title: A novel L-aspartate dehydrogenase from the mesophilic bacterium Pseudomonas aeruginosa PAO1: molecular characterization and application for L-aspartate production.
abstract: L-aspartate dehydrogenase (EC 1.4.1.21; L: -AspDH) is a rare member of amino acid dehydrogenase superfamily and so far, two thermophilic enzymes have been reported. In our study, an ORF PA3505 encoding for a putative L-AspDH in the mesophilic bacterium Pseudomonas aeruginosa PAO1 was identified, cloned, and overexpressed in Escherichia coli. The homogeneously purified enzyme (PaeAspDH) was a dimeric protein with a molecular mass of about 28 kDa exhibiting a very high specific activity for L-aspartate (L-Asp) and oxaloacetate (OAA) of 127 and 147 U mg(-1), respectively. The enzyme was capable of utilizing both nicotinamide adenine dinucleotide (NAD) and nicotinamide adenine dinucleotide phosphate (NADP) as coenzyme. PaeAspDH showed a T (m) value of 48°C for 20 min that was improved to approximately 60°C by the addition of 0.4 M NaCl or 30% glycerol. The apparent K (m) values for OAA, NADH, and ammonia were 2.12, 0.045, and 10.1 mM, respectively; comparable results were observed with NADPH. The L-Asp production system B consisting of PaeAspDH, Bacillus subtilis malate dehydrogenase and E. coli fumarase, achieved a high level of L-Asp production (625 mM) from fumarate in fed-batch process with a molar conversion yield of 89.4%. Furthermore, the fermentative production system C released 33 mM of L-Asp after 50 h by using succinate as carbon source. This study represented an extensive characterization of the mesophilic AspDH and its potential applicability for efficient and attractive production of L-Asp. Our novel production systems are also hopeful for developing the new processes for other compounds production.
authors.affiliation: Department of Life Science and Biotechnology, Faculty of Life and Environmental Science, Shimane University, 1060 Nishikawatsu, Matsue, Shimane, Japan.
authors.forename: Yinxia
authors.initials: Y
authors.lastname: Li
authors.forename: Norika
authors.initials: N
authors.lastname: Kawakami
authors.forename: Henry Joseph Oduor
authors.initials: HJ
authors.lastname: Ogola
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Ashida
authors.forename: Takahiro
authors.initials: T
authors.lastname: Ishikawa
authors.forename: Hitoshi
authors.initials: H
authors.lastname: Shibata
authors.forename: Yoshihiro
authors.initials: Y
authors.lastname: Sawa
mesh_terms: D000594:Amino Acid Oxidoreductases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D001224:Aspartic Acid / Q000378:metabolism*; D003001:Cloning, Molecular; D003067:Coenzymes / Q000378:metabolism; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D007700:Kinetics; D008970:Molecular Weight; D009243:NAD / Q000378:metabolism; D009249:NADP / Q000378:metabolism; D062907:Oxaloacetic Acid / Q000378:metabolism; D055503:Protein Multimerization; D011550:Pseudomonas aeruginosa / Q000201:enzymology* / Q000235:genetics*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity; D013696:Temperature
chemical_list: D003067:Coenzymes; D011994:Recombinant Proteins; D009243:NAD; D062907:Oxaloacetic Acid; D001224:Aspartic Acid; D009249:NADP; D000594:Amino Acid Oxidoreductases; C025745:aspartate dehydrogenase