WO2001019982A2

WO2001019982A2 - Micro-organisms, their use and method for producing d-amino acids

Info

Publication number: WO2001019982A2
Application number: PCT/ZA2000/000173
Authority: WO
Inventors: Stephanie Gail Burton; Rosemary Ann Dorrington; Carol Janet Hartley
Original assignee: Sa Bioproducts (Proprietary) Limited; Rhodes University
Priority date: 1999-09-17
Filing date: 2000-09-18
Publication date: 2001-03-22
Also published as: EP1220904A2; CA2385241A1; JP2005518179A; WO2001019982A3; HK1044173A1; AU7887000A

Abstract

The invention relates to novel micro-organisms which are simple to cultivate and their use in the production of D-amino acids, particularly micro-organisms suitable for the production of D-amino acids from corresponding hyantoins of N-carbamoylamino acids.

Description

NOVEL MICRO-ORGANISMS. THEIR USE AND METHOD FOR PRODUCING D-AMINO ACIDS

FIELD OF THE INVENTION

The invention relates to novel micro-organisms and their use in the production of D- amino acids In particular, micro-organisms suitable for the production of D-amino acids from corresponding hydantoins or N-carbamoylamino acids These novel micro-organisms are simple to cultivate and make possible high D-amino acids yields from different substrates

BACKGROUND OF THE INVENTION

The importance of optically pure amino acids is pπmaπlv due to the use of D-amino acids, e g D-/?-hydroxyphenylglycιne, as side chains in semi-synthetic penicillins and cephalospoπns (Syldatk et al, 1990) Optically pure amino acids also have applications in the production of other pharmaceuticals and flavourants (e g D- alanine in sweetners), pesticides (D-vahne in the synthesis of insecticide fluvamlate), and as additives in animal feedstock (Polastro, 1989) Conventionally, D, L-5- substituted hvdantoins have been used as starting materials for the chemical synthesis of D-ammo acids This process is cumbersome and inefficient since chemical synthesis results in an equimolar mixture of D- and L-ammo acids requiring racemate resolution to obtain optically pure D-ammo acids (Syldatk et al , 1990) An alternative to chemical synthesis is the use of enzvmatic conversion of hydantoins to their respective amino acids (Olivieπ et al 1979) Biocatalytic conversions have major advantages the enzvme svstems are stereoselective and mild reaction conditions result in a cheap industrial process with environmentally benign by-products and effluents (Santaniello et al , 1992) The biocatalytic conversion of D,L-p- hydroxyphenylhydantoin to D-p-hydroxyphenylglycine has been listed as one of the main biocatalytic processes in the world market (Polastro, 1989)

The biocatalytic conversion of hydantoins to their corresponding ammo acids is catalysed by two enzymes first, an hydantoinase catalyses the ring-opening hydrolysis of the 5-substituted hydantoin to produce an N-carbamylarmno acid in a reversible reaction Classified as cyclic amidases (E C 3 5 2), hydantoinases may be D-, L- or non-stereoselective In the second reaction, the N-carbamylamino acid is converted to its corresponding amino acid either chemically, or through the action of a second enzyme, an N-carbamylamino acid amidohydrolase (E C 3 5 1 6), which is usually stereoselective (Olivieri et al , 1979) While racemization of the hydantoins occurs spontaneously at alkaline pH, certain microbial systems include a D-racemase which converts L-5-substituted hydantoins to the corresponding D-enantiomers (Runser et al , 1990, Hartley et al , 1998)

D-selective hydantoin-hydrolysing enzyme systems have been identified in a variety of bacteria, including a Pseudomonas isolate (Ikenaka et al , 1998), Bacillus stearothermophilus (Lee et al , 1996), Bacillus circulans (Luksa et al , 1997) and several Agrobacterium strains (Olivieri et al , 1981 , Runser et al , 1990, Hartley et al , 1998, Νanba et al , 1998) The genes encoding one hydantoinase and three N- carbamylamino acid amidohydrolase enzymes from the Agrobacterium strains have been cloned and over-expressed in Eschenchia coli (Durham and Weber, 1995, Buson et al 1996, Grifantini et al , 1998, Νanba et al , 1998) DΝA sequence analysis has revealed a high degree of amino acid homology between N-carbamylamino acid amidohydrolases from the Agrobacteria (Νanba et al , 1998)

