A 1440bp open-reading frame encoding D-hydantoinase from Pseudomonas putida YZ-26 was cloned and sequenced（ GenBank AY387829）. The DNA fragment was inserted into Nde and BamHI sites of vector pET-3a, yielding a reco...A 1440bp open-reading frame encoding D-hydantoinase from Pseudomonas putida YZ-26 was cloned and sequenced（ GenBank AY387829）. The DNA fragment was inserted into Nde and BamHI sites of vector pET-3a, yielding a recombinant plasmid, pET-HDT. After being transferred into the host strain, the artificial strain, pET-HDT/ E. coli BL21 can express the D-hydantoinase as the soluble form in the Lura-Bertani medium without addition of any inducers. The activity of the enzyme toward substrate DL-hydantoin can reach 3000-4000 IU per cells from one-liter bacterial culture incubated at 30 ℃ for 10-12 h. By the comparison of amino acid sequence homology, hydrophobic residues analysis and secondary structure prediction, it was found that D-hydantoinase reported herein is quite similar to that from Pseudomonas putdia CCRC12857, and alike to that from Pseudomonas putdia DSM84 or other bacteria. A rapid and efficient purification procedure of the enzyme was performed by a three-step procedure： ammonium sulfate fractionation, phenyl Sepharose hydrophobic interaction chromatography and Sephacryl S-200 gel filtration. The molecular mass of the monomeric enzyme is 52042 Da as determined by MALDI-TOF mass spectrometry.展开更多
Hydantoinase and N-carbamoylase play important rol es in the production of optically pure amino acids from racemic 5-monosubstitut ed hydantoins. In this report, hydantoinase and the N-carbamoylase from Burkh olderia ...Hydantoinase and N-carbamoylase play important rol es in the production of optically pure amino acids from racemic 5-monosubstitut ed hydantoins. In this report, hydantoinase and the N-carbamoylase from Burkh olderia cepecia.njut01 were purified to homogeneity by chromatography (Pharma cia Explorer 100 system). The substrate specificity, enantioselectivity, pH depe ndence of activity and temperature stability of the activity were characterized. The results show that the hydantoinase and N-carbamoylase induced from Burkh olderia cepecia.njut01 are both strict D-stereo selective enzymes. They both hydrolyze substrates with side chains containing aliphatic and aromatic res idues with higher activity and affinity toward aromatic than aliphatic substitu ted substrates. The hydantoinase is a homotetramer with subunit molecular weight near 52,000 and is active between pH 6.5 and 10 with an optimum near pH 9.0. The en zyme is active at temperatures up to 60°C, however,it appears instable at h ig her temperatures. The subunit molecular weight of N-carbamoylase is about 35KD. The N-carbamoylase is active in the pH range from 6.0 to 9.5. The optim-pH is 7.2 and the optimizing bioconversion temperature of the N-carbamyolase is 52 °C.展开更多
基金Supported by the Natural Science Foundation of Shanxi Province(No.20031042).
文摘A 1440bp open-reading frame encoding D-hydantoinase from Pseudomonas putida YZ-26 was cloned and sequenced（ GenBank AY387829）. The DNA fragment was inserted into Nde and BamHI sites of vector pET-3a, yielding a recombinant plasmid, pET-HDT. After being transferred into the host strain, the artificial strain, pET-HDT/ E. coli BL21 can express the D-hydantoinase as the soluble form in the Lura-Bertani medium without addition of any inducers. The activity of the enzyme toward substrate DL-hydantoin can reach 3000-4000 IU per cells from one-liter bacterial culture incubated at 30 ℃ for 10-12 h. By the comparison of amino acid sequence homology, hydrophobic residues analysis and secondary structure prediction, it was found that D-hydantoinase reported herein is quite similar to that from Pseudomonas putdia CCRC12857, and alike to that from Pseudomonas putdia DSM84 or other bacteria. A rapid and efficient purification procedure of the enzyme was performed by a three-step procedure： ammonium sulfate fractionation, phenyl Sepharose hydrophobic interaction chromatography and Sephacryl S-200 gel filtration. The molecular mass of the monomeric enzyme is 52042 Da as determined by MALDI-TOF mass spectrometry.
文摘Hydantoinase and N-carbamoylase play important rol es in the production of optically pure amino acids from racemic 5-monosubstitut ed hydantoins. In this report, hydantoinase and the N-carbamoylase from Burkh olderia cepecia.njut01 were purified to homogeneity by chromatography (Pharma cia Explorer 100 system). The substrate specificity, enantioselectivity, pH depe ndence of activity and temperature stability of the activity were characterized. The results show that the hydantoinase and N-carbamoylase induced from Burkh olderia cepecia.njut01 are both strict D-stereo selective enzymes. They both hydrolyze substrates with side chains containing aliphatic and aromatic res idues with higher activity and affinity toward aromatic than aliphatic substitu ted substrates. The hydantoinase is a homotetramer with subunit molecular weight near 52,000 and is active between pH 6.5 and 10 with an optimum near pH 9.0. The en zyme is active at temperatures up to 60°C, however,it appears instable at h ig her temperatures. The subunit molecular weight of N-carbamoylase is about 35KD. The N-carbamoylase is active in the pH range from 6.0 to 9.5. The optim-pH is 7.2 and the optimizing bioconversion temperature of the N-carbamyolase is 52 °C.
