compound to form a treated solution; and initiating the
enzymatic reaction; wherein the aldehyde compound is a substrate
for the enzymatic reaction.
23. The method of claim 22, wherein initiating the enzymatic
reaction comprises adding an enzyme and hydrogen cyanide to the
treated solution.
24. The method of claim 22, wherein the treated solution comprises
0.1 weight percent or less of a carboxylic acid, based on the total
weight of the solution.
25. The method of claim 22, wherein treating with an alkaline
compound comprises: combining the solution comprising the aldehyde
compound with an alkaline aqueous solution to form an aqueous
phase; and separating the aqueous phase from the solution
comprising the aldehyde compound.
26. The method of claim 24, wherein treating with an alkaline
compound comprises: combining the solution comprising the aldehyde
compound with an alkaline aqueous solution to form an aqueous
phase; and separating the aqueous phase from the solution
comprising the aldehyde compound.
27. The method of claim 22, wherein the enzymatic reaction
comprises the reaction of the aldehyde compound and hydrogen
cyanide to form a corresponding optically active cyanohydrin
wherein the reaction is catalyzed by hydroxynitrile lyase.
28. The method of claim 24, wherein the enzymatic reaction
comprises the reaction of the aldehyde compound and hydrogen
cyanide to form a corresponding optically active cyanohydrin
wherein the reaction is catalyzed by hydroxynitrile lyase.
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 wherein
hydroxynitrile lyase catalyzes the synthesis of an optically active
cyanohydrin from hydrogen cyanide and a carbonyl compound as
substrates, this enzyme is relatively stable in an organic solvent,
but it provides a significant decrease in reaction rate when water
is absent from the reaction 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.
[0003] With regard to the synthesis of an optically active
cyanohydrin in the presence of hydroxynitrile lyase, the following
reaction systems have been reported: an aqueous system, i.e., a
system which uses water or an aqueous buffer containing an enzyme
and a substrate dissolved therein (Japanese Patent Examined
Publication No. 07-53116); a mixed solvent system comprising a
mixture of a polar solvent and water (Appl. Microbiol. Biotechnol.,
Vol. 29, 419-425, 1988); an organic solvent system saturated with
water or an aqueous buffer (Japanese Patent Laid-Open Publication
No. 63-219388); and a two-phase system comprising a mixture of an
organic solvent with water or an aqueous buffer at a volume ratio
of 1/5 to 5/1 (Japanese Patent Laid-Open Publication No. 05-317065;
Biocatal. Biotrans. Vol. 12, 255-266, 1995; Japanese Patent
Laid-Open Publication No. 11-243983).
[0004] 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. A two-phase system
comprising an organic solvent and water is advantageous in
improving a reaction rate and in increasing substrate and product
concentrations. 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 denatured proteins may affect separation at the
interface between organic and aqueous phases and, in some cases,
may result in an emulsified reaction mixture. The two-phase system
therefore involves difficulties of the separation of a reaction
mixture into two phases.
[0005] An enzyme reaction using an aldehyde compound as a substrate
is a widely carried out reaction. In particular, an enzyme reaction
for synthesizing an optically active cyanohydrin from an aldehyde
compound and hydrogen cyanide as substrates is useful because this
reaction enables efficient synthesis of an optically active
cyanohydrin which is difficult to chemically synthesize.
[0006] Thus, the above reaction is very advantageous, but it is
known that, for example, the synthesis of (R)-mandelonitrile from
benzaldehyde in the presence of (R)-hydroxynitrile lyase as a
catalyst is inhibited when benzaldehyde as a starting material is
contaminated by benzoic acid. However, there is no knowledge
regarding acceptable concentration and effective removal of
impurities such as benzoic acid, which act as inhibitors against
enzyme reactions.
[0007] Impurities present in a starting aldehyde compound may be
removed by distillation. 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. It has been therefore desirable to develop an
enzyme reaction method that achieves simple and effective removal
of impurities and thereby provides a product of interest in high
yield.
[0008] There have been many reports about reactions for the
synthesis of an optically active cyanohydrin from a carbonyl
compound and prussic acid in the presence of an enzyme catalyst
such as hydroxynitrile lyase. However, none of these reports has
mention an industrially produced prussic acid containing a
stabilizer which seriously affects the activity of hydroxynitrile
lyase. There are two possible reasons for this. 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. Second, the above fact did not present a problem in
cases where prussic acid was added to a reaction system at low
concentration the stabilizer hardly affected the reaction.
[0009] Further, when an optically active cyanohydrin is synthesized
from a carbonyl compound and prussic acid (starting materials) in
an organic solvent (reaction solvent) in the presence of
hydroxynitrile lyase as a catalyst, the resulting reaction product
is dissolved in the organic solvent used as a reaction solvent.
[0010] To collect the optically active cyanohydrin produced in this
reaction, in general, there is a need to remove low-boiling
solvents by distillation from the reaction product solution
containing the optically active cyanohydrin. 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.
OBJECTS AND SUMMARY OF THE INVENTION
[0011] In the first aspect, the present invention provides an
immobilized enzyme reaction system in which a product of interest
can be synthesized at a high concentration and at a sufficient
reaction rate.
[0012] In the second aspect, the present invention provides an
enzyme reaction method using an aldehyde compound as a substrate,
which achieves removal of an inhibitor against the enzyme reaction
and thereby provides a product of interest in high yield.
[0013] In the third aspect, the present invention provides an
industrially advantageous method for synthesizing an optically
active cyanohydrin from a carbonyl compound and prussic acid in the
presence of an enzyme catalyst such as hydroxynitrile lyase.
[0014] In the fourth aspect, the present invention provides the
effective use of a reaction solvent and unreacted prussic acid in a
method for producing an optically active cyanohydrin.
[0015] Our research efforts were directed to overcoming the above
problems, and we have found that the use of an immobilized enzyme
having a water content of 10% by weight or more in combination with
a substrate-containing organic solvent saturated with water or an
aqueous buffer enables the construction of a reaction system which
permits a reaction at a high reaction rate and at a high
concentration, and very simple separation and recovery of the
enzyme from the reaction mixture after the reaction, thereby
finally completing the first aspect of the present invention.
[0016] Namely, the first aspect of the present invention
encompasses the following embodiments.
[0017] (1) 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.
[0018] (2) A method according to (1) above, wherein an enzyme
comprising immobilized hydroxynitrile lyase which catalyzes the
synthesis of a cyanohydrin from hydrogen cyanide and a carbonyl
compound is used as the immobilized enzyme to convert the carbonyl
compound into the corresponding optically active cyanohydrin.
[0019] (3) A method according to (2) above, wherein hydroxynitrile
lyase is (R)-hydroxynitrile lyase or (S)-hydroxynitrile lyase, and
an asymmetric carbonyl compound is converted into the corresponding
optically active cyanohydrin.
[0020] (4) A method according to any one of (1) to (3) above,
wherein a carrier capable of retaining water is used as a carrier
for the immobilized enzyme.
[0021] (5) A method according to any one of (1) to (4) above,
wherein the reaction is carried out under such conditions that a
liquid phase contains water in a saturation amount in order to
prevent the release of water from the immobilized enzyme into the
liquid phase during the reaction.
[0022] In the first aspect, the present invention not only
increases the substrate and product concentrations because the
reaction uses a homogeneous system of organic solvent, but also
improves the activity and stability of the enzyme because the
enzyme is present within a carrier for immobilization having a
sufficient water content. Further, the present invention is
characterized by a simple separation of the enzyme from the
reaction mixture through solid-liquid separation since the reaction
mixture can be kept clear without emulsification. A reaction in an
organic solvent system containing water in a trace amount is
generally called an enzyme reaction in an organic solvent-trace
aqueous system. The water content in this case is normally at most
several percent. In contrast, in the present invention, there is no
upper limit on water content relative to the whole reaction system,
so long as water is not released from the immobilized enzyme to
form an aqueous phase in the reaction mixture and thereby produce
two separated phases. Unlike the above organic solvent-trace
aqueous system, an enzyme reaction in the present invention favours
a higher water content of 10% by weight or more in the immobilized
enzyme. The present invention is therefore completely different
from the conventional organic solvent-trace aqueous system.
[0023] In the first aspect, the present invention can also be
particularly and preferably applied to a reaction in which
hydroxynitrile lyase catalyzes the synthesis of an optically active
cyanohydrin from a carbonyl compound and hydrogen cyanide.
[0024] In addition, we have found that in an enzyme reaction using
an aldehyde compound as a substrate, the reaction is inhibited by a
carboxylic acid compound which is present in and corresponds to the
aldehyde compound.
[0025] We have now found that an alkaline treatment of an aldehyde
compound achieves simpler and more certain removal of such an
inhibitor ensuring successful use of a lower-grade aldehyde
compound in the enzyme reaction, thereby finally completing the
second aspect of the present invention.
[0026] Namely, the second aspect of the present invention
encompasses the following embodiments.
[0027] (1) 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.
[0028] (2) 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.
[0029] (3) A method according to (1) or (2) above, wherein the
alkaline treatment comprises mixing the aldehyde compound with an
alkaline aqueous solution and then separating the aldehyde compound
from the aqueous phase.
[0030] (4) A method according to any one of (1) to (3) above,
wherein the enzyme reaction is the synthesis of an optically active
cyanohydrin from the aldehyde compound and hydrogen cyanide in the
presence of hydroxynitrile lyase as a catalyst.
[0031] Further, our research efforts were directed to overcoming
the above problems, and we have found that an acidic substance
(e.g., sulfurous acid or sulfuric acid) included as a stabilizer in
an industrially available prussic acid inhibits the activity of an
enzyme such as hydroxynitrile lyase and that the reduction of the
stabilizer's inhibitory effect provides an significantly extended
life-time of an enzyme such as hydroxynitrile lyase, thereby
finally completing the third aspect of the present invention.
[0032] Namely, the third aspect of the present invention
encompasses the following embodiments.
[0033] (1) A method for enzymatically producing an optically active
cyanohydrin from a carbonyl compound and prussic acid containing an
acidic substance as a stabilizer, said prussic acid providing an
aqueous phase with pH 5 or less when dissolved at a concentration
of 1.5 M in an organic solvent substantially immiscible with water,
mixed with pure water at such a ratio that the mixture separates
into organic and aqueous phases, and then allowed to stand, wherein
said method comprises:
[0034] subjecting the prussic acid to a treatment for reducing
inhibitory effect of the stabilizer on an enzyme; and
[0035] performing an enzyme reaction to synthesize the optically
active cyanohydrin using the treated prussic acid.
[0036] (2) A method for enzymatically producing an optically active
cyanohydrin from prussic acid and a carbonyl compound, which
comprises:
[0037] dissolving prussic acid in an organic solvent substantially
immiscible with water to give an organic solution of prussic
acid;
[0038] adding a buffer to this solution in a saturation amount or
more;
[0039] mixing;
[0040] collecting the organic phase; and
[0041] performing an enzyme reaction to synthesize the optically
active cyanohydrin using the organic phase as prussic acid.
[0042] (3) The method according to (2) above, wherein the buffer
has buffering ability in a range of pH 4 to pH 7.
[0043] (4) The method according to any one of (1) to (3) above,
wherein the enzyme reaction is catalyzed by hydroxynitrile
lyase.
[0044] Furthermore, we have found that reduced-pressure
distillation of a reaction solution containing an optically active
cyanohydrin synthesized in an enzyme reaction using hydroxynitrile
lyase can provide very effective collection of not only a reaction
solvent but also unreacted prussic acid and that the solution thus
collected can be re-used for the enzyme reaction, thereby finally
completing the fourth aspect of the present invention.
[0045] Namely, the fourth aspect of the present invention
encompasses the following embodiments.
[0046] (1) A method for enzymatically producing an optically active
cyanohydrin from prussic acid and a carbonyl compound, which
comprises:
[0047] performing distillation of a reaction solution after
completion of an enzyme reaction to separate and collect unreacted
prussic acid and organic solvent therefrom; and
[0048] repeatedly using the collected prussic acid and organic
solvent at least once.
[0049] (2) The method according to (1) above, wherein the reaction
solution after completion of an enzyme reaction is obtained from
the method of the first, second or third aspect of the present
invention.