Characterisation of the enzyme system of A tumefaciens RU-OR showed that enzymes activity was induced at high levels only when cells were grown in the presence of 2-thiouracil or hydantoin Furthermore, maximum enzyme activity in cells grown in complete medium was detected in early stationary phase (Hartley et al , 1988) Similar observations have been made for hydantoin-hydrolysing enzyme systems from A. radiobacter (Deepa et al . 1993), Agrobacterium sp IP 1-671 (Meyer & Runser, 1993) and those of other bacteria with L-selective enzyme systems, such as Arthrobacter crystallopoietes (Moller et al , 1988) An A. tumefaciens mutant, with inducer-independent production of hydantoinase and NCAAH, has been isolated by Hartley et al (1998) and a similar mutant strain, Arthrobacter sp DSM 9771 , has been isolated by Wagner et al (1996)

In this invention the word "constitutive" is to be understood to mean unregulated expression of enzymes, the word "expression" is understood to mean the production of a protein from a DNA template via transcription and translation, the word "activity" is understood to mean the ability of the hydantoinase and N-carbamylamino acid aminohydrolase enzymes to hydrolyse hydantoins to N-carbamylamino acids and amino acids and vice versa, respectively, the phrase "over-express" to mean levels of enzyme production in excess of those under the same conditions in the original isolate, and the phrase "enzyme system" is to be understood to include hydantoinase, N-carbamylamino acid amidohydrolase and hydantoin racemase enzymes which are capable of converting D- or L- or D,L-5-monosubstituted hydantoins or D- or L- or D,L- N-carbamoylamino acids to their corresponding, optically pure D-amino acids

Recombinant systems for the over-expression of both hydantoinase and ΝCAAH enzymes in E. coli are known However, reports of the production of insoluble aggregates and plasmid instability in cells over-expressing the ΝCAAH indicate that heterologous expression of these enzymes in E. coli may not be the ideal system This has led to renewed interest in the use of homologous hosts for hydantoinase and ΝCAAH production, where the main problem is that enzyme activity needs to be induced and is confined to stationary growth phase under optimum growth conditions This means that the levels of enzyme production per unit biomass in commercial strains remain relatively low The re-introduction of a recombinant ΝCAAH gene under control of a constitutive promoter into Agrobacterium 80/44-2A resulted in high levels of biocatalytic activity

The problems relating to genetically modified organisms and the obvious economic advantages of industrial strains that are not genetically modified, have led to the examination of the potential of mutant bacterial strains in the high-level production of hydantoinase and ΝCAAH enzymes OBJECT OF THE INVENTION

An object of the invention is the isolation of micro-organisms able constitutively to produce enzymes which convert racemic mixtures of 5-substιtuted hydantoins or N- carbamyl amino acids to D-amino acids and thereby, at least partially, to alleviate the problems associated with chemical synthesis of D-amino acids

SUMMARY OF THE INVENTION

In accordance with the invention there is provided a biologically pure culture of a mutant strain of Agi obacterium RU-OR which constitutively expresses a stereoselective enzyme system which may be used in the enzymatic synthesis of D- amino acids

Further in accordance with the invention there is provided a biologically pure culture of a glutamine synthesis-deficient micro-organism able constitutively to produce enzymes which convert racemic mixtures of 5-substιtuted hydantoins to D-amino acids

Furthermore in accordance with the invention there are provided micro-organisms which are able to constitutively produce enzymes which convert racemic mixtures of N-carbamylamino acids to D-amino acids

Further in accordance with the invention there is provided an isolated and purified enzyme system able to convert racemic mixtures of 5-substιtuted hydantoins to D- ammo acids

Still further in accordance with the invention there is provided an isolated and purified enzyme system able to convert racemic mixtures of N-carbamylammo acids to D- amino acids

Furthermore in accordance with the invention there is provided a micro-organism for use in the production of D-amino acids for the production ot pharmaceuticals, alternatively agrochemicals, further alternatively for use in the production of D-amino acids for the production of pesticides, and still further alternatively for use in the production of D-amino acids for the production of feedstock additives.

