Production of amino acids and enzymes used therefor

Details Production of amino acids and enzymes used therefor Production of amino acids and enzymes used therefor

1999-05-26

2001-07-31

Lilling, Herbert J. (Department: 1651)

Chemistry: molecular biology and microbiology

Micro-organism, tissue cell culture or enzyme using process...

Preparing alpha or beta amino acid or substituted amino acid...

C435S252100, C435S280000, C435S822000

Reexamination Certificate

active

06268185

ABSTRACT:

The invention relates to new processes of enantioselective conversion of &agr;-amino amides, &agr;-methyl amides and &agr;-aminomethyl amides to their corresponding acids and to new microorganisms and enzymes useful in these processes.
It is known that &agr;-amino amides may be converted to &agr;-amino acids by hydrolysis. This hydrolysis may be catalyzed by an amidase enzyme. It is also known to convert &agr;-amino nitriles to &agr;-amino acids by first converting the nitrile to an amide by its hydration. This nitrile-to-amide hydration may be carried out using a nitrile hydratase enzyme catalyst.
It is known that these transformations may be carried is out in an enantioselective fashion (that is, the &agr;-amino acid produced has an excess of one enantiomer) by choice of suitable enzymes. For instance, Bauer et al in Appl. Microbiol. Biotechnol. (1994) 42:1-7 describes the conversion of &agr;-amino phenyl acetonitrile to &agr;-amino phenyl acetic acid. The conversion is catalyzed by a strain of
Agrobacterium tumefaciens.
The nitrile is converted in almost stoichiometric amounts to the amide. The amide is then slowly hydrolyzed to the acid. The S enantiomer is preferentially formed. The best enantiomeric excess (97%) appears to be achieved after 43% conversion of the amide.
It appears that it is necessary to terminate the reaction at this point in order to achieve the high enantiomeric excess, ie the conversion does not give time-independent enantioselectivity.
This requirement to stop the reaction at a particular point in order to achieve the best enantiomeric excess is well known in enantioselective hydrolysis of amides to acids. In general, known catalysts of the above mentioned amides to their corresponding acids hydrolyse one amide enantiomer more rapidly than the other and eventually tend to convert a significant amount of the less preferred enantiomer.
Other conversion reactions have been described which appear to give high conversion and enantioselectivity, but do not appear to exhibit tame independent enantioselectivity. For instance, EP-A-332,379 discloses production of various amino acids. These are produced from a nitrile by exposure to various microorganisms. It is not stated whether the effective enzyme catalyst is a nitrilase or a combination of nitrile hydratase and amidase. It is stated that it is essential to carry out the reaction either at a pH between 8 and 12 or in the presence of an aldehyde. U.S. Pat. No. 4,080,259 describes amidase enzymes which effect hydrolysis of various amides to produce amino acids. Where the pH of the reaction mixture is given it appears to be from around 8 to around 10. U.S. Pat. No. 4,366,250 and FR-A-2,626,287 describe enzymic hydrolysis of various natural amino acids. U.S. Pat. No. 3,971,700 describes selective hydrolysis of phenyl glycine amide to give phenyl glycine. None of these references alleges that the reaction described gives time independent enantioselectivity.
U.S. Pat. No. 5,248,608 describes an enantioselective hydrolysis of various &agr;-substituted carboxylic acid amides. The hydrolysis is carried out using an amidase enzyme produced by
Ochrobactrum anthropi
or a Klebsiella sp. The citation alleges that very high conversion and enantiomeric excess are obtained. It explains that the general theory regarding enantioselective conversions described in past publications applies to the process described in U.S. Pat. No. 5,248,608. The publications referred to are those which rely upon stopping the reaction at a particular time in order to obtain a high enantiomeric excess i.e. reactions which do not give time independent enantioselectivity.
This document describes various examples. Some examples are of hydrolysis of &agr;-amino amides but the majority are of other &agr;-substituted amides. Only one of the examples demonstrates time independent enantioselectivity. This single example demonstrates hydrolysis of an N-hydroxy substituted &agr;-amino amide.
Some examples demonstrate hydrolysis of &agr;-amino amides, but none of these show time independent enantioselectivity. Further, the maximum time for reaction which is demonstrated in these examples is 8 hours. The reactions are all carried out as batch reactions.
U.S. Pat. No. 5,215,897 also describes an enantioselective hydrolysis of amino acid amide, which produces mainly L-amino acid. Again the only reactions described are batch reactions which are carried out for a maximum of three hours. The yield in the majority of the reactions is below 50% of the starting mixture and there is no demonstration that the reaction is or could be such that it gives time independent enantioselectivity.
It would be desirable to be able to produce &agr;-amino is amides having a high enantiomeric excess. This is particularly desirable for the production of enantiomerically pure unnatural &agr;-amino acids. It would also be desirable to achieve such enantiomeric purity together with high conversion of the relevant amide enantiomer. It would also be desirable to be able to do this in a manner which provides production processes which are conveniently adaptable to an industrial scale.
According to a first aspect of the invention we provide a process of converting an &agr;-amino amide to an &agr;-amino acid, comprising conducting a conversion reaction catalyzed by an amidase enzyme, in which the &agr;-amino amide starting material comprises amide enantiomers (A) and (B) and in the conversion reaction enantiomer (A) is converted preferentially over enantiomer (B),
Characterized in that the amidase enzyme is capable of converting enantiomer (A) such that it gives an enantiomeric excess of at least 90% independently of the conversion time.
In this specification, when we say that the enantiomeric excess is given independently of conversion time, we mean chat the high enantiomeric excess is retained throughout the time there is sufficient of enantiomer (A) to dominate the reaction, that is until most of enantiomer (A) is converted. For instance this can be up to 90% conversion of enantiomer (A). Generally time independent enantiomeric excess is maintained up to 95% and often 100% conversion of enantiomer IA). In some cases time independent enantiomeric excess is remained beyond 100% conversion of enantiomer (A), but this is not essential. The enantiomeric excess is that of the acid product.
Thus in the invention we achieve high selectivity for one enantiomer. In the majority of known processes the enantiomeric excess in the product varies as the conversion reaction progresses and it is often necessary to stop the reaction at a suitable point in order to obtain the best enantiomeric excess. In the invention, however, we achieve high enantiomeric excess at all times during the reaction, ie independently of the conversion time. This is believed to be due to very high selectivity of the amidase for a single enantiomer of the starting material. In known reactions it is usually observed that the amidase converts one enantiomer faster than the other, so that as the reaction progresses the enantiomeric excess tends to decrease. The present process is selective to such an extent that the enantiomeric excess remains at least 90% throughout the reaction.
The starting material in the conversion reaction of the process of the invention is an &agr;-amino amide. The amide is chosen so that it gives on hydrolysis the required &agr;-amino acid.
Suitable &agr;-amino amides used as starting materials have the formula I as follows:
In this preferred formula R is suitably alkyl, cycloalkyl, alkenyl, cycloalkenyl, aryl, alkaryl, aralkyl, R
1
NHCOR
1
, R
1
CONHR
1
, SO
2
R
1
or SO
2
NHR
1
in which R
1
is alkyl, cycloalkyl, alkenyl, cycloalkenyl, aryl, alkaryl or aralkyl, or substituted versions of any of these. In particular, R can be C
4
to C
9
, for instance C
4
to C
7
, linear or branched alkyl or alkenyl, cyclic alkyl or alkenyl, phenyl, or substituted phenyl in which the substituent is selected from para-CH
3
, meta-CH
3
, ortho-CH
3
, para-CF
3
, para-Et, para-(CH
3
)
3
C, para-Cl, para-CH
3
(CH
2

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