[0050] Any enzyme may be used in the first aspect of the present
invention without particular limitations, so long as it can provide
an improved reaction efficiency in a reaction system in which an
organic solvent is used as a reaction solvent. Such an enzyme may
be an enzyme catalyst whose substrate is a compound
hard-to-dissolve or insoluble in water. Illustrative examples
includes oxidase or reductase, such as monooxygenase, which
catalyzes the oxidation-reduction of an aromatic compound; an
ester-hydrolyzing enzyme, such as esterase, which catalyzes the
synthesis and/or substitution of an ester compound;
glycosyltransferase, such as glucosidase, which catalyzes the
glycosylation of a compound hard-to-dissolve or insoluble in water;
nitrile hydratase which hydrolyzes a nitrile compound; and
hydroxynitrile lyase which catalyzes the synthesis of an optically
active cyanohydrin from a carbonyl substrate easily soluble in an
organic solvent. In particular, it is preferable to use
hydroxynitrile lyase which catalyzes the synthesis of an optically
active cyanohydrin.
[0051] The above-mentioned hydroxynitrile lyase means an enzyme
catalyzing the synthesis of an optically active cyanohydrin from
hydrogen cyanide and a carbonyl compound. Hydroxynitrile lyase for
the synthesis of (R)-cyanohydrin (hereinafter, (R)-hydroxynitrile
lyase) includes those derived from Rosaceae plants such as almond
(Prunus amygdalus) and Linaceae plants. Hydroxynitrile lyase for
the synthesis of (S)-cyanohydrin (hereinafter, (S)-hydroxynitrile
lyase) includes those derived from Gramineae plants such as sorghum
(Sorghum bicolor), Euphorbiaceae plants such as Manihot esculenta
and Hevea brasiliensis, and Olacaceae plants such as Xinienia
americana.
[0052] The above enzyme may be prepared by extraction from
organism's tissues containing the enzyme. Alternatively, the enzyme
may also be produced from a recombinant organism into which a
cloned gene of the enzyme has been introduced. Furthermore, any
hydroxynitrile lyase having an altered enzyme action, which is
created by modifying the wild-type hydroxynitrile lyase gene, may
be used in the present invention, so long as it retains the above
activity.
[0053] In the first aspect, the present invention uses a carrier
for immobilization as a support to hold enzyme molecules and to
retain water. Any carrier may be used without particular
limitations, so long as it can hold enzyme molecules and retain
water. A preferred carrier may be one which is hydrophilic or one
which retains water or an aqueous buffer therein, for example, a
porous inorganic carrier, a water-retaining carrier based on fibers
such as cellulose, or a carrier made of a polymer compound(s).
Specific examples include, but are not limited to, inorganic
carriers such as porous ceramic particles, porous silica gel
particles and zeolite particles; natural polymer gels such as agar,
calcium alginate and chitosan; and synthetic polymer gels such as
polyacrylic acid, polyacrylamide and polyvinyl alcohol.
[0054] In the first aspect of the present invention, enzyme
molecules may be immobilized in any manner, for example, by
allowing carriers to absorb an enzyme solution, by mixing carriers
with an enzyme solution to immobilize enzyme molecules on/in the
carriers by absorption, by entrapping and immobilizing enzyme
molecules within carriers, or by cross-linking enzyme molecules via
crosslinkers.
[0055] In the first aspect of the present invention, it is
important to adjust the water content of the immobilized enzyme
such that the water content (%) relative to the whole reaction
system (comprising the immobilized enzyme, water or an aqueous
buffer, a solvent, a substrate and a product) is greater than a
saturation amount (%) of water dissolvable in a reaction solvent
containing the substrate and/or product.
[0056] At the water content as little as the above saturation
amount, the enzyme is not substantially surrounded with water,
leading to a significant decrease in reaction rate. On the other
hand, when water is given in an amount far in excess of that which
can be retained within the immobilized enzyme, a liquid phase is
separated into two phases, i.e., organic and aqueous phases. This
makes the immobilized enzyme less dispersible in the reaction
solvent, leading to a decrease in reaction efficiency.
[0057] In practice, the amount of water soluble in a solvent will
vary depending on the type of solvent to be used and conditions for
temperature and substrate concentration. It is therefore desirable
to select the water content appropriately according to reaction
conditions to be applied.
[0058] In the first aspect of the present invention, an organic
solvent substantially immiscible with water is used as a reaction
solvent in order to improve the concentration of starting materials
and productivity. As used herein, an "organic solvent substantially
immiscible with water" means an organic solvent except for those
soluble in water in any proportion. Any solvent may be used as an
organic solvent without particular limitations, so long as it is
substantially immiscible with water and has no influence upon an
enzyme reaction. For example, a solvent to be used in the synthesis
of an optically active cyanohydrin catalyzed by hydroxynitrile
lyase may be selected appropriately according to the nature of
aldehyde or ketone to be used as a starting material for the
synthesis and the nature of cyanohydrin obtainable as a reaction
product.
[0059] Specific examples of an organic solvent substantially
immiscible with water include optionally halogenated hydrocarbon
solvents such as saturated or unsaturated linear, branched or
cyclic aliphatic hydrocarbons and aromatic hydrocarbons, for
example, pentane, hexane, cyclohexane, benzene, toluene, xylene,
methylene chloride and chloroform; optionally halogenated alcoholic
solvents such as saturated or unsaturated linear, branched or
cyclic aliphatic alcohols and aralkyl alcohols, for example,
n-butanol, isobutanol, t-butanol, hexanol, cyclohexanol and n-amyl
alcohol; optionally halogenated ether solvents such as saturated or
unsaturated linear, branched or cyclic aliphatic ethers and
aromatic ethers, for example, diethyl ether, dipropyl ether,
diisopropyl ether, dibutyl ether, t-butyl methyl ether and
dimethoxyethane; and optionally halogenated ester solvents such as
saturated or unsaturated linear, branched or cyclic aliphatic
esters and aromatic esters, for example, methyl formate, methyl
acetate, ethyl acetate, butyl acetate and methyl propionate. These
solvents may be used alone or in combination.
[0060] In some cases, an organic solvent merely saturated with
water or an aqueous buffer may increase its solubility in water
upon a substrate is dissolved in this solvent, resulting in the
loss of water from the immobilized enzyme. In such a case where a
substrate having the nature as mentioned above is used for the
reaction, the saturation treatment with water or an aqueous buffer
may be preferably carried out after the substrate is dissolved in
the organic solvent.
[0061] For example, in the synthesis of an optically active
cyanohydrin using prussic acid as a substrate, the saturation
treatment with water or an aqueous buffer may be preferably carried
out after prussic acid is dissolved in an organic solvent. That is,
prussic acid may be dissolved in the organic solvent at a given
concentration, which may then be mixed with a saturation amount of
water or an aqueous buffer. Alternatively, the organic solvent and
the substrate may be added to the reaction system before the
saturation treatment, followed by addition of water or an aqueous
buffer in a saturation amount which has been determined by
measuring an amount of water soluble in the organic solvent
containing the substrate dissolved therein. An aqueous buffer used
here may be any buffer adjusted to around an optimum pH of an
enzyme reaction, for example, a buffer prepared from a salt such as
phosphate, citrate, glutarate, malate, malonate, o-phthalate or
succinate.
[0062] By way of example, the construction of a reaction system for
synthesizing an optically active cyanohydrin will be presented
below. This reaction system uses t-butyl methyl ether as a reaction
solvent, 1 M benzaldehyde and 1.5 M prussic acid (hydrocyanic acid)
as substrates, and immobilized hydroxynitrile lyase as a catalyst.
The synthesis is carried out at a reaction temperature of about
20.degree. C.
[0063] 1. Preparation of Immobilized Enzyme
[0064] An immobilized enzyme having a water content of 10% by
weight or more is prepared by introducing carriers for
immobilization into an enzyme solution to immobilize enzyme
molecules on/in the carriers by absorption, or by mixing the
carriers with the enzyme solution in an amount equal to or less
than that of water which can be retained within the carriers. The
water content in this case may vary widely, so long as water can be
retained within the immobilized enzyme, that is, a liquid phase can
form a homogeneous system of organic solvent. It preferably ranges
from 10 to 60% by weight, more preferably 20 to 50% by weight. The
water content of the immobilized enzyme per unit (U) of enzyme
preferably ranges from 0.1 to 100 .mu.L/U, more preferably 1 to 50
.mu.L/U.
[0065] 2. Construction of Reaction System
[0066] A mixture of the solvent and prussic acid is saturated with
water or an aqueous buffer at a temperature of around the reaction
temperature. Since the saturation solubility of water under this
condition is about 2% by weight, it is desirable to incorporate
water or the aqueous buffer into the reaction system at least in
the same amount. On the other hand, the immobilized enzyme should
have a high water content enough to ensure its sufficient activity
and easy dispersion in the reaction solvent. When water is present
in an excess amount, the water content can be adjusted by reducing
the amount of water added to the reaction solvent. When the carrier
contains slightly less water, the water content can also be
adjusted by adding water or the aqueous buffer as such to the
reaction system containing the immobilized enzyme.
[0067] 3. Reaction
[0068] In a batch fashion, the immobilized enzyme should be
dispersed over the reaction system by stirring or the like. If the
immobilized enzyme is filled into a column or the like, the
reaction can be carried out by passing a solution containing the
substrates through the column at an appropriate flow rate, and then
collecting the effluent. In a batch reaction, the product can be
collected by stopping the mixing at the time when the reaction is
completed, precipitating the immobilized enzyme, and then removing
the organic phase containing the product dissolved therein in a
general manner. This immobilized enzyme may be re-used by mixing it
with a substrate-containing solution prepared in the same manner as
the first round.
[0069] A carbonyl compound and hydrogen cyanide are used as
substrates for the synthesis of an optically active cyanohydrin
which can be synthesized according to the first aspect of the
present invention.
[0070] As used herein, a carbonyl compound means aldehyde or
ketone, which is specifically represented by the following formula
(I):
R.sup.1--CO--R.sup.2 (I)
[0071] wherein
[0072] R.sup.1 and R.sup.2 are different from each other, each of
which is a hydrogen atom or a monovalent hydrocarbon group
containing at most 22 carbon atoms, in which each of --CH.sub.2--
and CH.sub.2 in --CH.sub.3 may be replaced by a carbonyl group, a
sulfonyl group, --O-- or --S--; .dbd.CH.sub.2 may be replaced by
.dbd.O or .dbd.S; or each C--H in --CH.sub.2--, --CH.sub.3,
>CH--, .dbd.CH-- and .dbd.CH.sub.2 may be replaced by N or a
C-halogen group; or R.sup.1 and R.sup.2 may together form an
asymmetric divalent group.
