| 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
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| 2011-12-01 | |
| 发表期刊 | APPLIED BIOCHEMISTRY AND BIOTECHNOLOGY
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| 卷号 | 165期号:7-8页码:1770-1778 |
| 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.; 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. |
| 文章类型 | Article |
| 关键词 | Propionyl-coa Dehydrogenase Gene Expression Characterization Candida Rugosa Pichia Pastoris |
| 学科领域 | 生物基化学品 |
| WOS标题词 | Science & Technology ; Life Sciences & Biomedicine |
| DOI | 10.1007/s12010-011-9393-x |
| 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研究方向 | Biochemistry & Molecular Biology ; Biotechnology & Applied Microbiology |
| WOS类目 | Biochemistry & Molecular Biology ; Biotechnology & Applied Microbiology |
| WOS记录号 | WOS:000297222900027 |
| 引用统计 | |
| 文献类型 | 期刊论文 |
| 条目标识符 | http://ir.qibebt.ac.cn/handle/337004/1115 |
| 专题 | 生物基材料组群 |
| 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. |
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