Creation of a thermostable NADP+-dependent d-amino acid dehydrogenase from Ureibacillus thermosphaericus strain A1 meso-diaminopimelate dehydrogenase by site-directed mutagenesis

Hironaga Akita, Katsumi Doi, Yutaka Kawarabayasi, Toshihisa Ohshima

Research output: Contribution to journalArticle

18 Citations (Scopus)

Abstract

A thermostable, NADP+-dependent d-amino acid dehydrogenase (DAADH) was created from the meso-diaminopimelate dehydrogenase of Ureibacillus thermosphaericus strain A1 by introducing five point mutations into amino acid residues located in the active site. The recombinant protein, expressed in Escherichia coli, was purified to homogeneity using a two-step separation procedure and then characterized. In the presence of NADP+, the protein catalyzed the oxidative deamination of several d-amino acids, including d-cyclohexylalanine, d-isoleucine and d-2-aminooctanoate, but not meso-diaminopimelate, confirming the creation of a NADP+-dependent DAADH. For the reverse reaction, the corresponding 2-oxo acids were aminated in the presence of NADPH and ammonia. In addition, the d-amino acid dehydrogenase showed no loss of activity at 65 °C, indicating the mutant enzyme was more thermostable than its parental meso-diaminopimelate dehydrogenase.

Original languageEnglish
Pages (from-to)1693-1699
Number of pages7
JournalBiotechnology letters
Volume34
Issue number9
DOIs
Publication statusPublished - Jan 1 2012

Fingerprint

diaminopimelate dehydrogenase
Mutagenesis
Site-Directed Mutagenesis
NADP
Amino acids
Oxidoreductases
Amino Acids
Keto Acids
Recombinant proteins
Deamination
Isoleucine
Ammonia
Point Mutation
Recombinant Proteins
Escherichia coli
Catalytic Domain
Enzymes
Proteins

All Science Journal Classification (ASJC) codes

  • Biotechnology
  • Bioengineering
  • Applied Microbiology and Biotechnology

Cite this

Creation of a thermostable NADP+-dependent d-amino acid dehydrogenase from Ureibacillus thermosphaericus strain A1 meso-diaminopimelate dehydrogenase by site-directed mutagenesis. / Akita, Hironaga; Doi, Katsumi; Kawarabayasi, Yutaka; Ohshima, Toshihisa.

In: Biotechnology letters, Vol. 34, No. 9, 01.01.2012, p. 1693-1699.

Research output: Contribution to journalArticle

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