[0073] In the above formula (I), a monovalent hydrocarbon group
containing at most 22 carbon atoms includes a linear or branched
chain hydrocarbon group, a monocyclic hydrocarbon group with or
without side chain, a polycyclic hydrocarbon group with or without
side chain, a spiro hydrocarbon group with or without side chain, a
ring-assembled hydrocarbon group with or without side chain, or a
chain hydrocarbon group substituted with the above cyclic
hydrocarbon groups. It also includes both saturated and unsaturated
hydrocarbon groups, provided that unsaturated hydrocarbon groups
having an allene structure (C.dbd.C.dbd.C) are excluded. A linear
or branched chain hydrocarbon group includes, for example,
saturated chain hydrocarbon groups such as a linear alkyl group
containing at least one carbon atom and a branched alkyl group
containing at least 3 carbon atoms; and unsaturated chain
hydrocarbon groups such as a linear alkenyl group containing at
least 2 carbon atoms, a branched alkenyl group containing at least
3 carbon atoms, a linear alkynyl group containing at least 3 carbon
atoms, a branched alkynyl group containing at least 4 carbon atoms,
a linear alkadienyl group containing at least 4 carbon atoms and a
branched alkadienyl group containing at least 5 carbon atoms. A
monocyclic hydrocarbon group includes, for example, saturated
monocyclic hydrocarbon groups such as a cycloalkyl group without
side chain which contains at least 3 carbon atoms and a cycloalkyl
group with side chain which contains at least 4 carbon atoms in
total; and unsaturated monocyclic hydrocarbon groups such as a
cycloalkenyl group without side chain which contains at least 4
carbon atoms, a cycloalkynyl group with side chain which contains
at least 5 carbon atoms in total, a cycloalkadienyl group without
side chain which contains at least 5 carbon atoms and a
cycloalkadienyl group with side chain which contains at least 6
carbon atoms in total. An unsaturated monocyclic or polycyclic
hydrocarbon group includes aromatic hydrocarbon groups such as an
aromatic group without side chain which contains 6 to 22 carbon
atoms in total, for example, a phenyl group, a 1-naphthyl group, a
2-naphthyl group and a 9-anthryl group; an aromatic group with side
chain which contains at least 7 carbon atoms in total; and
furthermore ring-assembled hydrocarbon groups such as a
phenylphenyl group containing 12 carbon atoms and a phenylphenyl
group with side chain which contains at least 13 carbon atoms in
total. A polycyclic hydrocarbon group includes a condensed cyclic
hydrocarbon group without side chain which contains at least 6
carbon atoms, a condensed cyclic hydrocarbon group with side chain
which contains at least 7 carbon atoms in total, a bridged cyclic
hydrocarbon group without side chain which contains at least 7
carbon atoms, a bridged cyclic hydrocarbon group with side chain
which contains at least 8 carbon atoms in total, a spiro
hydrocarbon group without side chain which contains at least 9
carbon atoms in total and a spiro hydrocarbon group with side chain
which contains at least 10 carbon atoms in total. In addition, the
above condensed cyclic hydrocarbon group without side chain
includes those which contain at least 9 carbon atoms in total when
one of their condensed rings is a benzene ring, and the above
condensed cyclic hydrocarbon group with side chain includes those
which contain at least 10 carbon atoms in total when one of their
condensed rings is a benzene ring. A ring-assembled hydrocarbon
group includes a cycloalkyl-cycloalkyl group without side chain
which contains at least 6 carbon atoms in total, a
cycloalkyl-cycloalkyl group with side chain which contains at least
7 carbon atoms in total, a cycloalkylidene-cycloalkyl group without
side chain which contains at least 6 carbon atoms in total and a
cycloalkylidene-cycloalkyl group with side chain which contains at
least 7 carbon atoms in total. As used herein, a "cyclic
hydrocarbon with side chain" means a cyclic hydrocarbon having a
chain hydrocarbon group attached to its ring. Such a chain
hydrocarbon group attached to a cyclic hydrocarbon group includes a
linear alkyl group which is substituted with an aromatic group
without side chain and contains at least 7 carbon atoms in total, a
linear alkyl group which is substituted with an aromatic group with
side chain and contains at least 8 carbon atoms in total, a
branched alkyl group which is substituted with an aromatic group
without side chain and contains at least 9 carbon atoms in total, a
branched alkyl group which is substituted with an aromatic group
with side chain and contains at least 10 carbon atoms in total, a
linear alkenyl group which is substituted with an aromatic group
without side chain and contains at least 8 carbon atoms in total, a
linear alkenyl group which is substituted with an aromatic group
with side chain and contains at least 9 carbon atoms in total, a
branched alkenyl group which is substituted with an aromatic group
without side chain and contains at least 9 carbon atoms in total, a
branched alkenyl group which is substituted with an aromatic group
with side chain and contains at least 10 carbon atoms in total, a
linear alkynyl group which is substituted with an aromatic group
without side chain and contains at least 8 carbon atoms in total, a
linear alkynyl group which is substituted with an aromatic group
with side chain and contains at least 9 carbon atoms in total, a
branched alkynyl group which is substituted with an aromatic group
without side chain and contains at least 10 carbon atoms in total,
a branched alkynyl group which is substituted with an aromatic
group with side chain and contains at least 11 carbon atoms in
total, a linear alkadienyl group which is substituted with an
aromatic group without side chain and contains at least 10 carbon
atoms in total, a linear alkadienyl group which is substituted with
an aromatic group with side chain and contains at least 11 carbon
atoms in total, a branched alkadienyl group which is substituted
with an aromatic group without side chain and contains at least 11
carbon atoms in total, a branched alkadienyl group which is
substituted with an aromatic group with side chain and contains at
least 12 carbon atoms in total, a linear alkyl group which is
substituted with a cycloalkyl group without side chain and contains
at least 4 carbon atoms in total, a linear alkyl group which is
substituted with a cycloalkyl group with side chain and contains at
least 5 carbon atoms in total, a branched alkyl group which is
substituted with a cycloalkyl group without side chain and contains
at least 6 carbon atoms in total, a branched alkyl group which is
substituted with a cycloalkyl group with side chain and contains at
least 7 carbon atoms in total, a linear alkenyl group which is
substituted with a cycloalkyl group without side chain and contains
at least 5 carbon atoms in total, a linear alkenyl group which is
substituted with a cycloalkyl group with side chain and contains at
least 6 carbon atoms in total, a branched alkenyl group which is
substituted with a cycloalkyl group without side chain and contains
at least 6 carbon atoms in total, a branched alkenyl group which is
substituted with a cycloalkyl group with side chain and contains at
least 7 carbon atoms in total, a linear alkynyl group which is
substituted with a cycloalkyl group without side chain and contains
at least 5 carbon atoms in total, a linear alkynyl group which is
substituted with a cycloalkyl group with side chain and contains at
least 6 carbon atoms in total, a branched alkynyl group which is
substituted with a cycloalkyl group without side chain and contains
at least 7 carbon atoms in total, a branched alkynyl group which is
substituted with a cycloalkyl group with side chain and contains at
least 8 carbon atoms in total, a branched alkadienyl group which is
substituted with a cycloalkyl group without side chain and contains
at least 8 carbon atoms in total, a branched alkadienyl group which
is substituted with a cycloalkyl group with side chain and contains
at least 9 carbon atoms in total.
[0074] Hereinafter, an aromatic group with or without side chain
and a phenylphenyl group with or without side chain are
collectively referred to as aryl groups, and a linear or branched
alkyl group substituted with the aryl group is referred to as an
aralkyl group. Other cyclic hydrocarbon groups including both of
those having side chain on their ring and those having no side
chain are simply referred to as, for example, cycloalkyl groups,
unless otherwise specified. Further, chain hydrocarbon groups
including both linear and branched ones are also simply referred to
as, for example, alkyl groups.
[0075] In the above hydrocarbon group, when --CH.sub.2-- is
replaced by a carbonyl group, sulfonyl group, --O-- or --S--, a
ketone, sulfone, ether or thioether structure is introduced
thereinto, respectively. When --CH.sub.2-- in --CH.sub.3 is
replaced by a carbonyl group, --O-- or --S--, it converts into a
formyl(aldehyde) group, a hydroxyl group or a mercapto group,
respectively. When a terminal .dbd.CH.sub.2 is replaced by .dbd.O
or .dbd.S, a ketone or thioketone structure is introduced
thereinto, respectively. When C--H in --CH.sub.2-- is replaced by
N, it converts into --NH--. When C--H in >CH-- is replaced by N,
it converts into >N--. When C--H in .dbd.CH-- is replaced by N,
it converts into .dbd.N--. When C--H in a terminal --CH.sub.3 is
replaced by N, --NH.sub.2 is introduced thereinto. When C--H in
.dbd.CH.sub.2 is replaced by N, it converts into .dbd.NH. Further,
when C--H in --CH.sub.3, --CH.sub.2--, .dbd.CH--, .dbd.CH or
>CH-- is replaced by a C-halogen group, that carbon has a
halogen atom attached thereto. Furthermore, the replacement by
--O--, --S-- or N in a carbon chain corresponds to oxa-, thia- or
aza-substitution of the hydrocarbon group, respectively. For
example, when such a substitution takes place at a ring carbon of
the hydrocarbon ring, the hydrocarbon ring converts into a
heterocyclic ring containing oxygen, sulfur or nitrogen. The
replacement of CH.sub.2 and C--H in the hydrocarbon group may
independently take place and it may further take place when
CH.sub.2 or C--H still remains on the carbon after the previous
replacement. Furthermore, such a replacement may convert
--CH.sub.2--CH.sub.3 into --CO--O--H (carboxylic structure).
[0076] As used herein, a halogen atom refers to a fluorine atom, a
chlorine atom, a bromine atom and an iodine atom, with a fluorine
atom, a chlorine atom and a bromine atom being preferred.
[0077] Accordingly, the above hydrocarbon group may be selected
from chain hydrocarbon groups and ring-containing hydrocarbon
groups including cyclic hydrocarbon groups, for example, saturated
chain hydrocarbon groups such as a linear or branched alkyl group;
unsaturated chain hydrocarbon groups such as a linear or branched
alkenyl group, a linear or branched alkynyl group and a linear or
branched alkadienyl group; saturated cyclic hydrocarbon groups such
as a cycloalkyl group; unsaturated cyclic hydrocarbon groups such
as a cycloalkenyl group, a cycloalkynyl group and a cycloalkadienyl
group; and aromatic hydrocarbon groups such as an aryl group, an
aralkyl group and an arylalkenyl group.
[0078] In more detail, a linear or branched alkyl group includes a
methyl group, an ethyl group, a propyl group, an isopropyl group, a
butyl group, a 1-methylpropyl group, a pentyl group, a
1-methylbutyl group, a hexyl group, a 1-methylpentyl group, a
heptyl group, a 1-methylhexyl group, a 1-ethylpentyl group, a octyl
group, a nonyl group, a decyl group, an undecyl group, a dodecyl
group, a tridecyl group, a tetradecyl group, a 2-methylpropyl
group, a 2-methylbutyl group, a 3-methylbutyl group, a
2-methylpentyl group, a 3-methylpentyl group, a 4-methylpentyl
group, a methylhexyl group, a methylheptyl group, a methyloctyl
group, a methylnonyl group, a 1,1-dimethylethyl group, a
1,1-dimethylpropyl group, a 2,6-dimethylheptyl group, a
3,7-dimethyloctyl group and a 2-ethylhexyl group; a cycloalkylalkyl
group includes a cyclopentylmethyl group and a cyclohexylmethyl
group; a cycloalkyl group includes a cyclopropyl group, a
cyclobutyl group, a cyclopentyl group, a methylcyclopentyl group, a
cyclohexyl group, a methylcyclohexyl group, a cycloheptyl group and
a cyclooctyl group; and a bicycloalkyl group includes a norbornyl
group, a bicyclo[2.2.2]octyl group and an adamantyl group. A linear
or branched alkenyl group includes a vinyl group, an allyl group, a
crotyl(2-butenyl) group and an isopropenyl(1-methylvinyl) group; a
cycloalkenyl or cycloalkadienyl group includes a cyclopentenyl
group, a cyclopentadienyl group, a cyclohexenyl group and a
cyclohexadienyl group. A linear or branched alkynyl group includes
an ethynyl group, a propynyl group and a butynyl group. An aryl
group includes a phenyl group, a 1-naphthyl group, a 2-naphthyl
group, a 2-phenylphenyl group, a 3-phenylphenyl group, a
4-phenylphenyl group, a 9-anthryl group, a methylphenyl group, a
dimethylphenyl group, a trimethylphenyl group, an ethylphenyl
group, a methylethylphenyl group, a diethylphenyl group, a
propylphenyl group and a butylphenyl group. An aralkyl group
includes a benzyl group, a 1-naphthylmethyl group, a
2-naphthylmethyl group, a phenethyl(2-phenylethyl) group, a
1-phenylethyl group, a phenylpropyl group, a phenylbutyl group, a
phenylpentyl group, a phenylhexyl group, a methylbenzyl group, a
methylphenethyl group, a dimethylbenzyl group, a dimethylphenethyl
group, a trimethylbenzyl group, an ethylbenzyl group and a
diethylbenzyl group. An arylalkenyl group includes a styryl group,
a methylstyryl group, an ethylstyryl group, a dimethylstyryl group
and a 3-phenyl-2-propenyl group.
