Relationship between an increase in thermostability and amino acid substitutions in N-carbamyl-D-amino acid amidohydrolase.
For the production of D-amino acids using stable N-carbamyl-D-amino acid amidohydrolase (DCase) in an immobilized form, the DCase gene of Agrobacterium sp. KNK712 was mutagenized to increase its enzymatic thermostability. In a search for thermostability-related amino acid sites besides the two known sites of DCase, i.e., the 57th and 203rd amino acids, the new mutant enzyme found, in which the 236th amino acid, valine, had been changed to alanine, showed a 10 degrees C increase in thermostability. These known three thermostability-related amino acids were changed to other amino acids by the PCR technique, and it was proved that the thermostability of the DCase increased when the 57th amino acid of DCase, histidine, was changed to leucine, the 203rd amino acid, proline, to asparagine, glutamate, alanine, isoleucine, histidine, or threonine, and the 236th amino acid, valine, to threonine or serine, in addition to the known mutations.
Ikenaka
Y
Y
Fine Chemicals Research Laboratories, Kaneka Corporation, Hyogo, Japan. Yasuhiro.Ikenaka@kaneka.co.jp
Nanba
H
H
Yajima
K
K
Yamada
Y
Y
Takano
M
M
Takahashi
S
S
D000581:Amidohydrolases / Q000737:chemistry* / Q000235:genetics; D019943:Amino Acid Substitution; D001665:Binding Sites; D004795:Enzyme Stability; D005798:Genes, Bacterial; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D016296:Mutagenesis; D012231:Rhizobium / Q000201:enzymology* / Q000235:genetics; D013329:Structure-Activity Relationship
D000581:Amidohydrolases; C112401:N-carbamyl-D-amino acid amidohydrolase