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`IPR2014-01126-1033 p. 1
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`1258 O. KEIL et al. In typical experiments, 7 mmol of (+)-1-14 were added to 70 ml of a thermostated (50°C) buffer solution [0.1 M glycine/NaOH, pH 8.5,1 mM Mn 2+] under nitrogen, followed by the addition of 70 lal D-HYD 1 or 14 pl D-HYD 2 solution. The beginning enzymatic hydrolysis is indicated by a decrease of pH which is kept constant at pH 8.5 by continuous addition of 1 N NaOH-solution using an autotitrator. The specific activity of the enzymes 6 was calculated from the initial rate of the transformation. After completion the obtained reaction mixtures were filtered over Celite ®, concentrated (vacuum) to 10 -20 ml and then acidified with HC1 to pH 2-3. With the exception of the products resulting from (+)-9 and (+)-10, the N-carbamoyl-D-amino acids were isolated by filtration after ice cooling, washed with a small amount of water and then dried. They were further converted into the corresponding D-amino acids by diazotation. For this 1-3 mmol of the corresponding carbamoylate were dissolved in 50 ml 3.5 N HCI to which at 0°C an equimolar quantity of NaNO2, dissolved in 10 ml H20 was added under vigorous stirring. The resulting D- amino acids were purified by ion exchange chromatography (25 g DOWEX ® 50). Their enantiomeric purities were determined via derivatisation with 2,3,4,6-tetra-O-benzoyl-13-D-glucopyranosylisothiocyanate BGIT 7 followed by HPLC analysis [LiChrospher 100 RP-18; acetonitrile/0.1% trifluoroacetic acid] of the resulting diastereomeric thiourethanes. The optical purities of D-23-27 were determined by HPLC using a chiral column [Chrownpack ® CR(+), Daicel]. The results, also showing the broad substrate tolerance, are summarized in the table. Using (+)-2 and (+)-3 as model substrates the dependence of the specific activities from (a) buffer, (b) pH (Fig.2) and temperature (Fig.3) were determined for both enzymes. Glycine/NaOH buffer proved to be superior to TRIS/I-ICI, a pH 8.5 resulting in the highest activity for both D-HYD 1 (85 U/mg protein) and D-HYD 2 (200 U/mg protein) (Fig.2). A ten fold increase was observed in changing the temperature from 37°C to 80°C (Fig.3). Both enzymes display a remarkable temperature stability and can be used for several hours at temperatures as high as 70°C, while at T>80°C increasing loss of activity is observed. A similar stability was reported previously only in one case (hydantoinase from Agrobacterium sp. IP-6718). 200.00 i5000 m 10000
`0.00 t J [ 700 8.00 9.00 1000 II 00 pH D-Hydaatoinase-2 3oo.oo ................. D-Hydantoinu¢-I 1 TRIS/HCI 0.I N ~ GIy/NaOH 0.1 N 0". I I ' I ' T 20.00 40.00 60.00 80.00 100,00 temperature [~l Fig.2: Effect of the pH on the specific activity in 0.1 N glycine/NaOH and 0.1 N TRIS/HCI buffer using DL-n-butylhydantoin (+)-2 as substrate Fig.3: Effect of temperature on the specific activity of hydantoinase catalysed hydrolysis of (:t=)-5-(J3-phenylethyl)hydantoin (+)-3 with D-HYD 2
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`IPR2014-01126-1033 p. 2
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`New hydantoinases 1259 Table Specific activity, reaction time, isolated yield, optical rotation of the N-carbamoyl-amino acids and the enantiomeric purities of the isolated amino acids Substratc Enzyme Specific Reaction Isolated [OqD 20 cnanfiomcric (5-substituted hydantoin) Activity time [h] yield of (c=l in excess of the [Ulm~] carbamoylate MeOH) amino acid [%] DL-5-Methyl (+)-1 D-HYD-1 133 22.8 71% -4.0 Alanine 94 D-HYD-2 769 7.6 73% -1.8 D-15 34 DL-5-n-Butyl 0:)-2 D-HYD-1 82 23.3 67% -14.0 Norleucine >99 D-HYD-2 127 21.5 70% -11.5 D-16 83 DL-5 -(]3-Phenylethyl) D-HYD-1 63 41.2 53% -19.0 Homophenyl- >99 (+)-3 DoHYD-2 70 27.2 77% - 17.8 alanine D-17 >99 DL-5-Isobutyl (+)-4 D-HYD-1 94 19.2 89% +5.8 a) Leucine >99 D-HYD-2 156 18.0 93% +4.7 a) D-18 >99 DL-5-Benzyl (+)-5 D-HYD-1 47 39.2 67% -46.7 