The invention also extends to a growth medium to achieve over-expressed levels of hydantoinase and/or NCAAH enzyme activity during optimum culture conditions.

The invention also provides for a N-carbamylamino acid produced in accordance with the invention.

The invention also provides for a D-amino acid produced in accordance with the invention.

BRIEF DESCRIPTION OF THE FIGURES

In the accompanying Figures:

Figure 1 shows the DNA sequence of the 16S rRNA gene i Agrobacterium RTJ-OR;

Figure 2 shows hydantoinase and N-carbamylamino acid amidohydrolase activity in Agrobacterium RU-OR cells during mid-logarithmic phase during growth in HMM;

Figure 3 shows the effect of carbon and nitrogen source on hydantoinase and N- carbamylamino acid amidohydrolase activities in RU-OR cells;

Figure 4 shows that ammonia shock represses enzyme activity in wild-type Agrobacterium RU-OR cells;

Figure 5 shows that RU-ORPΝ1 cells constitutively express hydantoinase enzyme, but that the hydantoinase enzyme is inactive due to repression by ammonium in the growth medium;

Figure 6 shows that RU-ORPΝ1 cells constitutively express active N-carbamylamino acid amidohydrolase enzyme, while the wild type enzyme is repressed; Figure 7 shows that hydantoinase activity in RU-ORPN l F9 cells is not sensitive to ammonia shock,

Figure 8 shows the levels of hydantoinase activity in RU-ORPNl F9 cells during mid- logarithmic growth phase compared with the levels in the wild-type RU-OR and mutant RU-ORPNl , when cells are grown under optimal growth conditions,

Figure 9 shows the levels of N-carbamylamino acid amidohydrolase activity in both RU-ORPΝ1 and RU-ORPΝ1F9 cells during mid-logarithmic growth phase compared with the levels in the wild-type RU-OR, when cells are grown under optimal growth conditions, and

Figure 10 shows the increase in specific hydantoinase activity per unit biomass in RU-ORPNl F9 cells in mid-logarithmic growth phase, with D,L-p- hydroxyphenylhydantoin as substrate, as compared with the specific hydantoinase activity in the wild-type RU-OR cells and RU-ORPNl cells achieved during stationary phase

DESCRIPTION OF ONE EMBODIMENT OF THE INVENTION

Several Agrobacterium strains have been reported to have hydantoin-hydrolysing activity Among these are Agrobacterium tumefaciens 47 C, Agrobacterium radiobacter B 1 1291 and Agtobacterinm sp IP 1-671 Agrobacterium radiobacter B11291 and Agrobacterium sp IP 1-671 also have N-carbamylamino acid and amidohydrolase activity In the present invention, a novel Agrobacterium species (RU-OR) was isolated which is capable of producing a number of enzymes in amounts such that the cell mass has a high activity for the methods described herein

CULTURE AND BIOCATALYTIC ASSAY CONDITIONS

Agrobacterium RU-OR and RU-ORPNl cells grown to saturation in hydantoin minimal medium (HMM) broth, are diluted to ODδoo_n = 0 02 in standard minimal medium (MM) (MM per litre l Og glucose, O Ol lg CaC l₂, 0 02g M_gCl₂, 60g Na₂HP0_4, 30g KH₂ P0₄, 5g NaC l , 0 04g boric acid, 0 04g MnS0₄, 0 02g (NH₄)₆Mo₂0₂₄ 4H₂o, O Olg KI, 0 004g CuS0₄) supplemented with 1% hydantoin (HMM), 0 01% casamino acids (SMM), or (NH₄)₂S0₄ (AMM) Strain RU-ORPN1F9 cells are grown in HMM or SMM or AMM supplemented with 0 002% glutamine. Enzyme activity in Agrobacterium RU-OR cells was induced by growth in medium containing 0 1% thiouracil Cells are harvested at ODsoonm = 0 5 - 0 8, pelleted by centrifugation, washed in 0 1 M P0₄ buffer pH 8 0 and resuspended in hydantoin or N-carbamylglycine reaction buffer at a final hydrated biomass concentration of 20 mg/ml (reaction buffer either 50 mM hydantoin or 25 mM N-carbamylglycine in 0 1 M P0₄ buffer pH 8 0) Hydantoinase activity is measured as the sum of the concentration of N-carbamylglycine (μmol/ml) and glycine (μmol/ml) produced from 50 μmol/ml hydantoin in a 5 ml reaction volume after 6 h, shaking, at 40°C N- carbamylamino acid amidohydrolase activity is measured as the concentration of glycine (μmol/ml) produced from 25 μmol/ml N-carbamylglycine in a 5 ml reaction volume after 6 h, shaking, at 40°C

