Overexpression and characterization of hydantoin racemase from Agrobacterium tumefaciens C58
Hydantoin racemase enzyme together with a stereoselective hydantoinase and a stereospecific d-carbamoylase guarantee the total conversion from d, l-5-monosubstituted hydantoins with a low velocity of racemization to optically pure d-amino acids. In this work we have cloned and expressed the hydantoi...
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Veröffentlicht in: | Biochemical and biophysical research communications 2003-04, Vol.303 (2), p.541-547 |
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Sprache: | eng |
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Zusammenfassung: | Hydantoin racemase enzyme together with a stereoselective hydantoinase and a stereospecific
d-carbamoylase guarantee the total conversion from
d,
l-5-monosubstituted hydantoins with a low velocity of racemization to optically pure
d-amino acids. In this work we have cloned and expressed the hydantoin racemase gene from two strains of
Agrobacterium tumefaciens, C58 and LBA4404, in
Escherichia coli BL21. The recombinant protein was purified in a one-step procedure by using immobilized cobalt affinity chromatography and showed an apparent molecular mass of 32,000
Da in SDS–gel electrophoresis. Size exclusion chromatography analysis determined a molecular mass of about 100,000
Da, suggesting that the native enzyme is a tetramer. The optimal conditions for hydantoin racemase activity were pH 7.5 and 55
°C with
l-5-ethylhydantoin as substrate. Enzyme activity was slightly affected by the addition of Ni
2+ and Co
2+ and strongly inhibited by Cu
2+ and Hg
2+. No effect on enzyme activity was detected with Mn
2+, EDTA, or DTT. Kinetic studies showed the preference of the enzyme for hydantoins with short rather than long aliphatic side chains or hydantoins with aromatic rings. |
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ISSN: | 0006-291X 1090-2104 |
DOI: | 10.1016/S0006-291X(03)00377-2 |