Phenylalanine >99 D-HYD-2 5 29.2 12% -43.7 D-19 >99 DL-5-Isopropyl (+)-6 D-HYD-1 61 45.0 66% -15.6 Valine >99 D-HYD-2 65 43.7 71% -16.0 D-20 >99 DL-5-sec-Butyl (-1-)-7 D-HYD-1 29 24.2 65% -23.1 Isoleucine >99, >99 b) D-I-IYD-2 23 41.0 71% -23.0 D-21a,b b) >99, >99 b) DL-5-Methylthioethyl D-HYD-1 71 12.7 81% -1.7 Methionine >99 (+)-8 D-I--IYD-2 358 14.1 75% -1.4 D-22 99 DL-5-Hydroxymethyl D-I--IYD- 1 27 16.6 Serine 98 (+)-9 D-I--IYD-2 156 10.2 D-23 97 DL-5 -(2-Hydroxyethyl), D-HYD-1 61 21.0 Threonine >98 (+)-10 (allo-free) D-HYD-2 78 21.2 D-24a,b b) >98 DL-5-(2-Thienyl) D-I--IYD- 1 612 0.5 42% -110.4 (2-Thienyl)glycine 97 (±)-11 D-HYD-2 1351 0.3 50% -111.9 D-25 96 DL-5-Phenyl D-HYD-1 620 0.75 95% -143.3 Phenylglycine 96 (+)-12 D-HYD-2 1221 0.6 90% -152.5 D-26 >99 DL-5-(p-Hydroxyphenyl) D-HYD-1 302 5.8 65% -172.0 (p-Hydroxyphenyl) >99 (±)-13 D-HYD-2 52 11.0 78% -167.5 ~lycine D-27 >99 DL-5-Methyl-5-phenyl D-HYD-1 <1 72 3 2-Phenylalanine >99 (5:)-14 D-HYD-2 <1 72 7 D-28 97 a) c=0.5 in MeOH b) allo-configurafion; enantiomeric purity of a/to-threonJne not determined D-HYD 2 usually displays higher activities with aliphatic substituted hydantoins (_+)-1-7 than D-HYD 1. Considerable activity differences were observed for compounds (_+)-1,2,4 which have two or more protons at C(1) next to the ring system, and also for the aliphatic heterosubstituted hydantoins (_+)-8- 10. The specific activity of both D-hydantoinases seems to decrease with increasing steric bulk in (_+)-1-7. In the hydrolysis of the aromatic substituted compounds (_+)-11-13 remarkably high specific activities were observed, most likely due to the high acidity of the proton on C(5) of the hydantoin ring.
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`IPR2014-01126-1033 p. 3
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`1260 O. KE1L et al. In contrast to this, both hydantoinases display only a very small activity towards the 5,5-disubstituted hydantoin (+)-14. The resulting N-carbamoyl-D-amino acids were isolated in high yields (>75%) and further converted into the corresponding D-amino acids, which were obtained with excellent enantiomeric purities. D-HYD 1 usually leads to higher enantiomeric purities for aliphatic amino acids D-15-24 as compared to D- HYD 2, which in turn is the preferred enzyme for the produced D-amino acids with aromatic substituents D- 25-27. Both enzymes display no diastereoselectivity, hydrolysis of(+)-7 and (+)-10 produces diastereomeric product mixtures. In summary, the described thermostable D-hydantoinases catalyze the highly enantioselective hydrolysis of hydantoins with great structural variety. They thus allow the synthesis of numerous a-D-amino acids in high chemical and optical yields. REFERENCES 1. J. Kamphuis, W.H.J. Boesten, B. Kaptein, H.F.M. Hermes, T. Sonke, Q.B. Broxtermann, W.J.J. van den Tweel, H.E. Schoemaker; "Chirality in industry", Eds. A.N. Collins, G.N. Sheldrake, J. Crosby, Wiley & Sons Ltd. (1992) 187-208 2. W. Diarckheimer, J. Blumbach, R. Lattrell, K.H. Scheunemann, Angew. Chem. 97 (1985) 183-205 3. A.A. Patchett, Nature (London) 288 (1980) 280 4. R.M. Williams, "Synthesis of Optically Active ct-Amino Acids", Organic Chemistry Series, Eds. J.E. Baldwin, FRS & P D Magnus, FRS, Vol.7, Pergamon Press (1989) 5. H.T. Bucherer, W. Steiner, J. prakt. Chem. 140 (1934) 291-316 6. D-HYD 1 (14 nag protein/ml); specific activity 2.4 kU/ml; [1% (w/v) 5-phenylhydantoin, 1 mM MnC12, pH 8.5, 37°C] and D-HYD-2 (55 mg protein/ml); specific activity 12 kU/ml [1 °A (w/v) 5-phenylhydantoin, 1 mM MnCI2, pH 8.5, 37°C]. Both enzymes are originally derived from thermophilic microorganisms and are expressed in Escherichia coll. They are commercially available from Boehringer Mannheim. 7. M. Lobell, M.P. Schneider, J Chromatogr. 633 (1993) 287-294 8. S. Runser, E. Ohleyer, Biotechnol. Left. 12(4) (1990) 259-264 (Received in UK 11 May 1995)
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`IPR2014-01126-1033 p. 4