ISOLATION OF AGROBACTERIUM RU-OR, RU-ORPNl and RU-ORPN1F9

Soil samples from the Eastern Cape environment were inoculated into hydantoin minimal medium (HMM) broth (per litre l Og glucose, 0 01 lg CaCl₂, 0 02g MgCl₂, 60g Na₂HP0₄, 30g KH₂ P0₄, 5g NaCl, 0 04g boric acid, 0 04g MnS0₄ 0 02g (NH₄)₆Mo₂0₂₄ 4H₂0, 0 Olg KI, 0 004g CuS0₄, 1% hydantoin) and incubated, shaking at 25°C for 24 hours, after which serial dilutions were plated onto HMM agar and incubated for 5 days at 25°C Resulting colonies, which utilised hydantoins as a sole nitrogen source, were purified by re-streaking onto HMM agar Isolated strains were examined for the presence of hydantoinase and N-carbamylamino acid amidohydrolase activity using resting cell biocatalytic assays The wild-type Agrobacterium sp strain RU-OR, which was among these isolates, was identified through determination of its 16S rRΝA gene sequence (shown in Figure 1) as described in Hartley et al. (1998)

Mutant RU-ORPΝ1 was selected as follows Agrobacterium RU-OR cells were cultured in HMM broth to mid-log phase and then subjected to mutagenesis using ethylmethane sulfonate (EMS) according to the method described in Miller (1992) Mutated cells were plated onto MM agar supplemented with 0 1% (NH₄) S0 and 0 1% 5-fluorouracil Strain RU-ORPNl was isolated from these plates and evaluated under standard culture and assay conditions for enzyme activity in the absence of inducer Strain RU-ORPNl F9 was isolated by mutagenizing RU-ORPNl cells as described above and after penicillin-enrichment for glutamine-dependent growth, cells were plated onto HMM agar supplemented with 0 002% glutamine Gin mutants were selected by relica plating to HMM without supplementation with glutamine

GLUTAMINE SYNTHETASE ASSAYS.

Total glutamine synthetase activity was measured using the γ-glutamyl transferase assay Cells were prepared by treatment with 0 01% cetyl-trimethylammonium bromide for 10 minutes before harvesting. The cells were then washed twice with 0.1M phosphate buffer pH 9 0 before being suspended in 50 times less volume of resuspension buffer, and assayed according to the method of Bender et al (1977). Protein concentration was determined by the method of Bradford (1976). Activity is expressed as μmoles of γ-glutamyl hydroxamate generated per minute per milligram protein The percentage adenylation of the glutamine synthetase enzyme subunits was measured using the method of Magasanik et al. (1995), which compares γ-glutamyl transferase in the presence and absence of magnesium ions Magnesium ions inhibit the activity of adenylated enzyme subunits and the difference can then be used to calculate the percentage adenylation of the glutamine synthetase enzyme

REGULATION OF HYDANTOINASE AND NCAAH ACTIVITY

Hydantoinase and NCAAH activities in A. tumefaciens RU-OR cells could be detected only in early stationary phase during batch culture in a complete growth medium (nutrient broth) Furthermore, enzyme activity was dependent upon growth in the presence of the hydantoin-analogue 2-thiouracil The nutritional factors responsible for regulating enzyme activity were identified by establishing standard culture conditions under which enzyme activity was not limited to stationary phase Hydantoinase and NCAAH activities were measured during growth of RU-OR cells in a chemically defined minimal medium containing hydantoin and glucose as sole nitrogen and carbon sources, respectively (MM plus 0 1 % hydantoin) Activity of both enzymes was low in early exponential phase and after the cells reached stationary phase, with highest activity detected during mid to late exponential phase (Figure 2)

