Expression and Characterization of a Novel Propionyl-CoA Dehydrogenase Gene from Candida rugosa in Pichia pastoris

CORC > 青岛生物能源与过程研究所 > 中国科学院青岛生物能源与过程研究所 > 材料生物技术研究中心

	Expression and Characterization of a Novel Propionyl-CoA Dehydrogenase Gene from Candida rugosa in Pichia pastoris
	Zhou, Feng-li; Zhang, Yong-guang; Zhang, Ru-bing; Liu, Wei; Xian, Mo
刊名	APPLIED BIOCHEMISTRY AND BIOTECHNOLOGY
	2011-12-01
卷号	165 期号:7-8 页码:1770-1778
关键词	Propionyl-CoA dehydrogenase Gene expression Characterization Candida rugosa Pichia pastoris
中文摘要	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. Mg²⁺ had an activating effect on the enzyme, while Mn²⁺, Ca²⁺, Zn²⁺, and Cu²⁺ 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.
英文摘要	The propionyl-CoA dehydrogenase (PACD) gene was firstly cloned from Candida rugosa by the cDNA RACE technique. The 6x 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 mu M, 0.566 s(-1) and 0.693 U mg(-1) min(-1). The optimal temperature and pH of the purified PACD were 30 A degrees 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 A degrees 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 beta-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.
学科主题	生物基化学品
WOS标题词	Science & Technology ; Life Sciences & Biomedicine
类目[WOS]	Biochemistry & Molecular Biology ; Biotechnology & Applied Microbiology
研究领域[WOS]	Biochemistry & Molecular Biology ; Biotechnology & Applied Microbiology
关键词[WOS]	3-HYDROXYPROPIONIC ACID ; ESCHERICHIA-COLI ; BETA-OXIDATION ; REDUCTASE ; PATHWAY ; CLONING ; COENZYME ; COMPLEX
收录类别	SCI
语种	英语
WOS记录号	WOS:000297222900027
公开日期	2012-11-07
内容类型	期刊论文
源URL	[http://ir.qibebt.ac.cn:8080/handle/337004/1115]
专题	青岛生物能源与过程研究所_材料生物技术研究中心
作者单位	Chinese Acad Sci, Qingdao Inst Bioenergy & Bioproc Technol, Qingdao 266101, Peoples R China
推荐引用方式 GB/T 7714	Zhou, Feng-li,Zhang, Yong-guang,Zhang, Ru-bing,et al. Expression and Characterization of a Novel Propionyl-CoA Dehydrogenase Gene from Candida rugosa in Pichia pastoris[J]. APPLIED BIOCHEMISTRY AND BIOTECHNOLOGY,2011,165(7-8):1770-1778.
APA	Zhou, Feng-li,Zhang, Yong-guang,Zhang, Ru-bing,Liu, Wei,&Xian, Mo.(2011).Expression and Characterization of a Novel Propionyl-CoA Dehydrogenase Gene from Candida rugosa in Pichia pastoris.APPLIED BIOCHEMISTRY AND BIOTECHNOLOGY,165(7-8),1770-1778.
MLA	Zhou, Feng-li,et al."Expression and Characterization of a Novel Propionyl-CoA Dehydrogenase Gene from Candida rugosa in Pichia pastoris".APPLIED BIOCHEMISTRY AND BIOTECHNOLOGY 165.7-8(2011):1770-1778.