• Microbiology and Biotechnology Letters
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• v.18 no.5
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• pp.476-483
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• 1990

In order to investigate hydantoinase-producing strain of the genus Streptomyces, 523 strains of Streptomycee sp. isolated from soils were cultivated in various media and conversion activity of the enzyme was measured to DL-5-phenylhydantoin. A number of strains producing hydantoinase were detected and among them, the strain of Streptomyces sp. Y-183 was selected as a most powerful strain to producing the enzyme. The optimal culture conditions for the production of hydantoinase of the strain were studied, and it was found that almost all hydantoinase activity was produced in the cell fraction. The maximum activity of the enzyme, 17.8 unitstg of dried cells weight, was obtained when the strain was cultured at $30^{\circ}C$ for 72 hr in a medium containing 1.0% of glycerol. 0.5% of yeast extract. 0.5% of soytone, 0.5% of beef extract, 0.6% of KCI, 0.002% of $K_2HP0_4, 0.25% \;of \;CaC0_3, \;0.0002% \; of \; ZnSO_4, \; 0.0002%\; of\; FeS0_4$, and 0.4% of uracil as an inducer, and the pH of culture broth was adjusted ranging from 7.0 to 7.5.

• Journal of Microbiology and Biotechnology
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• v.12 no.2
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• pp.242-248
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• 2002

Previously, it was reported that the nonhomologous C-terminal regions of the D-hydantoinases are nonessential for catalysis, but affect the oligomeric structure of the enzyme [3]. In an effort to further confirm the above observation, the C-terminal region-inserted enzyme was constructed by attaching a peptide (22 residues) at the C-terminal of the D-hydantoinase from Bacillus thermocatenulatus GH2, and its structural and biochemical properties were compared with both the wild-type and C-terminal region-truncated enzymes. As a result, native tetrameric D-hydantoinase was dimerized as the truncated enzyme, and the inserted mutant with a new sequence was expressed as a catalytically active form in E. coli. Expression level of the inserted and truncated enzymes were found to be significantly increased compared to the level of the wild-type enzyme, and this appears to be due to the reduced toxic effect of the mutant enzymes on host cells. Dimerized enzymes exhibited increased thermo- and pH stabilities considerably when compared with the corresponding wild-type enzyme. Comparison of the substrate specificity between the mutant and wild-type enzymes suggests that the substrate specificity of the D-hydantoinase is closely linked with the oligomeric structure.

• 한국생물공학회:학술대회논문집
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• 2000.11a
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• pp.604-607
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• 2000

Preparation for the D-phenylalanine using chemo-enzymatic reaction was investigated. D,L-5-benzylhydantoin was synthesized chemically from L-phenylalanine and converted to N-carbamoyl-D-phenylalanine by the immobilized hydantoinase. The pH and temperature affected the solubility and racemization rate of benzylhydantoin. The optimal temperature and pH of the process were $50^{\circ}C$, 8.5, respectively and the conversion yield hadn't much difference with the hydantoinase content in 10hrs. This produced N-carbamoyl-D-phenylalanine was transformed chemically into D-phenylalanine.

• Journal of Microbiology and Biotechnology
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• v.19 no.12
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• pp.1497-1505
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• 2009

The thermophile Bacillus fordii MH602 was screened for stereospecifically hydrolyzing DL-5-substituted hydantoins to L-$\alpha$-amino acids. Since the reaction occurs at higher temperature, the advantages for enhancement of substrate solubility and for racemization of DL-5-substituted hydantoins during the conversion were achieved. The hydantoin metabolism gene cluster from thermophile is firstly reported in this paper. The genes involved in hydantoin utilization (hyu) were isolated on an 8.2-kb DNA fragment by restriction site-dependent PCR, and six ORFs were identified by DNA sequence analysis. The hyu gene cluster contained four genes with novel cluster organization characteristics: the hydantoinase gene hyuH, putative transport protein gene hyuP, hyperprotein gene hyuHP, and L-carbamoylase gene hyuC. The hyuH and hyuC genes were heterogeneously expressed in E. coli. The results indicated that hyuH and hyuC are involved in the conversion of DL-5-substituted hydantoins to an N-carbamyl intermediate that is subsequently converted to L-$\alpha$-amino acids. Hydantoinase and carbamoylase from B. fordii MH602 compared respectively with reported hydantoinase and carbamoylase showed the highest identities of 71% and 39%. The novel cluster organization characteristics and the difference of the key enzymes between thermopile B. fordii MH602 and other mesophiles were presumed to be related to the evolutionary origins of concerned metabolism.

• Biotechnology and Bioprocess Engineering:BBE
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• v.7 no.3
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• pp.155-162
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• 2002

The cyclic amidohydrolase family enzymes, which include allantoinase, dihydroorotase, dihydropyrimidinase and (phenyl)hydantoinase, are metal-dependent hydrolases and play a crucial role in the metabolism of purine and pyrimidine in vivo. Each enzyme has been independently characterized, and thus well documented, but studies on the higher structural traits shared by members of this enzyme family are rare due to the lack of comparative study. Here, we report upon the expression in E. coli cells of maltose-binding protein (MBP)- and glutathione S-transferase (GST)-fused cyclic amidohydrolase family enzymes, facilitating also for both simple purification and high-level expression. Interestingly, the native quaternary structure of each enzyme was maintained even when fused with MBP and GST. We also found that in fusion proteins the favorable biochemical properties of family enzymes such as, their optimal pHs, specific activities and kinetic properties were conserved compared to the native enzymes. In addition, MBP-fused enzymes showed remarkable folding ability in-vitro. Our findings, therefore, suggest that a previously unrecognized trait of this family, namely the ability to functional fusion with some other protein but yet to retain innate properties, is conserved. We described here the structural and evolutionary implications of the properties in this family enzyme.

• Proceedings of the Korean Society for Applied Microbiology Conference
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• 2003.06a
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• pp.147-147
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• 2003

• Analytical Science and Technology
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• v.8 no.4
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• pp.465-468
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• 1995

Matrix assisted laser desorption ionization in mass spectrometry is a fast and accurate method to determine the molecular weight of natural and synthetic polymers. Unknown peptides such as elastase inhibitor and $\small{D}$-hydantoinase were analyzed using sinapinic acid as matrix and their molecular weights were compared with the results from protein sequencer and gel filtration chomatography, respectively. Synthetic polymers such as polyethyleneglycol, polypropyleneglycol, polydimethylsiloxane, and polystyrene were analyzed using matrices such as 2,5-dihydroxybenzoic acid, 4-hdroxyazobenzenecarboxylic acid, and 2-nitrophenyl octyl ether. Average molecular weights of polystyrene were compared with molecular weights by gel permeation chromatography.