In all subsequent experiments, enzyme activities were determined in cells harvested during mid-exponential phase at ODβoo = 0 5 - 0 8

The effect of different carbon and nitrogen sources upon hydantoin-hydrolysing enzyme activity was determined by examining growth-rate and assaying for biocatalytic activity at mid-exponential growth phase Cells were grown in minimal medium containing either glucose or glycerol as carbon source and hydantoin as nitrogen source. The growth-rate of RU-OR cells was not significantly affected by either carbon source (Figure 3) and there was also little difference in hydantoinase and NCAAH activity (Table 1 )

Table 1 Hydantoin-hydrolysing activity in RU-OR cells grown with different carbon and nitrogen sources.

Carbon Source Nitrogen Source Hydantoinase Activity NCAAH Activity (μmol/ml) (μmol/ml)

1% glucose 1 % hydantoin 4 87 ± 0 400 5 77 ± 0 55 1% glycerol 1 % hydantoin 3 97 ± 0 58 5 85 ± 0 58 1% glucose 0 1% (NH₄)₂S0₄ 1 15 + 0 2 1.09 ± 0 16 1%) glucose 0 1% serine 4 70 ± 0 26 3 70 ± 0 56* 1% glucose 0 01% CAA 10 87 ± 0 43 8 68 ± 0 61

± - SEM (n = 3) * Measured as the amount of glycine generated from hydantoin as substrate CAA - casamino acids

In contrast, the growth rate of RU-OR cells appeared to be dramatically affected by the choice of nitrogen source Hydantoin was the most growth-rate-limiting while 0 1% (NH₄)₂S0₄ and 0 1% serine were the least growth-rate limiting sources of nitrogen (Figure 3) Cells in medium containing 0 01% casamino acids, grew at an intermediate rate The highest enzyme activity was detected in cells growing in 0 01%) casamino acids and the lowest in (NH )₂Sθ4 Cells grown with serine or hydantoin as a nitrogen source showed intermediate levels of enzyme activity (Table 1) growth of cells in medium containing (NH₄)₂S04 had a repressive effect upon hydantoinase and NCAAH activity (nitrogen repression)

Induced RU-OR cells (grown in SMM plus 0 1% thiouracil) were resuspended and grown in AMM plus 2-thiouracil (ammonia shock) Within 30 minutes, the hydantoinase activity had dropped three-fold, and a corresponding two-fold drop in NCAAH activity was observed (Figure 4)

When induced cells were resuspended and grown in AMM containing the glutamine synthetase inhibitor, D,L-methionine D,L-sulfoximine (MSX), there was very little drop in both hydantoinase and NCAAH activities (Figure 4), indicating that the loss of hydantoinase and NCAAH activity in RU-OR cells after ammonia shock is dependent upon glutamine synthetase activity Induced cells were subjected to ammonia shock for 30 minutes, after which they were washed and resuspended in SMM plus thiouracil and grown for a further 60 minutes before assaying for enzyme activity Hydantoinase and NCAAH activity returned to levels observed before ammonia shock suggesting that the ammonia shock effect could be reversed rapidly in the absence of ( H₄)₂S0₄ Together, this data indicates that hydantoinase and NCAAH activity in wild-type Agrobacterium RU-OR is dependent upon the presence of a) inducer and b) the nitrogen source in the growth medium

CHARACTERIZATION OF MUTANT STRAINS.

