This invention relates to a process for the biocatalyst-mediated preparation of optically active α-amino acids by deracemisation technique.
Amino acids are a growing class of intermediates required for pharmaceuticals, agrochemicals and other industrial products. Optically pure amino acids can be obtained by using (a) resolution (results in < 50% yield) (b) asymmetric transformation of a prochiral substrate or by a dynamic kinetic resolution and (c) deracemisation of a mixture of enantiomers in which a racemic mixture is converted to the single enantiomer form. Deracemisation has been reportedly achieved in two ways, either (i) by using two or more enzymes/whole cells (e.g. dehydrogenases) with complementary enantioselectivity or (ii) by combining an enantioselective enzyme (povane kidney D-amino acid oxidase)-catalysed oxidation reaction with a non-selective, non-enzymatic reduction reaction using reagents like sodium cyanoborohydride.

Kinetic resolution methods (both chemical and enzymatic) end up with maximum of 50 % yield of each enantiomer which is a major disadvantage if only one enantiomer is desired.
Asymmetric hydrogenation of a-oxo acids by transamination requires optically active a-phenylglycine as a chiral reagent. Many other reported methods like Schiff base complex catalyzed enantioselective nitrile addition to imines, ring opening of aziridine-2-carboxylates and lactones result in optically pure amino acids but the reaction conditions are generally harsh. Dynamic kinetic resolution is a tedious method which requires repeated resolution and racemisation cycles. All these require long reaction times (24-48 hrs) and toxic transition metals like [RuCl2(p-cymene)] or isolated enzymes (D and L-amino acid oxidases and Novozyme). Deracemisation by stereo-inversion using a combination of biocatalysts and chemocatalysts for natural and unnatural amino acids have been reported. Deracemisation of a-amino acids using various biocatalysts (e.g Nocardia

diaphanozonaria JCM 3208) which utilizes the cell-free system was reported and it was found α-amino acids, when whole cells were used as biocatalyst. Two hydantoinases of different origins and stereoselectivities and one L-N-carbamoylase were used for the stereoselective bioconversion of (dimethyl)phenylsilyl and 1-methyl-1-silacyclopentyl substituted alanine derivatives. Whole resting cells of immobilized Agrobacterium sp. IP 1-671 were used for the synthesis of D-amino acids from DL-hydantoines with 88% yield and 95% ee. Deracemisation of the DL-hydantoins using D-hydantoinase activities of microorganisms or purified enzymes is reported.
The reported deracemisation of a-amino acids via their ketene derivatives and by enantioselective carboxylation, alkylation, metalation and protonation of Schiff bases yields optically active amino acids with an enantiomeric excess of only up to 76%. The reported deracemisation method using the chiral auxiliary (3S)-N,N'-bis-(p-methoxybenzyl)-3-isopropylpiperazine-2,5-dione gives

optically active phenyl alanine (yield 70 %).
The problems associated with the known art are:
1. Longer reaction times
2. Chiral reagents are required
3. Harsh and hazardous chemicals are required
4. Isolated enzymes are required.
5. Yields are lower
6. Degradation of the starting materials
7. Multistep reactions
8. Higher temperatures are needed.
9. The major problem of all these reported biocatalytic methods is that they are not applicable for preparation of optically pure α-amino acids in general. This invention intends to overcome the above mentioned problems.
So far, no single method has been proven sufficiently versatile to prepare optically pure amino acids. To address this need, we have developed a general biocatalytic process to prepare optically pure, amino acids in high yields by deracemisation of racemic starting materials using the

whole cells of Candida parapsilosis ATCC 7330. Our invention relates to the preparation of optically active a-aminoacids by deracemisation of DL-α-aminoacids using whole cells of Candida parapsilosis ATCC 7330 without any formation of side products in very mild and environment friendly conditions. Our invention is simple, efficient and requires short reaction times followed by simple isolation of product.
The process, according to this invention, for the biocatalyst mediated preparation of optically pure α-amino acids comprises the deracemisation of the racemic a-amino acids


The Racemic α-amino, is selected from natural and synthetic a-amino acid and its derivatives such as a-amino amides, a-amino esters.

heteroaryl, mono cyclic and poly cyclic rings)
Example: DL-a-phenyl alanine (24 mg, 0.124x10-3 moles) dissolved in water (1ml) was added to the harvested cells, (24.0 g)

suspended in distilled water [16.2 ml]. The biotransformation reaction was carried out for 4 h at 150 rpm and 25°C in water bath shaker. Control experiments were done wherein only the solvent (water) was added to the cell suspension. After 4 h, the product was obtained by evaporation of the solvent (water) under high vaccum which was collected by centrifugation of the reaction mass.

In the above mentioned description the ranges, substitutes of process parameters/chemicals that are employed are as follows:
1. Temperature will be 24-30 °C
2. Water bath shaker rpm will be 50-400 rpm

r
1 water.
6. The supernatant can be lyophilized instead of concentrating.
7. Racemic a-amino acid can be any natural and unnatural a-amino acid and its derivatives such as a-amino amides, a-amino esters.
As opposed to the reported methods for the preparation of optically active a-amino acids which involve the use of harsh reagents, multi-step reactions and severe conditions or the use of multienzyme systems, buffer medium, tedious isolation of the product, the process proposed by us is
• Efficient as it involves the preparation of optically active a-amino acids by dereacemisation from their racemates to give high yields (79-92%) and ee (91->99 %)
• Whole cells and no additional cofactors are used


Environment friendly - 'Green' because the biotransformation is carried out in water at 25°C The isolation of the product is simple
Some of the noteworthy features of this invention are:
? It is a one pot reaction
? Aqueous medium reaction
? Environment friendly conditions
? Short reaction times
? Simple work up
? Selective for one enantiomer [high ee 91 ->99 %]
? High yield [79-92 % ]
Optically pure amino acids are useful chiral synthons
Given the
Simplicity of the proposed 'green' process developed by us it will find use in preparing optically pure α-amino acids.
The proposed process can be scaled up to industrial levels for the manufacture of optically pure α-amino acids


• These optically pure α-amino acids can be used to prepare peptides and proteins by polycondensation process
• These optically pure α-amino acids are used to prepare many important drugs like Lisinopril, Captopril and Penicillin G and also the artificial sweetner Aspartame in the food industry.
The terms and expressions herein are not of limitation, since various other ways of carrying out this invention are possible without departing from the scope and ambit thereof

We Claim:
1. A process for the biocatalyst mediated preparation of
optically pure a-amino acids comprises the deracemisation
of the racemic α -amino acids using the wet cells of
Candida parapsilosis ATCC 7330 suspended in Buffer,
distilled water of pH 5-8 for 2-6 hours at-24-
-28oC, the reaction flask being placed in a shaker
or stirrer 50 - 400 rpm the
progress of the reaction followed by chiral HPLC after
separating the cells, using a centrifuge;
and concentration of the supernatant to
obtain the products of 79-92%.
2. A process as claimed in Claim 1 wherein the Racemic α -
amino is selected from natural and synthetic α -amino acid
and its derivatives such as α -amino amides, α -amino esters.

3. A process as claimed in Claim 1 OR claim 2 wherein
racemic α -amino acids of 0.124x10 moles and wet cells
of Candida parapsilosis ATCC 7330 24.0 g are suspended in distilled water of pH 6.8 [16.2 ml] for 4 hours
4, A process for the biocatalyst mediated preparation of
optically pure α -amino acids substantially as herein
described with reference to and as illustrated by the
Examples.