BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 234, 485–488 (1997) ARTICLE NO. RC976659 A Thermostable D-Hydantoinase Isolated from a Mesophilic Bacillus sp.AR9 Rakesh Sharma and Rakesh M. Vohra 1 Biochemical Engineering Research and Process Development Centre, Institute of Microbial Technology, Sector 39-A, Chandigarh-160036, India Received April 1, 1997 D-hydantoinase from a thermophilic Bacillus stearoth- A thermostable hydantoinase has been character- ermophilus SD1 which has a half life of 20 min at 80°C. ized from a mesophilic Bacillus sp.AR9. The hydantoi- In this paper we report a D-hydantoinase from a meso- nase produced by this Bacillus sp.AR9 is strictly D- philic Bacillus sp.AR9 which is not only alkalo- and specific and is constitutively produced with high thermostable but also has a pH and temperature opti- yields (4500 U/ml) in this strain. The enzyme is not only mum in the desired range. alkalo- and thermostable but has a pH and tempera- ture optimum of 9.5 and 65°C, respectively, which is MATERIALS AND METHODS advantageous for the bioconversion of DL-5-monosub- stituted-hydantoin derivatives. The enzyme has a half Chemicals life of 80 minutes at 70°C and loses only 33% of its activ- ity in 4 hr at 60°C. The enzyme has a broad substrate D- and L-p-Hydroxyphenylglycine, Phenylglycine, Hydantoin, Hy- dantoic acid, Dihydrouracil, p-Dimethylaminobenzaldehyde and Chi- specificity with a maximum of 100% with hydantoin ral TLC plates were purchased from Sigma. DL-5-monosubstituted- and about 26% with dihydrouracil. Co// ions enhance hydantoin derivatives were synthesized from corresponding alde- the activity of the enzyme by more than 60%.  1997 hydes (10) or from corresponding amino acids (11). All other reagents Academic Press used were of analytical grade. Production of Biocatalyst D-Hydantoinase (dihydropyrimidinase E.C. 3.5.2.2), Bacillus sp.AR9 was grown in a medium having the following com- has been exploited for stereoselective hydrolysis of DL- position (in gram per liter) Peptone 10g, Meat Extract 5g, KCl 2g, Ca(NO 3 ) 2 0.2g, MgSO 4 .7H 2 O 0.5g, MnCl 2 .2H 2 O 0.02g, FeSO 4 .7H 2 O 5-monosubstituted hydantoin derivatives to N-carbam- 0.002g with a pH of 7.2. The cells were cultured in 200 ml of medium oyl-D-amino acids (1,2) which are further hydrolyzed in one liter flasks at 37°C at 200 r.p.m. for 18-20h. Cells were har- chemically or enzymatically to D-amino acids(3,4). A vested by centrifugation at 8000g for 10 min. These cells were then thermostable D-hydantoinase with a pH optimum in suspended in 100mM phosphate buffer pH 8.0 (40% w/v) and ultra- sonically disrupted and centrifuged at 14000g for 1 h. The superna- the alkaline range is desirable for two reasons one, the tant thus obtained was used as enzyme in all subsequent experi- substrate solubility increases at higher temperatures ments. two,the process of self-racemization takes place under alkaline conditions (5) thereby resulting in increased Assay of Hydantoinase productivity. A predetermined amount of cells or 50 ml of enzyme was incubated The D-hydantoinases characterized so far have low with 50 mM hydantoin in 100 mM phosphate buffer pH 8.0 for 15 thermostability and pH optima near neutrality. Of the min at 50°C in a total volume of 500 ml. The reaction was terminated five D-hydantoinases isolated from Pseudomonas sps. by the addition of 125 ml 12 % (w/v) trichloroacetic acid followed by all were stable only up to 45°C (6). A D-hydantoinase the addition of 125 ml of p-dimethylaminobenzaldehyde (10 % w/v in from Peptococcus anaerobius had a half life of 1 h at 6N HCl). This was then diluted to 1.5 ml by the addition of distilled water and centrifuged to remove the precipitate. Absorbance of the 60°C (7). D-hydantoinases reported from Agrobacter- supernatent was determined at 420 n.m.. One unit of enzyme activity ium sps. (1,8) were found to be relatively stable how- was defined as one mmole of product formed per minute under the ever, little if any data has been provided for their ther- assay condition. mostability. A recent study (9) reports a thermostable Determination of Stereoselectivity Chiral thin layer chromatography. N-carbamoyl-p-hydroxyphe- 1 Corresponding author. Fax: (91)-172-690585, 690632. E-mail: vohra@koel.imtech.ernet.in. nylglycine produced from DL-p-hydroxyphenylhydantoin was con- 0006-291X/97 $25.00 Copyright  1997 by Academic Press All rights of reproduction in any form reserved. 485