Inducer-independent hydantoinase and N-carbamylamino acid amidohydrolase, activity was assessed by measuring enzyme activity in cells grown in SMM without 2- thiouracil RU-ORPΝ1 cells showed a significant (three-fold) increase in hydantoinase activity and ΝCAAH activity was equivalent to induced levels in Agrobacterium RU-OR cells Table 2. Hydantoin-hydrolysing activity of mutant RU-OR strains

Strain

HYDANTOINASE ΝCAAH

N-carbamylglycine plus glycine Glycine

(μmol/ml) (μmol/ml) no inducer 2-thiouracil no inducer 2-thiouracil

RU-OR (wt) 1 98±0 65 7 51±0 37 2 62±0 15 1 1 74+0 80

RU-ORPNl 21.8±0 78 nd 8 04±0 35 nd

± - SEM (n = 3) nd - not determined

RU-ORPΝ1 cells grown in minimal medium with (ΝH₄)₂S0₄ as nitrogen source had repressed levels of hydantoinase activity, as observed in the wild-type, RU-OR cells (Figure 5), but, in contrast to the RU-OR, NCAAH activity in RU-ORPNl cells was elevated to wild-type, induced levels (Figure 6) After growth in SMM for 60 minutes, hydantoinase activity in mutant RU-ORPNl cells recovered to levels normally observed in induced wild-type cells (see table 2) while there was no increase in hydantoinase activity in the wild-type Agrobacterium RU-OR cells after growth in SMM Thus, unlike the wild-type, the mutant strain expresses both hydantoinase and N-carbamylamino acid amidohydrolase enzymes even under nitrogen repression conditions, but the hydantoinase enzyme is inactive in the presence of (ΝH₄)₂S0₄

Inhibition of glutamine synthesis reduces the sensitivity of hydantoinase activity to ammonia shock in RU-OR cells (Figure 4) Therefore, the gin auxotrophic mutant RU-ORPN1F9 was subjected to ammonia shock and hydantoinase activity in the auxotrophic mutant Figure 7 shows that hydantoinase activity in mutant RU- ORPNl F9 is no longer sensitive to ammonia shock as compared to that of the wild- type Agrobacterium RU-OR and mutant RU-ORPNl

Glutamine synthetase assays of all three strains before and after ammonia shock showed that glutamine synthesis was reduced by 60% in RU-ORPN1F9 when compared to that in Agrobacterium RU-OR and RU-ORPN 1 cells Thus a reduction in glutamine synthesis when RU-ORPN 1F9 cells are grown in (NH₄) Sθ₄, results in insensitivity of hydantoinase activity to ammonia shock HYDANTOINASE AND NCAAH ACTIVITY IN REGULATORY MUTANTS DURING GROWTH IN (NH₄)?SO.ι.

The hydantoinase and NCAAH activity of RU-ORPN l and RU-ORPN1F9 cells were assessed during batch culture in SMM and compared with enzyme activity of the wild-type Agrobacterium RU-OR grown in the same medium, supplemented with 2- thiouracil

Hydantoinase activity in mutant strain RU-ORPNl followed the same trend as in the wild-type Agrobacterium RU-OR (Figure 8), but high levels of activity were detected in exponential growth phase in RU-ORPN 1F9 cells NCAAH activities in strains RU-ORPNl and RU-ORPN 1F9 were highest in exponential growth phase and these levels declined during stationary phase. RU-ORPN 1F9 cells achieved the highest overall hydantoin-hydrolyzing activity of all three strains during exponential growth phase (Figures 8 and 9) indicating that the gin phenotype does not have a deleterious effect upon hydantoinase or NCAAH production in this strain Strain Agrobacterium RU-OR was selected for its efficient conversion of D,L-p-hydroxyphenylhydantoin to D-/?-hydroxyphenylglycine High levels of D,L- ?-hydroxyphenylhydantoin- hydrolysis were also achieved The highest D,L-/?-hydroxyphenylhydantoin conversion by the wild-type Agrobacterium RU-OR and RU-ORPNl cells was detected during stationary growth phase In strain RU-ORPN 1F9 both hydantoinase and NCAAH activity during exponential growth phase exceeded that detected in either Agrobacterium RU-OR or RU-ORPNl cells Up to 45 % of D,L-/?- hydroxyphenylhydantoin was converted to either N-carbamyl-p- hydroxyphenylglycine or D- ?-hydroxyphenylglycine by RU-ORPΝ1F9 cells within six hours. RU-ORPN 1F9 cells produced approximately 6 μmoles/ml D-p- hydroxyphenylglycine after six hours, which corresponds to 25 % conversion of D,L- -hydroxyphenylhydantoin