[0079] The hydrocarbon groups comprising the replacement of
CH.sub.2 by a carbonyl group, a sulfonyl group, O or S, or
comprising the replacement of C--H by N or a C-halogen group
include those having one or more structures such as ketone,
aldehyde, carboxylic acid, sulfone, ether, thioether, amine,
alcohol, thiol, halogen and heterocycles (e.g., oxygen-containing
heterocycle, sulfur-containing heterocycle, nitrogen-containing
heterocycle). As used herein, an oxygen-containing heterocycle, a
sulfur-containing heterocycle and a nitrogen-containing heterocycle
mean cyclic hydrocarbon groups whose ring carbon is replaced by
oxygen, sulfur and nitrogen, respectively. Further, these
heterocycles may contain two or more heteroatoms. Illustrative
examples of the hydrocarbon groups comprising the above
replacements include those having a ketone structure, such as an
acetylmethyl group and an acetylphenyl group; those having a
sulfone structure, such as a methanesulfonylmethyl group; those
having an ether structure, such as a methoxymethyl group, a
methoxyethyl group, an ethoxyethyl group, a methoxypropyl group, a
butoxyethyl group, an ethoxyethoxyethyl group, a methoxyphenyl
group, a dimethoxyphenyl group and a phenoxymethyl group; those
having a thioether structure, such as a methylthiomethyl group and
a methylthiophenyl group; those having an amine structure, such as
an aminomethyl group, a 2-aminoethyl group, a 2-aminopropyl group,
a 3-aminopropyl group, a 2,3-diaminopropyl group, a 2-aminobutyl
group, a 3-aminobutyl group, a 4-aminobutyl group, a
2,3-diaminobutyl group, a 2,4-diaminobutyl group, a
3,4-diaminobutyl group, a 2,3,4-triaminobutyl group, a
methylaminomethyl group, a dimethylaminomethyl group, a
methylaminoethyl group, a propylaminomethyl group, a
cyclopentylaminomethyl group, an aminophenyl group, a diaminophenyl
group, and an aminomethylphenyl group; those having an
oxygen-containing heterocycle, such as a tetrahydrofuranyl group, a
tetrahydropyranyl group and a morphorylethyl group; those having an
oxygen-containing aromatic heterocycle, such as a furyl group, a
furfuryl group, a benzofuryl group and a benzofurfuryl group; those
having a sulfur-containing aromatic heterocycle, such as a thienyl
group; those having a nitrogen-containing aromatic heterocycle,
such as a pyrrolyl group, an imidazolyl group, an oxazolyl group, a
thiadiazolyl group, a pyridyl group, a pyrimidinyl group, a
pyridazinyl group, a pyrazinyl group, a tetrazinyl group, a
quinolyl group, an isoquinolyl group and a pyridylmethyl group;
those having an alcohol structure, such as a 2-hydroxyethyl group,
a 2-hydroxypropyl group, a 3-hydroxypropyl group, a
2,3-dihydroxypropyl group, a 2-hydroxybutyl group, a 3-hydroxybutyl
group, a 4-hydroxybutyl group, a 2,3-dihydroxybutyl group, a
2,4-dihydroxybutyl group, a 3,4-dihydroxybutyl group, a
2,3,4-trihydroxybutyl group, a hydroxyphenyl group, a
dihydroxyphenyl group, a hydroxymethylphenyl group and a
hydroxyethylphenyl group; those having a thiol structure, such as a
2-mercaptoethyl group, a 2-mercaptopropyl group, a 3-mercaptopropyl
group, a 2,3-dimercaptopropyl group, a 2-mercaptobutyl group, a
3-mercaptobutyl group, a 4-mercaptobutyl group and a mercaptophenyl
group; those which are halogenated, such as a 2-chloroethyl group,
a 2-chloropropyl group, a 3-chloropropyl group, a 2-chlorobutyl
group, a 3-chlorobutyl group, a 4-chlorobutyl group, a fluorophenyl
group, a chlorophenyl group, a bromophenyl group, a difluorophenyl
group, a dichlorophenyl group, a dibromophenyl group, a
chlorofluorophenyl group, a trifluorophenyl group, a
trichlorophenyl group, a fluoromethylphenyl group and a
trifluoromethylphenyl group; those having an amine structure and an
alcohol structure, such as a 2-amino-3-hydroxypropyl group, a
3-amino-2-hydroxypropyl group, a 2-amino-3-hydroxybutyl group, a
3-amino-2-hydroxybutyl group, a 2-amino-4-hydroxybutyl group, a
4-amino-2-hydroxybutyl group, a 3-amino-4-hydroxybutyl group, a
4-amino-3-hydroxybutyl group, a 2,4-diamino-3-hydroxybutyl group, a
3-amino-2,4-dihydroxybutyl group, a 2,3-diamino-4-hydroxybutyl
group, a 4-amino-2,3-dihydroxybutyl group, a
3,4-diamino-2-hydroxybutyl group, a 2-amino-3,4-dihydroxybutyl
group and an aminohydroxyphenyl group; those which are substituted
with a halogen atom and a hydroxyl group, such as a
fluorohydroxyphenyl group and a chlorohydroxyphenyl group; and
those having a carboxylic structure, such as a carboxyphenyl
group.
[0080] An asymmetric divalent group mentioned under R.sup.1 and
R.sup.2 includes, but is not limited to, norbornan-2-ylidene and
2-norbornen-5-ylidene.
[0081] A carbonyl compound of the above formula (I) includes, for
example, an aromatic aldehyde such as benzaldehyde,
3-phenoxybenzaldehyde, 4-methylbenzaldehyde, 2-chlorobenzaldehyde,
3-chlorobenzaldehyde, 4-chlorobenzaldehyde, 3-nitrobenzaldehyde,
3,4-methylenedioxybenzaldehyde- , 2,3-methylenedioxybenzaldehyde,
phenylacetaldehyde and furfural; an aliphatic aldehyde such as
acetaldehyde, butylaldehyde, isobutylaldehyde, valeraldehyde and
cyclohexanealdehyde; a saturated aliphatic ketone such as ethyl
methyl ketone, butyl methyl ketone, methyl propyl ketone, isopropyl
methyl ketone, methyl pentyl ketone, methyl(2-methylpropyl)keto- ne
and methyl(3-methylbutyl)ketone; an unsaturated aliphatic ketone
such as methyl(2-propenyl)ketone and (3-butenyl)methyl ketone; an
alkyl(haloalkyl)ketone such as (3-chloropropyl)methyl ketone; a
2-(protected amino) aldehyde such as
2-(alkoxycarbonylamino)-3-cyclohexyl- propionaldehyde; and an
alkylthio aliphatic aldehyde such as
3-methylthiopropionaldehyde.
[0082] To convert the above aldehyde or ketone into an optically
active cyanohydrin, hydrogen cyanide is used as a starting
material, which may be supplied in liquid or gaseous form. In
addition to hydrogen cyanide, hydrocyanic acid, i.e., prussic acid,
which is an aqueous solution of hydrogen cyanide, may also be used
in exactly the same manner. Further, any substance which can
produce a cyanide ion (CN.sup.-) when added to the reaction system
may also be used, including salts of hydrocyanic acid (e.g., sodium
cyanide or potassium cyanide) and cyanohydrins (e.g., acetone
cyanohydrin).
[0083] The preparation of hydroxynitrile lyase enzyme used in the
first aspect of the present invention may be carried out by
extraction from plant tissues containing the enzyme or by
extraction from cultures of recombinant organisms into which a gene
of the enzyme has been introduced. The extraction may be
accomplished in a general manner. The resultant enzyme preparation
may be used without purification unless its components other than
hydroxynitrile lyase, if any, affect the enzyme reaction. The
hydroxynitrile lyase enzyme thus prepared is immobilized on/in
carriers to obtain the immobilized enzyme.
[0084] The water content of the immobilized enzyme may be
determined from the weight ratio between dried carriers before used
for immobilization and the immobilized enzyme prepared.
Alternatively, it may also be determined using a Karl Fischer
moisture meter or the like.
[0085] The amounts of the immobilized enzyme and substrate to be
used and a reaction temperature are determined appropriately
depending on the type of substrate to be used. In general, the
immobilized enzyme may be used in an amount of 1 to 1000 units,
preferably 10 to 500 units, relative to 50 mmol of the carbonyl
compound as a substrate. The substrate concentration may be
generally set between 0.1 and 10 mol/L when the carbonyl compound
is used. Hydrogen cyanide may be added at a concentration of 1- to
5-fold molar excess, preferably 1.1- to 3-fold molar excess,
relative to the carbonyl compound. Since the enzyme activity and
reaction rate in this reaction will vary depending on the substrate
concentration, the substrate concentration should be determined
appropriately according to the type of carbonyl compound to be
used. The reaction is preferably, but not always, continued until
the conversion ratio of carbonyl compound reaches 80% or more,
preferably 90% or more. The reaction may be carried out at any
temperature at which the enzyme sufficiently catalyzes the
reaction, generally at 0 to 40.degree. C., preferably at 4 to
30.degree. C.
[0086] The product produced in the first aspect of the present
invention, such as an optically active cyanohydrin, may be measured
and assayed by, for example, high performance liquid chromatography
(HPLC) and, if necessary, may be separated and purified by a
standard procedure such as extraction, distillation under reduced
pressure, or column separation. When the product is to be stored
for a long time, a stabilizing agent may be added thereto.
[0087] Any enzyme reaction which uses an aldehyde compound as a
substrate may be used in the second aspect of the present
invention. A preferred enzyme reaction uses both hydrogen cyanide
and an aldehyde compound as substrates.
[0088] In addition, any enzyme may be used in the second aspect of
the present invention without particular limitations, so long as it
can be used for the above enzyme reaction. Hydroxynitrile lyase may
be preferably used, which catalyzes the synthesis of an optically
active cyanohydrin from an aldehyde compound and hydrogen
cyanide.
[0089] As used herein, a "reaction inhibitor" means a substance
that inhibits the enzyme reaction so as to decrease its reaction
rate and/or so as to reduce the yield of a target product. In the
above enzyme reaction using an aldehyde compound as a substrate,
the reaction inhibitor includes, for example, a carboxylic acid
compound corresponding to the aldehyde compound.
[0090] In the second aspect, the present invention uses
hydroxynitrile lyase as described in connection with the first
aspect.
[0091] Any aldehyde compound may be used in the present invention
without particular limitations. A preferred aldehyde compound can
provide phase separation when mixed with water.
[0092] Specifically, it has the following formula (II):
R--CHO (II)
[0093] wherein
[0094] R is a monovalent hydrocarbon group containing at most 22
carbon atoms, in which each CH.sub.2 in --CH.sub.2-- and --CH.sub.3
may be replaced by a carbonyl group, a sulfonyl group, --O-- or
--S--; .dbd.CH.sub.2 may be replaced by .dbd.O or .dbd.S; or each
C--H in --CH.sub.2--, --CH.sub.3, >CH--, .dbd.CH-- and
.dbd.CH.sub.2 may be replaced by N or a C-halogen group.
