title: Gene cloning, expression, and characterization of a novel acetaldehyde dehydrogenase from Issatchenkia terricola strain XJ-2.
abstract: Acetaldehyde is a known mutagen and carcinogen. Active aldehyde dehydrogenase (ALDH) represents an important mechanism for acetaldehyde detoxification. A yeast strain XJ-2 isolated from grape samples was found to produce acetaldehyde dehydrogenase with a high activity of 2.28 U/mg and identified as Issatchenkia terricola. The enzyme activity was validated by oxidizing acetaldehyde to acetate with NAD(+) as coenzyme based on the headspace gas chromatography analysis. A novel acetaldehyde dehydrogenase gene (ist-ALD) was cloned by combining SiteFinding-PCR and self-formed adaptor PCR. The ist-ALD gene comprised an open reading frame of 1,578 bp and encoded a protein of 525 amino acids. The predicted protein of ist-ALD showed the highest identity (73%) to ALDH from Pichia angusta. The ist-ALD gene was expressed in Escherichia coli, and the gene product (ist-ALDH) presented a productivity of 442.3 U/mL cells. The purified ist-ALDH was a homotetramer of 232 kDa consisting of 57 kDa-subunit according to the SDS-PAGE and native PAGE analysis. Ist-ALDH exhibited the optimal activity at pH 9.0 and 40°C, respectively. The activity of ist-ALDH was enhanced by K(+), NH4(+), dithiothreitol, and 2-mercaptoethanol but strongly inhibited by Ag(+), Hg(2+), Cu(2+), and phenylmethyl sulfonylfluoride. In the presence of NAD(+), ist-ALDH could oxidize many aliphatic, aromatic, and heterocyclic aldehydes, preferably acetaldehyde. Kinetic study revealed that ist-ALDH had a k                         (cat) value of 27.71/s and a k                         (cat)/K                         (m) value of 26.80 × 10(3)/(mol s) on acetaldehyde, demonstrating ist-ALDH, a catalytically active enzyme by comparing with other ALDHs. These studies indicated that ist-ALDH was a potential enzymatic product for acetaldehyde detoxification.
authors.affiliation: College of Food Science and Technology, Nanjing Agricultural University, Nanjing, 210095, Jiangsu, People's Republic of China.
authors.forename: Zhengying
authors.initials: Z
authors.lastname: Yao
authors.forename: Chong
authors.initials: C
authors.lastname: Zhang
authors.forename: Fengxia
authors.initials: F
authors.lastname: Lu
authors.forename: Xiaomei
authors.initials: X
authors.lastname: Bie
authors.forename: Zhaoxin
authors.initials: Z
authors.lastname: Lu
mesh_terms: D000079:Acetaldehyde / Q000378:metabolism*; D000445:Aldehyde Oxidoreductases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D016000:Cluster Analysis; D004271:DNA, Fungal / Q000737:chemistry / Q000235:genetics; D020536:Enzyme Activators / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000378:metabolism; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009243:NAD / Q000378:metabolism; D016366:Open Reading Frames; D010802:Phylogeny; D010843:Pichia / Q000235:genetics; D055503:Protein Multimerization; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004718:Saccharomycetales / Q000145:classification / Q000201:enzymology* / Q000235:genetics / Q000302:isolation & purification; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D013696:Temperature; D027843:Vitis / Q000382:microbiology
chemical_list: D004271:DNA, Fungal; D020536:Enzyme Activators; D004791:Enzyme Inhibitors; D011994:Recombinant Proteins; D009243:NAD; D000445:Aldehyde Oxidoreductases; C018699:aldehyde dehydrogenase (NAD(P)+); D000079:Acetaldehyde