Publications (2)2.3 Total impact
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Article: Molecular cloning and expression of the complete DNA sequence encoding NAD+-dependent acetaldehyde dehydrogenase fromAcinetobacter sp. strain HBS-2
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ABSTRACT: The aldehyde dehydrogenase (ALDH) gene fromAcinetobacter sp. strain HBS-2 was cloned and characterized. The ORF of the gene was a 1467-bp sequence that encoded a 489-amino-acid protein. Strain HBS-2 carries a newALDH gene that differs from the knownALDH genes inAcinetobacter, and the product coded is a member of the NAD+-dependent ALDH family with 99% identity to several knownAcinetobacter baumannii orthologs. The determined sequence exhibited 56, 59 and 39% identity with human mitochondrial ALDH2, and ALDH fromPseudomonas aeruginosa andEscherichia coli, respectively. TheALDH gene was cloned into the pET28a(+) expression vector and expressed inE. coli BL21 cells under the control of the IPTG-inducible promoter T7. Two-thirds of the recombinant enzyme was available in soluble form, and its molecular mass was estimated at 57 kDa by SDS-PAGE. After purification on an Ni-sepharose column, the recombinant protein was found to have a specific activity of 60.6 U/mg protein. TheK m values of the enzyme were 15.18 μM for acetaldehyde, 2.12 μM for formaldehyde, and 0.49 μM for propionaldehyde. These results indicate that ALDH from strain HBS-2 preferentially reacts with propionaldehyde. However,V max of the enzyme for acetaldehyde was determined to be the highest. The recombinant enzyme exhibited novel biochemical characteristics. Its activity was significantly elevated in the presence of Mn2+. These findings are different from those reported for ALDHs from other microorganisms.Annals of Microbiology 04/2012; 59(1):97-104. · 0.69 Impact Factor -
Article: Efficient expression of codon-adapted human acetaldehyde dehydrogenase 2 cDNA with 6xHis tag in Pichia pastoris.
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ABSTRACT: Human mitochondrial acetaldehyde dehydrogenase 2 (ALDH2) catalyzes the oxidation of acetaldehyde to acetic acid. Therefore, ALDH2 has therapeutic potential in detoxification of acetaldehyde. Furthermore, ALDH2 catalyzes nitroglycerin to nitrate and 1, 2-glyceryldinitrate during therapy for angina pectoris, myocardial infarction, and heart failure. Large quantities of ALDH2 will be needed for potential clinical practice. In this study, Pichia pastoris was used as a platform for expression of human ALDH2. Based on the ALDH2*1 cDNA sequence, we designed ALDH2 cDNA by choosing the P. pastoris preferred codons and by decreasing the G + C content level. The sequence was synthesized using the overlap extension PCR method. The cDNA and 6xHis tags were subcloned into the plasmid pPIC9K. The recombinant protein was expressed in P. pastoris GS115 and purified using Ni(2+)-Sepharose affinity chromatography. The amount of secreted protein in the culture was 80 mg/L in shake-flask cultivation and 260 mg/L in high-density bioreactor fermentation. Secreted ALDH2 was easily purified from the culture supernatant by using Ni(2+)-Sepharose affinity chromatography. After purification of the fermentation supernatant, the enzyme had a specific activity of 1.2 U/mg protein. The yield was about 16 mg/L in a shake flask culture of P. pastoris GS115 which contained the original human ALDH2*1 cDNA.Science in China Series C Life Sciences 10/2009; 52(10):935-41. · 1.61 Impact Factor