[0095] In the above formula (II), a monovalent hydrocarbon group
containing at most 22 carbon atoms includes a linear or branched
chain hydrocarbon group, a monocyclic hydrocarbon group with or
without side chain, a polycyclic hydrocarbon group with or without
side chain, a spiro hydrocarbon group with or without side chain, a
ring-assembled hydrocarbon group with or without side chain, or a
chain hydrocarbon group substituted with the above cyclic
hydrocarbon groups. It also includes both saturated and unsaturated
hydrocarbon groups, provided that unsaturated hydrocarbon groups
having an allene structure (C.dbd.C.dbd.C) are excluded. A linear
or branched chain hydrocarbon group includes, for example,
saturated chain hydrocarbon groups such as a linear alkyl group
containing at least 2 carbon atoms and a branched alkyl group
containing at least 3 carbon atoms; and unsaturated chain
hydrocarbon groups such as a linear alkenyl group containing at
least 2 carbon atoms, a branched alkenyl group containing at least
3 carbon atoms, a linear alkynyl group containing at least 3 carbon
atoms, a branched alkynyl group containing at least 4 carbon atoms,
a linear alkadienyl group containing at least 4 carbon atoms and a
branched alkadienyl group containing at least 5 carbon atoms. A
monocyclic hydrocarbon group includes, for example, saturated
monocyclic hydrocarbon groups such as a cycloalkyl group without
side chain which contains at least 3 carbon atoms and a cycloalkyl
group with side chain which contains at least 4 carbon atoms in
total; and unsaturated monocyclic hydrocarbon groups such as a
cycloalkenyl group without side chain which contains at least 4
carbon atoms, a cycloalkynyl group with side chain which contains
at least 5 carbon atoms in total, a cycloalkadienyl group without
side chain which contains at least 5 carbon atoms and a
cycloalkadienyl group with side chain which contains at least 6
carbon atoms in total. An unsaturated monocyclic or polycyclic
hydrocarbon group includes aromatic hydrocarbon groups such as an
aromatic group without side chain which contains 6 to 22 carbon
atoms in total, for example, a phenyl group, a 1-naphthyl group, a
2-naphthyl group and a 9-anthryl group; an aromatic group with side
chain which contains at least 7 carbon atoms in total; and
furthermore ring-assembled hydrocarbon groups such as a
phenylphenyl group containing 12 carbon atoms and a phenylphenyl
group with side chain which contains at least 13 carbon atoms in
total. A polycyclic hydrocarbon group includes a condensed cyclic
hydrocarbon group without side chain which contains at least 6
carbon atoms, a condensed cyclic hydrocarbon group with side chain
which contains at least 7 carbon atoms in total, a bridged cyclic
hydrocarbon group without side chain which contains at least 7
carbon atoms, a bridged cyclic hydrocarbon group with side chain
which contains at least 8 carbon atoms in total, a spiro
hydrocarbon group without side chain which contains at least 9
carbon atoms in total and a spiro hydrocarbon group with side chain
which contains at least 10 carbon atoms in total. In addition, the
above condensed cyclic hydrocarbon group without side chain
includes those which contain at least 9 carbon atoms in total when
one of their condensed rings is a benzene ring, and the above
condensed cyclic hydrocarbon group with side chain includes those
which contain at least 10 carbon atoms in total when one of their
condensed rings is a benzene ring. A ring-assembled hydrocarbon
group includes a cycloalkyl-cycloalkyl group without side chain
which contains at least 6 carbon atoms in total, a
cycloalkyl-cycloalkyl group with side chain which contains at least
7 carbon atoms in total, a cycloalkylidene-cycloalkyl group without
side chain which contains at least 6 carbon atoms in total and a
cycloalkylidene-cycloalkyl group with side chain which contains at
least 7 carbon atoms in total. As used herein, a "cyclic
hydrocarbon with side chain" means a cyclic hydrocarbon having a
chain hydrocarbon group attached to its ring. Such a chain
hydrocarbon group attached to a cyclic hydrocarbon group includes a
linear alkyl group which is substituted with an aromatic group
without side chain and contains at least 7 carbon atoms in total, a
linear alkyl group which is substituted with an aromatic group with
side chain and contains at least 8 carbon atoms in total, a
branched alkyl group which is substituted with an aromatic group
without side chain and contains at least 9 carbon atoms in total, a
branched alkyl group which is substituted with an aromatic group
with side chain and contains at least 10 carbon atoms in total, a
linear alkenyl group which is substituted with an aromatic group
without side chain and contains at least 8 carbon atoms in total, a
linear alkenyl group which is substituted with an aromatic group
with side chain and contains at least 9 carbon atoms in total, a
branched alkenyl group which is substituted with an aromatic group
without side chain and contains at least 9 carbon atoms in total, a
branched alkenyl group which is substituted with an aromatic group
with side chain and contains at least 10 carbon atoms in total, a
linear alkynyl group which is substituted with an aromatic group
without side chain and contains at least 8 carbon atoms in total, a
linear alkynyl group which is substituted with an aromatic group
with side chain and contains at least 9 carbon atoms in total, a
branched alkynyl group which is substituted with an aromatic group
without side chain and contains at least 10 carbon atoms in total,
a branched alkynyl group which is substituted with an aromatic
group with side chain and contains at least 11 carbon atoms in
total, a linear alkadienyl group which is substituted with an
aromatic group without side chain and contains at least 10 carbon
atoms in total, a linear alkadienyl group which is substituted with
an aromatic group with side chain and contains at least 11 carbon
atoms in total, a branched alkadienyl group which is substituted
with an aromatic group without side chain and contains at least 11
carbon atoms in total, a branched alkadienyl group which is
substituted with an aromatic group with side chain and contains at
least 12 carbon atoms in total, a linear alkyl group which is
substituted with a cycloalkyl group without side chain and contains
at least 4 carbon atoms in total, a linear alkyl group which is
substituted with a cycloalkyl group with side chain and contains at
least 5 carbon atoms in total, a branched alkyl group which is
substituted with a cycloalkyl group without side chain and contains
at least 6 carbon atoms in total, a branched alkyl group which is
substituted with a cycloalkyl group with side chain and contains at
least 7 carbon atoms in total, a linear alkenyl group which is
substituted with a cycloalkyl group without side chain and contains
at least 5 carbon atoms in total, a linear alkenyl group which is
substituted with a cycloalkyl group with side chain and contains at
least 6 carbon atoms in total, a branched alkenyl group which is
substituted with a cycloalkyl group without side chain and contains
at least 6 carbon atoms in total, a branched alkenyl group which is
substituted with a cycloalkyl group with side chain and contains at
least 7 carbon atoms in total, a linear alkynyl group which is
substituted with a cycloalkyl group without side chain and contains
at least 5 carbon atoms in total, a linear alkynyl group which is
substituted with a cycloalkyl group with side chain and contains at
least 6 carbon atoms in total, a branched alkynyl group which is
substituted with a cycloalkyl group without side chain and contains
at least 7 carbon atoms in total, a branched alkynyl group which is
substituted with a cycloalkyl group with side chain and contains at
least 8 carbon atoms in total, a branched alkadienyl group which is
substituted with a cycloalkyl group without side chain and contains
at least 8 carbon atoms in total, a branched alkadienyl group which
is substituted with a cycloalkyl group with side chain and contains
at least 9 carbon atoms in total.
[0096] Hereinafter, an aromatic group with or without side chain
and a phenylphenyl group with or without side chain are
collectively referred to as aryl groups, and a linear or branched
alkyl group substituted with the aryl group is referred to as an
aralkyl group. Other cyclic hydrocarbon groups including both of
those having side chain on their ring and those having no side
chain are simply referred to as, for example, cycloalkyl groups,
unless otherwise specified. Further, chain hydrocarbon groups
including both linear and branched ones are also simply referred to
as, for example, alkyl groups.
[0097] In the above hydrocarbon group, when --CH.sub.2-- is
replaced by a carbonyl group, sulfonyl group, --O-- or --S--, a
ketone, sulfone, ether or thioether structure is introduced
thereinto, respectively. When --CH.sub.2-- in --CH.sub.3 is
replaced by a carbonyl group, --O-- or --S--, it converts into a
formyl(aldehyde) group, a hydroxyl group or a mercapto group,
respectively. When a terminal .dbd.CH.sub.2 is replaced by .dbd.O
or .dbd.S, a ketone or thioketone structure is introduced
thereinto, respectively. When C--H in --CH.sub.2-- is replaced by
N, it converts into --NH--. When C--H in >CH-- is replaced by N,
it converts into >N--. When C--H in .dbd.CH-- is replaced by N,
it converts into .dbd.N--. When C--H in a terminal --CH.sub.3 is
replaced by N, --NH.sub.2 is introduced thereinto. When C--H in
.dbd.CH.sub.2 is replaced by N, it converts into .dbd.NH. Further,
when C--H in --CH.sub.3, --CH.sub.2--, .dbd.CH--, .dbd.CH or
>CH-- is replaced by a C-halogen group, that carbon has a
halogen atom attached thereto. Furthermore, the replacement by
--O--, --S-- or N in a carbon chain corresponds to oxa-, thia- or
aza-substitution of the hydrocarbon group, respectively. For
example, when such a substitution takes place at a ring carbon of
the hydrocarbon ring, the hydrocarbon ring converts into a
heterocyclic ring containing oxygen, sulfur or nitrogen. The
replacement of CH.sub.2 and C--H in the hydrocarbon group may
independently take place and it may further take place when
CH.sub.2 or C--H still remains on the carbon after the previous
replacement.
[0098] As used herein, a halogen atom refers to a fluorine atom, a
chlorine atom, a bromine atom and an iodine atom, with a fluorine
atom, a chlorine atom and a bromine atom being preferred.
[0099] Accordingly, the above hydrocarbon group may be selected
from chain hydrocarbon groups and ring-containing hydrocarbon
groups including cyclic hydrocarbon groups, for example, saturated
chain hydrocarbon groups such as a linear or branched alkyl group;
unsaturated chain hydrocarbon groups such as a linear or branched
alkenyl group, a linear or branched alkynyl group and a linear or
branched alkadienyl group; saturated cyclic hydrocarbon groups such
as a cycloalkyl group; unsaturated cyclic hydrocarbon groups such
as a cycloalkenyl group, a cycloalkynyl group and a cycloalkadienyl
group; and aromatic hydrocarbon groups such as an aryl group, an
aralkyl group and an arylalkenyl group.
[0100] In more detail, a linear or branched alkyl group includes an
ethyl group, a propyl group, an isopropyl group, a butyl group, a
1-methylpropyl group, a pentyl group, a 1-methylbutyl group, a
hexyl group, a 1-methylpentyl group, a heptyl group, a
1-methylhexyl group, a 1-ethylpentyl group, a octyl group, a nonyl
group, a decyl group, an undecyl group, a dodecyl group, a tridecyl
group, a tetradecyl group, a 2-methylpropyl group, a 2-methylbutyl
group, a 3-methylbutyl group, a 2-methylpentyl group, a
3-methylpentyl group, a 4-methylpentyl group, a methylhexyl group,
a methylheptyl group, a methyloctyl group, a methylnonyl group, a
1,1-dimethylethyl group, a 1,1-dimethylpropyl group, a
2,6-dimethylheptyl group, a 3,7-dimethyloctyl group and a
2-ethylhexyl group; a cycloalkylalkyl group includes a
cyclopentylmethyl group and a cyclohexylmethyl group; a cycloalkyl
group includes a cyclopropyl group, a cyclobutyl group, a
cyclopentyl group, a methylcyclopentyl group, a cyclohexyl group, a
methylcyclohexyl group, a cycloheptyl group and a cyclooctyl group;
and a bicycloalkyl group includes a norbornyl group, a
bicyclo[2.2.2]octyl group and an adamantyl group. A linear or
branched alkenyl group includes a vinyl group, an allyl group, a
crotyl(2-butenyl) group and an isopropenyl(1-methylvinyl) group; a
cycloalkenyl or cycloalkadienyl group includes a cyclopentenyl
group, a cyclopentadienyl group, a cyclohexenyl group and a
cyclohexadienyl group. A linear or branched alkynyl group includes
an ethynyl group, a propynyl group and a butynyl group. An aryl
group includes a phenyl group, a 1-naphthyl group, a 2-naphthyl
group, a 2-phenylphenyl group, a 3-phenylphenyl group, a
4-phenylphenyl group, a 9-anthryl group, a methylphenyl group, a
dimethylphenyl group, a trimethylphenyl group, an ethylphenyl
group, a methylethylphenyl group, a diethylphenyl group, a
propylphenyl group and a butylphenyl group. An aralkyl group
includes a benzyl group, a 1-naphthylmethyl group, a
2-naphthylmethyl group, a phenethyl(2-phenylethyl) group, a
1-phenylethyl group, a phenylpropyl group, a phenylbutyl group, a
phenylpentyl group, a phenylhexyl group, a methylbenzyl group, a
methylphenethyl group, a dimethylbenzyl group, a dimethylphenethyl
group, a trimethylbenzyl group, an ethylbenzyl group and a
diethylbenzyl group. An arylalkenyl group includes a styryl group,
a methylstyryl group, an ethylstyryl group, a dimethylstyryl group
and a 3-phenyl-2-propenyl group.
