title: Expression and characterization of a novel propionyl-CoA dehydrogenase gene from Candida rugosa in Pichia pastoris.
abstract: The propionyl-CoA dehydrogenase (PACD) gene was firstly cloned from Candida rugosa by the cDNA RACE technique. The 6× His-tagged recombinant PACD gene was expressed in Pichia pastoris GS115 and purified with Ni-NTA affinity chromatography. SDS-PAGE analysis and Western blotting revealed that the molecular mass of the purified PACD was 49 kDa. The results showed that the recombinant protein had the activity of catalyzing propionyl-CoA to acrylyl-CoA. The K (m), k (cat), and V (max) values of the purified PACD were calculated to be 40.86 μM, 0.566 s(-1) and 0.693 U mg(-1) min(-1). The optimal temperature and pH of the purified PACD were 30 °C and 7.0, respectively. The recombinant PACD maintained 76.3%, 30.1%, and 4.3% of its original activity after 2 h incubation in standard buffer at 30, 40, and 50 °C, respectively. Mg(2+) had an activating effect on the enzyme, while Mn(2+), Ca(2+), Zn(2+), and Cu(2+) had weak inhibition. Since PACD catalyzed the key step (from propionyl-CoA to acrylyl-CoA) in the modified β-oxidation pathway from glucose to 3-hydroxypropionic acid (3-HP), the integration of recombinant PACD could benefit the engineered strains for effective production of 3-HP from the most abundant biomass-sugars.
authors.affiliation: Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences, Qingdao 266101, China.
authors.forename: Feng-Li
authors.initials: FL
authors.lastname: Zhou
authors.forename: Yong-Guang
authors.initials: YG
authors.lastname: Zhang
authors.forename: Ru-Bing
authors.initials: RB
authors.lastname: Zhang
authors.forename: Wei
authors.initials: W
authors.lastname: Liu
authors.forename: Mo
authors.initials: M
authors.lastname: Xian
mesh_terms: D044944:Acyl-CoA Dehydrogenases / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D002175:Candida / Q000201:enzymology*; D004795:Enzyme Stability; D005656:Fungal Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D015870:Gene Expression; D007700:Kinetics; D008969:Molecular Sequence Data; D010843:Pichia / Q000235:genetics* / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
chemical_list: D005656:Fungal Proteins; D011994:Recombinant Proteins; D044944:Acyl-CoA Dehydrogenases