Figure 10 (A - C) depicts the specific hydantoinase activity per milligram dry cell mass with D,L- -hydroxyphenylhydantoin as substrate Strain RU-ORPNl shows an overall increase of 50% in hydantoinase activity compared with wild-type Agrobacterium RU-OR Mutant RU-ORPN 1F9 showed the highest specific hydantoinase activity with a 300%> and 200%> increase over the wild-type Agrobacterium RU-OR and mutant RU-ORPNl respectively Most important, the highest specific hydantoinase activity per unit biomass was observed in RU- ORPN 1F9 cells during mid-logarithmic growth phase (0 015 units) versus 0.002 units and 0 003 units of activity in RU-OR and RU-ORPNl cells, respectively, during the same growth phase

Claims

1 A biologically pure culture of a mutant strain of micro-organism which constitutively expresses a stereoselective enzyme system for use in the enzymatic synthesis of D-amino acids

2 A biologically pure culture glutamine deficient micro-organism able constitutively to produce enzymes which convert racemic mixtures of 5- substituted hydantoins to D-amino acids

3 A micro-organism able constitutively to produce enzymes which convert racemic mixtures of N-carbamylamino acids to D-amino acids

4 A micro-organism able constitutively to produce enzymes which convert racemic mixtures of N-carbamylamino acids to D-amino acids

5 A micro-organism as claimed in any one of claims 1 to 3 wherein the microorganism is Agrobacterium sp

6 A micro-organism as claimed in any one of claims 1 to 4 wherein the microorganism is indistinguishable from Agrobacterium RU-OR based on its 16S rRNA gene sequence

7 An isolated and purified enzyme system able to convert racemic mixtures of 5- substituted hydantoins to D-amino acids where the enzyme system is isolated and purified from a micro-organism as claimed in any one of claims 1 to 3

8 An isolated and purified enzyme system able to convert racemic mixtures of N-carbamylamino acids to D-amino acids where the enzyme system is isolated and purified from a micro-organism as claimed in any one of claims 1 to 3

9 A micro-organism as claimed in any one of claims 1 to 3 for use in the production of D-amino acids for use in the production of pharmaceuticals

10 A micro-organism as claimed in any one of claims 1 to 3 for use in the production of D-amino acids for use in the production of agrochemicals

11 A micro-organism as claimed in any one of claims 1 to 3 for use in the production of D-amino acids for use in the production of pesticides

12 A micro-organism as claimed in any one of claims 1 to 3 for use in the production of D-amino acids for use in the production of feedstock additives 13 A growth medium for use in the production of a micro-organism constitutively producing an enzyme system catalysing the conversion of 5- substituted hydantoins to D-amino acids by a micro-organism as claimed in any one of claims 1 to 3 14 A growth medium for use in the production of a micro-organism constitutively producing an enzyme system catalysing the conversion of N-carbamylamino acids to D-amino acids by a micro-organism as claimed in any one of claims 1 to 3

15 A growth medium for use in the production of micro-organisms as claimed in any one of claims 1 to 4 producing an enzyme system as claimed in either one of claims 5 or 6

16 A growth medium as claimed in any one of claims 1 to 13 for the production of D-Amino acids from 5-substituted hydantoins during fermentation conditions 17 A growth medium as claimed in any one of claims 1 to 13 for the production of D-Amino acids from N-carbamoylamino acids during fermentation conditions 18. A growth medium for use under fermentation conditions to achieve over- expressed levels of enzyme activity for the conversion of racemic mixtures of 5-substituted hydantoins to D-amino acids by a micro-organism as claimed in any one of claims 1 to 3

19 A growth medium for use under fermentation conditions to achieve over- expressed levels of enzyme activity for the conversion of racemic mixtures of N-carbamylamino acids to D-amino acids by a micro-organism as claimed in any one of claims 1 to 3 0 A N-carbamylamino acid produced in accordance with the invention 1 A D-amino acid produced in accordance with the invention