[0101] The hydrocarbon groups comprising the replacement of
CH.sub.2 by a carbonyl group, a sulfonyl group, O or S, or
comprising the replacement of C--H by N or a C-halogen group
include those having one or more structures such as ketone,
aldehyde, sulfone, ether, thioether, amine, alcohol, thiol, halogen
and heterocycles (e.g., oxygen-containing heterocycle,
sulfur-containing heterocycle, nitrogen-containing heterocycle). As
used herein, an oxygen-containing heterocycle, a sulfur-containing
heterocycle and a nitrogen-containing heterocycle mean cyclic
hydrocarbon groups whose ring carbon is replaced by oxygen, sulfur
and nitrogen, respectively. Further, these heterocycles may contain
two or more heteroatoms. Illustrative examples of the hydrocarbon
groups comprising the above replacements include those having a
ketone structure, such as an acetylmethyl group and an acetylphenyl
group; those having a sulfone structure, such as a
methanesulfonylmethyl group; those having an ether structure, such
as a methoxymethyl group, a methoxyethyl group, an ethoxyethyl
group, a methoxypropyl group, a butoxyethyl group, an
ethoxyethoxyethyl group, a methoxyphenyl group, a dimethoxyphenyl
group and a phenoxymethyl group; those having a thioether
structure, such as a methylthiomethyl group and a methylthiophenyl
group; those having an amine structure, such as a 2-aminopropyl
group, a 3-aminopropyl group, a 2,3-diaminopropyl group, a
2-aminobutyl group, a 3-aminobutyl group, a 4-aminobutyl group, a
2,3-diaminobutyl group, a 2,4-diaminobutyl group, a
3,4-diaminobutyl group, a 2,3,4-triaminobutyl group, a
methylaminomethyl group, a dimethylaminomethyl group, a
methylaminoethyl group, a propylaminomethyl group, a
cyclopentylaminomethyl group, an aminophenyl group, a diaminophenyl
group, and an aminomethylphenyl group; those having an
oxygen-containing heterocycle, such as a tetrahydrofuranyl group, a
tetrahydropyranyl group and a morphorylethyl group; those having an
oxygen-containing aromatic heterocycle, such as a furyl group, a
furfuryl group, a benzofuryl group and a benzofurfuryl group; those
having a sulfur-containing aromatic heterocycle, such as a thienyl
group; those having a nitrogen-containing aromatic heterocycle,
such as a pyrrolyl group, an imidazolyl group, an oxazolyl group, a
thiadiazolyl group, a pyridyl group, a pyrimidinyl group, a
pyridazinyl group, a pyrazinyl group, a tetrazinyl group, a
quinolyl group, an isoquinolyl group and a pyridylmethyl group;
those having an alcohol structure, such as a 2-hydroxypropyl group,
a 3-hydroxypropyl group, a 2,3-dihydroxypropyl group, a
2-hydroxybutyl group, a 3-hydroxybutyl group, a 4-hydroxybutyl
group, a 2,3-dihydroxybutyl group, a 2,4-dihydroxybutyl group, a
3,4-dihydroxybutyl group, a 2,3,4-trihydroxybutyl group, a
hydroxyphenyl group, a dihydroxyphenyl group, a hydroxymethylphenyl
group and a hydroxyethylphenyl group; those having a thiol
structure, such as a 2-mercaptoethyl group, a 2-mercaptopropyl
group, a 3-mercaptopropyl group, a 2,3-dimercaptopropyl group, a
2-mercaptobutyl group, a 3-mercaptobutyl group, a 4-mercaptobutyl
group and a mercaptophenyl group; those which are halogenated, such
as a 2-chloroethyl group, a 2-chloropropyl group, a 3-chloropropyl
group, a 2-chlorobutyl group, a 3-chlorobutyl group, a
4-chlorobutyl group, a fluorophenyl group, a chlorophenyl group, a
bromophenyl group, a difluorophenyl group, a dichlorophenyl group,
a dibromophenyl group, a chlorofluorophenyl group, a
trifluorophenyl group, a trichlorophenyl group, a
fluoromethylphenyl group and a trifluoromethylphenyl group; those
having an amine structure and an alcohol structure, such as a
2-amino-3-hydroxypropyl group, a 3-amino-2-hydroxypropyl group, a
2-amino-3-hydroxybutyl group, a 3-amino-2-hydroxybutyl group, a
2-amino-4-hydroxybutyl group, a 4-amino-2-hydroxybutyl group, a
3-amino-4-hydroxybutyl group, a 4-amino-3-hydroxybutyl group, a
2,4-diamino-3-hydroxybutyl group, a 3-amino-2,4-dihydroxybutyl
group, a 2,3-diamino-4-hydroxybutyl group, a
4-amino-2,3-dihydroxybutyl group, a 3,4-diamino-2-hydroxybutyl
group, a 2-amino-3,4-dihydroxybutyl group and an aminohydroxyphenyl
group; and those which are substituted with a halogen atom and a
hydroxyl group, such as a fluorohydroxyphenyl group and a
chlorohydroxyphenyl group.
[0102] An aldehyde compound of the above formula (II) includes, for
example, an aromatic aldehyde such as benzaldehyde,
3-phenoxybenzaldehyde, 4-methylbenzaldehyde, 2-chlorobenzaldehyde,
3-chlorobenzaldehyde, 4-chlorobenzaldehyde, 3-nitrobenzaldehyde,
3,4-methylenedioxybenzaldehyde, 2,3-methylenedioxybenzaldehyde,
phenylacetaldehyde and furfural; an aliphatic aldehyde such as
butylaldehyde, isobutylaldehyde, valeraldehyde and
cyclohexanealdehyde; a 2-(protected amino) aldehyde such as
2-(alkoxycarbonylamino)-3-cyclohexyl- propionaldehyde; and an
alkylthio aliphatic aldehyde such as
3-methylthiopropionaldehyde.
[0103] To convert the above aldehyde compound into an optically
active cyanohydrin, hydrogen cyanide is used as a starting
material, which may be supplied in liquid or gaseous form in a
general manner. In addition to hydrogen cyanide, hydrocyanic acid,
which is an aqueous solution of hydrogen cyanide (i.e., aqueous
hydrogen cyanide), may also be used in exactly the same manner.
Further, any substance which can produce a cyanide ion (CN.sup.-)
when added to the reaction system may also be used, including salts
of hydrocyanic acid (e.g., sodium cyanide or potassium cyanide) or
cyanohydrins (e.g., acetone cyanohydrin).
[0104] In the second aspect of the present invention, there is no
particular limitation on the purity of the aldehyde compound to be
used as a starting material. An aldehyde compound containing a
carboxylic acid compound may be preferably used in the second
aspect of the present invention because it inhibits enzyme activity
when used as such for the enzyme reaction. In particular, in a case
where the enzyme reaction uses a starting aldehyde compound at a
high concentration of 1 M or more, it is desirable to apply the
second aspect of the present invention to the enzyme reaction
because such a concentrated aldehyde compound containing at least
0.1 wt % of a carboxylic acid compound causes a significant
inhibition of the enzyme reaction.
[0105] As used herein, an alkaline treatment means a procedure
which comprises mixing the aldehyde compound with an alkaline
aqueous solution and then separating the aqueous phase from the
aldehyde phase in a general manner. The aldehyde compound thus
alkaline-treated may be used for the enzyme reaction with or
without further general purification.
[0106] Any alkali which can give an alkaline aqueous solution when
dissolved in water may be used for the alkaline treatment in the
second aspect of the present invention. Illustrative examples
include, but are not limited to, inorganic base compounds such as
sodium hydroxide, potassium hydroxide, calcium hydroxide or ammonia
and organic base compounds such as amino compounds.
[0107] There is no particular limitation on the concentration of
the alkaline aqueous solution, but it preferably ranges from 0.001
N to 10 N, more preferably about 0.01 N to 5N. Alternatively, the
concentration of the alkaline aqueous solution may vary widely as
long as the alkaline-treated aldehyde compound, when added as is,
to the enzyme reaction mixture, does not provide a pH at which
there would be a loss of enzyme activity.
[0108] The amount of the alkaline aqueous solution to be added may
be an amount which permits phase separation between aqueous and
aldehyde phases when the alkaline aqueous solution is mixed with
the aldehyde compound, or may be determined appropriately depending
on the content of a reaction inhibitor such as a carboxylic acid
compound present in the aldehyde compound. Further, the alkaline
treatment may be carried out once or plural times until a reaction
inhibitor such as a carboxylic acid compound reaches a desired
concentration or below.
[0109] The above alkaline-treated aldehyde compound is almost free
from reaction inhibitors. Among reaction inhibitors, in particular,
a carboxylic acid compound corresponding to the aldehyde compound
can be almost completely removed, thereby resulting in a content
reduced to 0.1 wt % or less, preferably 0.05 wt % or less. Such an
alkaline-treated aldehyde compound may be used for the enzyme
reaction in which an aldehyde compound is used as a substrate.
[0110] The above alkaline treatment given to a starting aldehyde
compound achieves effective removal of reaction inhibitors
contained in the aldehyde compound, in particular, a carboxylic
acid compound corresponding to the aldehyde compound. Hence, the
use of such an alkaline-treated aldehyde compound can prevent the
enzyme reaction from being inhibited by reaction inhibitors such as
the above carboxylic acid compound, resulting in a significantly
improved yield of a target product.
[0111] In the third aspect, the present invention uses
hydroxynitrile lyase as described in connection with the first
aspect.
[0112] In the third aspect of the present invention, the above
enzyme may be used in any form such as powder, liquid, or
immobilized form on/in a suitable carrier. Various carriers may be
used for immobilizing the enzyme, for example, a porous inorganic
carrier, a fibrous carrier such as cellulose, or a carrier made of
a polymer compound(s). Specific examples include, but are not
limited to, porous ceramic particles, porous silica gel particles,
zeolite particles, natural polymer gels such as agar, calcium
alginate and chitosan, and synthetic polymer gels such as
polyacrylic acid, polyacrylamide and polyvinyl alcohol. Enzyme
molecules may be immobilized in any manner, for example, by
allowing carriers to absorb an enzyme solution, by mixing carriers
with an enzyme solution to immobilize enzyme molecules on/in the
carriers by absorption, by entrapping and immobilizing enzyme
molecules within carriers, or by cross-linking enzyme molecules via
crosslinkers.
[0113] In the third aspect of the present invention, a carbonyl
compound and prussic acid (hydrocyanic acid) are used as starting
materials for the production of an optically active
cyanohydrin.
[0114] As used herein, a carbonyl compound means aldehyde or
ketone, for example, those are specifically represented by formula
(I) mentioned above.
[0115] Prussic acid containing an acidic substance as a stabilizer
is used in the third aspect of the present invention. As used
herein, a stabilizer means an acidic substance, such as sulfurous
acid and sulfuric acid, which is added to prussic acid produced in
bulk in order to prevent prussic acid from being denatured by
polymerization etc. and in order to stabilize product quality.
[0116] Prussic acid used in the third aspect of the present
invention, which contains an acidic substance as a stabilizer,
means prussic acid that provides an aqueous phase with pH 5 or less
when dissolved at a concentration of 1.5 M in an organic solvent
substantially immiscible with water, mixed with pure water at such
a ratio that the mixture separates into organic and aqueous phases,
and then allowed to stand. Preferably, prussic acid which provides
an aqueous phase with pH 4 or less is applied in the method of the
third aspect of the present invention.
[0117] In the third aspect of the present invention, the inhibitory
effect on an enzyme caused by the stabilizer contained in prussic
acid may be reduced, for example, by dissolving prussic acid
containing an acidic substance as a stabilizer 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, and then collecting the organic
phase, which is used for the reaction; or by adjusting that prussic
acid between pH 5 and pH 6 by addition of an alkaline aqueous
solution or an aqueous buffer having buffering ability in a range
of pH 4 to pH 7.
[0118] By way of example, a procedure for reducing the above
stabilizer's inhibitory effect on an enzyme will be presented
below.
[0119] 1. Dissolve a given amount of prussic acid containing the
stabilizer into an organic solvent substantially immiscible with
water (optionally pre-saturated with water or an aqueous
buffer).
[0120] 2. Add a buffer in an excess amount greater than the amount
soluble in the above solution, mix and allow to stand.
[0121] 3. Collect the organic phase separated from the aqueous
phase. Use in the reaction.
[0122] Such a very simple procedure as mentioned above achieves
effective removal of an adverse effect caused by the stabilizer
contained in an industrially produced prussic acid.
[0123] Hydrogen cyanide may be supplied in liquid or gaseous
form.
[0124] A buffer used in the procedure for reducing the stabilizer's
inhibitory effect on an enzyme means a buffer exhibiting buffering
ability around the optimum pH for enzyme activity. Specific
examples include citrate, glutarate, malate, malonate, o-phthalate
and succinate buffers. In general, a buffer having a pH of 4 to 7,
preferably 5 to 7, is used. The buffer preferably has a
concentration sufficient to keep the aqueous phase at pH 5 to pH 7
after mixing with the organic solvent containing a given amount of
prussic acid dissolved therein.
[0125] In the third aspect of the present invention, an organic
solvent substantially immiscible with water is used as a reaction
solvent in order to improve the concentration of starting materials
and productivity. As used herein, an "organic solvent substantially
immiscible with water" means an organic solvent except for those
soluble in water in any proportion. Any solvent may be used as an
organic solvent without particular limitations, so long as it is
substantially immiscible with water, enables a substrate and a
product to be sufficiently dissolved therein and has no influence
upon an enzyme reaction. Such a solvent may be selected
appropriately according to the nature of aldehyde or ketone to be
used as a starting material for the synthesis, and the nature of
cyanohydrin obtainable as a reaction product.
[0126] Specific examples of an organic solvent substantially
immiscible with water include optionally halogenated hydrocarbon
solvents such as saturated or unsaturated linear, branched or
cyclic aliphatic hydrocarbons and aromatic hydrocarbons, for
example, pentane, hexane, cyclohexane, benzene, toluene, xylene,
methylene chloride and chloroform; optionally halogenated alcoholic
solvents such as saturated or unsaturated linear, branched or
cyclic aliphatic alcohols and aralkyl alcohols, for example,
n-butanol, isobutanol, t-butanol, hexanol, cyclohexanol and n-amyl
alcohol; optionally halogenated ether solvents such as saturated or
unsaturated linear, branched or cyclic aliphatic ethers and
aromatic ethers, for example, diethyl ether, dipropyl ether,
diisopropyl ether, dibutyl ether, t-butyl methyl ether and
dimethoxyethane; and optionally halogenated ester solvents such as
saturated or unsaturated linear, branched or cyclic aliphatic
esters and aromatic esters, for example, methyl formate, methyl
acetate, ethyl acetate, butyl acetate and methyl propionate. These
solvents may be used alone or in combination.
[0127] The organic solvent mentioned above may be saturated with
water or an aqueous buffer. Alternatively, water or an aqueous
buffer may be added in an excess amount to the organic solvent to
give a two-phase system comprising organic and aqueous phases. The
saturation of the organic solvent with water or an aqueous buffer
may be accomplished in any manner, for example, by mixing the
organic solvent with water or an aqueous buffer at such a ratio
that the mixture separates into two phases, followed by stirring
for a while and allowing to stand, to collect the organic phase,
which is then used for the reaction. Any aqueous buffer may be used
here, including the buffers mentioned above.
[0128] In a case where an industrially produced prussic acid
containing a stabilizer is used for the synthesis of an optically
active cyanohydrin, the third aspect of the present invention
provides a method for reducing an adverse effect on an enzyme
caused by the stabilizer contained in that prussic acid. The method
of the present invention does not depend on the intended form of
prussic acid. That is, prussic acid subjected to the method of the
third aspect of the present invention to reduce an adverse effect
of the stabilizer may be effectively used in any reaction system,
including a mixed system comprising water and an organic solvent,
an organic solvent system, a two-phase system comprising water and
an organic solvent, and a system using an immobilized enzyme.
[0129] The amounts of the immobilized enzyme and substrate to be
used and a reaction temperature are determined appropriately
depending on the type of substrate to be used. In general, the
immobilized enzyme may be used in an amount of 1 to 1000 units,
preferably 10 to 500 units, relative to 50 mmol of the carbonyl
compound as a substrate. The substrate concentration may be
generally set between 0.1 and 10 mol/L when the carbonyl compound
is used. Hydrogen cyanide may be added at a concentration of 1- to
5-fold molar excess, preferably 1.1- to 3-fold molar excess,
relative to the carbonyl compound. Since the enzyme activity and
reaction rate in this reaction will vary depending on the substrate
concentration, the substrate concentration should be determined
appropriately according to the type of carbonyl compound to be
used. The reaction is preferably, but not always, continued until
the conversion ratio of carbonyl compound reaches 80% or more,
preferably 90% or more. The reaction may be carried out at any
temperature at which the enzyme sufficiently catalyzes the
reaction, generally at 0 to 40.degree. C., preferably at 4 to
30.degree. C.
[0130] The optically active cyanohydrin produced in the third
aspect of the present invention may be measured and assayed by, for
example, high performance liquid chromatography (HPLC) and, if
necessary, may be separated and purified by a standard procedure
such as extraction, distillation under reduced pressure, or column
separation. When the product is to be stored for a long time, a
stabilizing agent may be added thereto.
[0131] In the fourth aspect of the present invention, a reaction
solvent and unreacted prussic acid may be collected by distillation
from a reaction solution containing an optically active cyanohydrin
after completion of an enzyme reaction. The fourth aspect of the
present invention can be applied in a case where a reaction solvent
has a lower boiling point than that of the cyanohydrin. The
distillation is preferably carried out at a relatively low
temperature and under reduced pressure, rather than at an elevated
temperature and under normal pressure, because an optically active
cyanohydrin is unstable at an elevated temperature.
[0132] Reduced pressure and temperature to be applied may be
determined appropriately according to the type of organic solvent
to be used. Generally, in a case where a solvent to be used has a
boiling point of about 30 to 100.degree. C., such as t-butyl methyl
ether or diisopropyl ether, distillation temperature and reduced
pressure may be preferably, but not always, set at 20 to 70.degree.
C., more preferably 20 to 60.degree. C., and at 1 to 600 torr,
preferably 5 to 400 torr, respectively. The distilled solvent and
prussic acid may be effectively collected, for example, by using a
condenser cooled to 10.degree. C. or below. In this process, water
contained in a reaction solution may also be azeotroped therefrom.
To recycle a reaction solvent and prussic acid, the aqueous phase
is separated off after the above distillation process and the
organic phase is collected for use in the next reaction. The
collected organic phase contains the greater part of prussic acid
contained in a reaction mixture.
[0133] In this distillation process, a stabilizer for an optically
active cyanohydrin may be added to a reaction solution containing
an optically active cyanohydrin collected after completion of an
enzyme reaction. Any stabilizer capable of keeping the above
reaction solution at an acidic pH may be used. For example, an
organic acid (e.g., p-toluenesulfonic acid and acetic acid) or an
inorganic acid (e.g., sulfuric acid) may be added in an amount of
1/200 to 1/10 mol per mol of cyanohydrin.
[0134] The collected organic solvent containing unreacted prussic
acid may then be used as a solvent for the next enzyme
reaction.
[0135] The first to fourth aspects of the present invention may be
performed in combination, if necessary.
[0136] According to the first aspect, the present invention can
provide highly efficient and stable synthesis of a target
product.
[0137] According to the second aspect of the present invention,
even an aldehyde altered to the extent that it affects enzyme
activity when used in an enzyme reaction can be almost completely
freed from reaction inhibitors present in the aldehyde by means of
a very simple alkaline treatment. Such an alkaline-treated aldehyde
can be used in an enzyme reaction as a starting material equivalent
to an unaltered aldehyde, resulting in a significantly improved
yield of a target product.
[0138] According to the third aspect, the present invention can
prevent an enzyme (e.g., hydroxynitrile lyase) from losing its
activity and can provide a significantly extended life-time for the
enzyme. Accordingly, the present invention achieves industrially
stable and low-cost production of an optically active cyanohydrin
using an industrially produced prussic acid containing a
stabilizer.
[0139] According to the fourth aspect, the present invention
achieves recycling of a reaction solvent and unreacted prussic acid
in a method for producing an optically active cyanohydrin.
[0140] This specification includes part or all of the contents as
disclosed in the specifications of Japanese Patent Applications
Nos. 2000-166578, 2000-166579 and 2000-206130, which are the bases
of the priority claim of the present application.
BRIEF DESCRIPTION OF THE DRAWINGS
[0141] FIG. 1 shows the relationship between water content and
reaction efficiency of the immobilized enzyme.
[0142] FIG. 2 shows the relationship between the conversion ratio
of 2-chlorobenzaldehyde (starting material) and the concentration
of 2-chlorobenzoic acid present in 2-chlorobenzaldehyde.
[0143] FIG. 3 shows the relationship between the conversion ratio
of aldehyde and the number of times a reaction was repeated.
Reference signs found in FIG. 3 have the following meanings:
[0144] .circle-solid. Example 8
[0145] .largecircle. Comparative Example 1
[0146] FIG. 4 shows the relationships between the number of
reaction batches and the conversion ratio of aldehyde and the
enantiomer excess. Reference signs found in FIG. 4 have the
following meanings:
[0147] .circle-solid. Conversion ratio of aldehyde
[0148] .tangle-solidup. Enantiomer excess
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0149] The present invention will be further described in the
following examples. The examples are provided for illustrative
purposes only, and are not intended to limit the scope of the
invention.
PREPARATION EXAMPLE 1
Preparation of (R)-hydroxynitrile Lyase
[0150] (1) 100 g of ground almond (Prunus amygdalus) seeds was
mixed and stirred with 200 ml acetone for 2 hours, and then
filtrated to collect the solids. After drying, the solids were
mixed with 600 g of water and adjusted to pH 7.5 with aqueous
ammonia, followed by overnight mixing with stirring. Subsequently,
the resulting slurry was centrifuged to collect the supernatant,
which was then adjusted to pH 5.5 and centrifuged to give a
solution free from insoluble components.
[0151] (2) The (R)-hydroxynitrile lyase enzyme solution prepared in
(1) above was assayed for its activity. The production rate of
benzaldehyde decomposed from DL-mandelonitrile (substrate) by the
action of the enzyme was measured as a change in absorbance at
249.6 nm to calculate enzyme activity. An enzyme activity at which
1 .mu.mol of benzaldehyde is produced per minute is defined as 1
unit (U). The enzyme solution prepared in (1) above was assayed in
this manner, indicating that 25,000 units of enzyme having an
activity of 60.57 U/ml can be collected.
PREPARATION EXAMPLE 2
Preparation of (S)-hydroxynitrile Lyase
[0152] (1) (S)-hydroxynitrile lyase was prepared using yeast,
Saccharomyces cerevisiae, as a host by introducing the
(S)-hydroxynitrile lyase gene cloned from Manihot esculenta into
the yeast and then culturing the resulting recombinant yeast. The
recombinant yeast was cultured in 1 L of YPD medium containing 1%
yeast extract, 2% peptone and 2% glucose for 24 hours to collect
the yeast cells, which were then homogenized and purified to give a
solution free from insoluble components.
[0153] (2) The (S)-hydroxynitrile lyase enzyme solution prepared in
(1) above was assayed in the same manner as described in
Preparation Example 1 (2), indicating that 9,000 units of enzyme
having an activity of 40 U/ml can be collected.
PREPARATION EXAMPLE 3
Preparation of Immobilized (R)-hydroxynitrile Lyase
[0154] The (R)-hydroxynitrile lyase enzyme solution prepared in
Preparation Example 1 was subjected to ammonium sulfate
precipitation to concentrate the enzyme, thereby providing a 1000
U/ml enzyme solution. Carriers for immobilization (porous silica
gel; microbead silicagel 300A, Fuji Silysia Chemical Ltd.) were
mixed with this enzyme solution in an amount of 1 g per ml of
enzyme solution. This mixture was used as such for the synthesis
reaction. This immobilized enzyme had a water content of 50% by
weight.
PREPARATION EXAMPLE 4
Preparation of Immobilized (S)-hydroxynitrile Lyase
[0155] The (S)-hydroxynitrile lyase enzyme solution prepared in
Preparation Example 2 was mixed with the same carriers as used in
Preparation Example 3 in an amount of 300 units per g of carrier,
and then gently mixed overnight. Next, the resulting immobilized
enzyme was collected by filtration and then used for the subsequent
reaction. The immobilized enzyme thus prepared was measured with a
Karl Fischer moisture meter, indicating that it had a water content
of 50% by weight.
EXAMPLE 1
Synthesis of (R)-cyanohydrin
[0156] 6 ml of 1000 U/ml (R)-hydroxynitrile lyase enzyme solution
prepared from almond (Prunus amygdalus) was mixed with 6 g of
porous silica gel (microbead silicagel 300A, Fuji Silysia Chemical
Ltd.) to prepare an immobilized enzyme. In order to examine the
relationship between water content and reaction efficiency, an
immobilized enzyme prepared in the same manner was dried under
reduced pressure for 3 hours using an evaporator, followed by
addition of 10 mM phosphate buffer (pH 5.5), to give an immobilized
enzyme having an adjusted water content. In the case of Condition 4
shown in Table 1, the enzyme solution was diluted two-fold with 10
mM phosphate buffer (pH 5.5) and then immobilized on twice as much
carrier as used under other conditions.
[0157] Untreated t-butyl methyl ether (TBME) used for the
examination had a water content of 0.03% by weight, while TBME
saturated with 10 mM phosphate buffer (pH 5.5) had a water content
of 1.34% by weight. Various conditions were set up using this
solvent non-saturated or saturated with the buffer to give reaction
systems of varying water contents.
[0158] Under each condition, the concentrations of the substrates,
2-chlorobenzaldehyde (o-chlorobenzaldehyde) and prussic acid, were
set at 1 M and 1.5 M, respectively. 600 units of each immobilized
enzyme prepared above was introduced into a 10 ml reaction bottle,
and then mixed with 4.143 ml of the solvent and 0.292 ml of prussic
acid. 0.565 ml of 2-chlorobenzaldehyde was added to the reaction
bottle to start the reaction. The reaction was carried out at
25.degree. C. while gently stirring the reaction bottle on a bottle
roller. The conditions and results are shown in Table 1.
1TABLE 1 Water content of Water content of Conversion ratio
immobilized reaction system* of aldehyde after Condition enzyme (wt
%) Reaction solvent (v/v %) 5 hours (mol %) 1 0.76 Untreated TBME
0.96 6.3 2 0.76 Buffer-saturated 1.86 6.7 TBME 3 50
Buffer-saturated 12.8 95.3 TBME 4 50 Buffer-saturated 24.8 96.8
TBME 1 * Water present in reaction system ( vol ) Reaction solution
( organic phase ) ( vol ) .times. 100
[0159] These results indicated that a high reaction efficiency is
achieved in the reaction system containing excess water as compared
with the saturation amount of water in the solvent, whereas the
solvent merely saturated with the buffer provides a low reaction
efficiency. In Conditions 3 and 4, the liquid phase was kept as a
single phase of organic solvent without two-phase separation, even
though water was given in an amount far in excess of that which was
soluble in the reaction solvent.
EXAMPLE 2
[0160] Since the carrier used in Preparation Example 1 could retain
as much water as its weight, the enzyme was used at the same units
and the carrier was used in different amounts to give reaction
systems of varying water contents in order to examine the
relationship between water content and reaction efficiency. The
immobilized enzyme prepared as described in Preparation Example 3
was dried under reduced pressure, followed by addition of 10 mM
phosphate buffer (pH 5.5), to give an immobilized enzyme having an
adjusted water content for examination of the relationship between
water content and reaction efficiency.
[0161] 600 units of (R)-hydroxynitrile lyase was used for
immobilization to prepare immobilized enzymes of varying water
contents. On the other hand, prussic acid was dissolved in t-butyl
methyl ether, followed by addition of 10 mM phosphate buffer (pH
5.5) in a saturation amount. Subsequently, each immobilized enzyme
was mixed with the above starting material solution, followed by
addition of 2-chlorobenzaldehyde. In this reaction system, the
organic phase volume was 5 ml, and the concentrations of prussic
acid and 2-chlorobenzaldehyde were 1.5 M and 1 M, respectively. The
reaction was continued at 25.degree. C. while gently mixing the
reaction mixture. After 5 hours, the conversion ratio of
2-chlorobenzaldehyde into (R)-2-chloromandelonitrile was measured,
thereby providing the result shown in FIG. 1. This result indicated
that the reaction efficiency increases with increase in water
content of the immobilized enzyme.
EXAMPLE 3
[0162] A solution of prussic acid (41 g) in t-butyl methyl ether
(610 g) was mixed and stirred with 40 ml of 0.2 M citrate buffer
(pH 7), and then allowed to stand, followed by collection of the
organic phase. The immobilized (S)-hydroxynitrile lyase prepared in
Preparation Example 4 (60,000 units) was added to the organic
phase. At this time, the immobilized enzyme had a water content of
50% by weight. After addition of benzaldehyde (106 g), the reaction
mixture was stirred to start the reaction. Reaction temperature was
set at 20.degree. C. The reaction mixture was assayed by HPLC,
indicating that 126 g of (S)-mandelonitrile was produced after one
hour. At this time, the conversion ratio of aldehyde was 93% and
(S)-mandelonitrile had an optical purity of 99% ee. After
completion of the reaction, the immobilized enzyme was recovered
and repeatedly used for the reaction under the same conditions.
When the reaction was repeated 16 times, (S)-mandelonitrile having
an optical purity of 99% ee or more could be synthesized at an
average conversion ratio of 94.2%. This indicated that the use of
an immobilized enzyme having a sufficient water content achieves a
highly efficient and stable synthesis of an optically active
cyanohydrin.
EXAMPLE 4
Effects on an Enzyme Reaction of Reaction Inhibitors Contained in
Aldehyde Compound
[0163] 2-chlorobenzaldehyde containing undetectable 2-chlorobenzoic
acid (reaction inhibitor) was dissolved in t-butyl methyl ether
saturated with 0.15 M citrate buffer (pH 5.5) to prepare a 1 M
solution of 2-chlorobenzaldehyde. To this solution, 2-chlorobenzoic
acid was added in different amounts to prepare aldehyde solutions
of varying 2-chlorobenzoic acid contents.
[0164] Immobilized (R)-hydroxynitrile lyase (600 units) was
introduced into 5 ml of each solution, and prussic acid was then
added to reach 1.5 M, followed by synthesis of
(R)-2-chloromandelonitrile. Five hours after initiation of the
reaction, an aliquot of the reaction mixture was sampled to measure
the conversion ratio of aldehyde by HPLC. FIG. 2 shows the reaction
rate of the reaction with the added 2-chlorobenzoic acid relative
to the reaction without the added 2-chlorobenzoic acid, which is
plotted against the concentration of 2-chlorobenzoic acid in
2-chlorobenzaldehyde.
[0165] As shown in FIG. 2, the enzyme reaction rate decreases with
increasing concentration of 2-chlorobenzoic acid (carboxylic acid),
indicating that the carboxylic acid compound acts as a reaction
inhibitor. This experiment showed that the addition of 0.5 wt % and
1.1 wt % of 2-chlorobenzoic acid to 2-chlorobenzaldehyde causes
about 30% and about 50% inhibition of the enzyme reaction,
respectively.
EXAMPLE 5
Removal of Reaction Inhibitors by Alkaline Treatment of Aldehyde
Compound
[0166] Benzaldehyde containing 0.2 wt % of benzoic acid (reaction
inhibitor) was used in the alkaline treatment to perform removal of
benzoic acid. Aqueous sodium hydroxide solutions of varying
concentrations were prepared and mixed with the above benzaldehyde
at a volume ratio of 1/1. After allowing to stand, the benzaldehyde
phase was sampled to measure the benzoic acid content by HPLC.
[0167] As shown in Table 2, the alkaline treatment was found to
achieve efficient removal of benzoic acid contained in
benzaldehyde.
2TABLE 2 Concentration of NaOH Benzoic acid content after Removal
ratio (N) alkaline treatment (wt %) (%) 0.1 N 0.022 91.0 0.01 N
0.16 27.9 untreated 0.20
EXAMPLE 6
Removal of Reaction Inhibitors by Alkaline Treatment of Aldehyde
Compound
[0168] In a manner similar to Example 5, benzaldehyde containing
0.2 wt % of benzoic acid was used to examine the removal of benzoic
acid. In this experiment, the concentration of aqueous sodium
hydroxide solution was kept constant at 0.1 N, while the volume of
alkaline aqueous solution to be mixed with benzaldehyde was
varied.
[0169] As shown in Table 3, the alkaline treatment was found to
have a sufficient effect on the removal of benzoic acid even at a
volume ratio of 1:1.
3TABLE 3 Benzaldehyde:0.1 N NaOH Benzoic acid content after Removal
ratio (v:v) alkaline treatment (wt %) (%) 1:1 0.043 86.0 10:1 0.21
13.7 1:10 0.008 96.9 untreated 0.22
EXAMPLE 7
Enzyme Reaction Using an Alkaline-Treated Aldehyde Compound
[0170] In a manner similar to Example 6,2-chlorobenzaldehyde was
repeatedly subjected to the alkaline treatment using an equal
volume of 0.1 N aqueous sodium hydroxide solution to reduce the
concentration of 2-chlorobenzoic acid from 1.4 wt % to 0.03 wt % or
less. This treated 2-chlorobenzaldehyde and an untreated
2-chlorobenzaldehyde originally containing 2-chlorobenzoic acid at
a low concentration of 0.03 wt % or less were used as substrates to
synthesize (R)-2-chloromandelonitrile.
[0171] The enzyme reaction was carried out under the same
conditions as described in Example 4, except for the aldehyde
compounds used. Three hours after initiation of the reaction, the
conversion ratio of aldehyde was measured, indicating that both the
treated and untreated aldehydes had the same conversion ratio of
69%. This showed that the alkaline treatment achieves the removal
of reaction inhibitors and the same reaction efficiency as in the
case where the untreated aldehyde originally containing benzoic
acid at a low concentration is used.
EXAMPLE 8
[0172] A mixture of t-butyl methyl ether (610 g) and prussic acid
containing sulfurous acid as a stabilizer (41 g) was stirred and
mixed with 40 ml of 0.2 M citrate buffer (pH 7). The mixture was
allowed to stand, and the organic phase separated. This organic
phase was mixed with the immobilized enzyme prepared in Preparation
Example 4 (60,000 units), followed by addition of benzaldehyde (106
g). This reaction mixture was stirred at room temperature to
synthesize (S)-mandelonitrile. After continuing the reaction for 30
minutes, the reaction solution was collected to measure the
conversion ratio of aldehyde by HPLC. After completion of the
reaction, the immobilized enzyme was recovered and then mixed again
with a substrate solution prepared in the same manner as described
above to repeat the reaction under the same conditions. As shown in
FIG. 3, enzyme activity was maintained stably even though the
reaction was repeated, and there was no loss of the enzyme activity
even when the reaction was repeated 11 times. A 2.5 ml aliquot was
taken from the 11th reaction and then mixed with 5 ml of pure
water, resulting in an aqueous phase having a pH of 5.
[0173] On the other hand, prussic acid containing sulfurous acid as
a stabilizer, which had been used as a starting material, was
dissolved at a concentration of 1.5 M in t-butyl methyl ether,
mixed with pure water at such a ratio that the mixture separated
into organic and aqueous phases (organic phase:water=1:2 (v/v)),
and then allowed to stand, resulting in an aqueous phase having a
pH of 2.9.
COMPARATIVE EXAMPLE 1
[0174] A mixture of t-butyl methyl ether saturated with 10 mM
phosphate buffer (pH 5) (610 g) and prussic acid containing
sulfurous acid as a stabilizer (41 g) was mixed with the
immobilized enzyme prepared in Preparation Example 4 (60,000
units), followed by addition of benzaldehyde (106 g). This reaction
mixture was stirred at room temperature to synthesize
(S)-mandelonitrile. After continuing the reaction for 30 minutes,
the reaction solution was collected to measure the conversion ratio
of aldehyde by HPLC. After completion of the reaction, the
immobilized enzyme was recovered and then mixed again with a
substrate solution prepared in the same manner as described above
to repeat the reaction under the same conditions. As shown in FIG.
3, enzyme activity significantly decreased when the reaction was
repeated 5 times, and was almost completely lost at the 6th
reaction. A 2.5 ml aliquot was taken from the 6th reaction and then
mixed with 5 ml of pure water, resulting in an aqueous phase having
a pH reduced to 3.5.
EXAMPLE 9
[0175] In a manner similar to Example 7, benzaldehyde was subjected
to the alkaline treatment using an equal volume of 0.1 N aqueous
sodium hydroxide solution. This treated benzaldehyde was used to
synthesize (S)-mandelonitrile by repeatedly using the immobilized
enzyme in the same manner as described in Example 8. After
completion of each batch reaction, the reaction solution was
collected and then mixed with p-toluenesulfonic acid monohydrate
(stabilizer) in an amount of 2.7 g per liter of reaction solution.
Each solution was then distilled under reduced pressure at 50 torr
and at 45.degree. C. to collect t-butyl methyl ether (solvent) and
unreacted prussic acid from the solution.
[0176] This experiment showed that 85% or more of t-butyl methyl
ether and prussic acid can be collected even though the
distillation was carried out under such conditions.
[0177] Further, additional prussic acid was added to the collected
t-butyl methyl ether containing prussic acid to prepare a 1.5 M
prussic acid solution in t-butyl methyl ether. This solution was
treated in the same manner as described in Example 3 and then used
to synthesize (S)-mandelonitrile. The result was shown in FIG. 4.
The first three batch reactions used fresh t-butyl methyl ether and
prussic acid, while other batch reactions used a recycled prussic
acid solution in t-butyl methyl ether which had been prepared by
collecting t-butyl methyl ether containing prussic acid through the
above distillation process, supplying it with fresh prussic acid in
order to adjust the concentration of prussic acid, and then
treating it in the same manner as described in Example 3.
[0178] As shown in FIG. 4, the use of the recycled t-butyl methyl
ether and prussic acid has no adverse effect on the enzyme reaction
and can provide the same result as in the case where fresh prussic
acid and t-butyl methyl ether are used.
[0179] All the publications, patents and patent applications cited
herein are incorporated herein by reference in their entirety.
* * * * *