菠菜乙醇酸氧化酶的酶学特性及同工酶电泳分析
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摘要
为了解析菠菜乙醇酸氧化酶(Sp GLO)的酶学特性及同工酶谱。首先提取菠菜的RNA并反转录成c DNA,通过NCBI数据库中提供的菠菜GLO基因mRNA序列信息,设计特异性引物,扩增目标序列并连接到p MD19-T载体上进行测序鉴定;随后再次设计引物并在上游引物加入His标签,克隆Sp GLO基因构建到p YES2载体上,将该载体转入酿酒酵母中进行表达并通过His-tag亲和柱纯化,然后在不同诱导时间点取样测定Sp GLO酶活。结果显示,转化后的酿酒酵母菌株在诱导发酵20 h后能得到最高的GLO活性。以乙醇酸为底物测定Sp GLO在不同p H、不同温度条件下的催化活性,其最适p H值为8. 0,最适温度为39℃。然后分别以乙醇酸、乙醛酸、甘油酸为反应底物,系统分析了Sp GLO的酶学特性,数据显示,Sp GLO对乙醇酸的亲和力最高,其Km为0. 41 mmol/L,Vm为45. 92μmol/(min·mg)。以乙醇酸、乙醛酸为底物,使用草酸抑制其催化活性,其Ki分别为4. 61,2. 09 mmol/L,表明以乙醛酸为底物时Sp GLO的催化活性更易被草酸抑制。同时将纯化后的Sp GLO通过Caps-氨水电泳体系进行同工酶电泳,经过染色后出现2条同工酶带,表明菠菜叶片中可能存在2种GLO同工酶。为将来深入研究植物GLO同工酶之间的生化特性差异并分析其不同生理功能奠定良好的基础。
The enzymatic properties of Spinacia oleracea glycolate oxidase( SpGLO) were studied in the paper. Firstly,the RNA of spinach was extracted and reversely transcribed into cDNA. Based on the mRNA sequence information of spinach GLO gene provided in NCBI database,the specific primers were designed,and the target sequences were cloned and connected to the pMD19-T vector for sequence identification. Then the primer was designed again and His label was added to the upstream primer,and SpGLO gene was cloned to build pYES2 vector.The SpGLO was heterologously expressed in Saccharomyces cerevisiae and purified by immobilized metal-affinity chromatography,and the enzyme activities were detected at different induction time. The results showed that the transformed S. cerevisiae strain presented the highest GLO activity after 20 hours of induction. The catalytic activities of SpGLO at different pHs and temperatures were determined by using glycolate as substrate,and the optimum pH was 8. 0 and the optimum temperature was 39 ℃. The enzymatic properties of SpGLO were analyzed with glycolate,glyoxylate and glycine as substrates respectively. Which showed that SpGLO displayed the highest affinity and activity with glycolate as substrate,and the Km of SpGLO for glycolate was 0. 41 mmol/L,and the Vm was 45. 92μmol/( min·mg). Oxalate could more strongly inhibit the glyoxylate-oxidizing activity of SpGLO,and the Ki values were 4. 61,2. 09 mmol/L,respectively. The purified SpGLO isozyme zymogram analysis was performed with a Caps-ammonium Clear Native-PAGE system,and two isozyme bands appeared after staining,indicating that there may be two GLO isozymes existed in spinach leaves. It provided a good foundation for future research on the biochemical difference between GLO isozyme in plant and the analysis of its physiological function.
The enzymatic properties of Spinacia oleracea glycolate oxidase( SpGLO) were studied in the paper. Firstly,the RNA of spinach was extracted and reversely transcribed into cDNA. Based on the mRNA sequence information of spinach GLO gene provided in NCBI database,the specific primers were designed,and the target sequences were cloned and connected to the pMD19-T vector for sequence identification. Then the primer was designed again and His label was added to the upstream primer,and SpGLO gene was cloned to build pYES2 vector.The SpGLO was heterologously expressed in Saccharomyces cerevisiae and purified by immobilized metal-affinity chromatography,and the enzyme activities were detected at different induction time. The results showed that the transformed S. cerevisiae strain presented the highest GLO activity after 20 hours of induction. The catalytic activities of SpGLO at different pHs and temperatures were determined by using glycolate as substrate,and the optimum pH was 8. 0 and the optimum temperature was 39 ℃. The enzymatic properties of SpGLO were analyzed with glycolate,glyoxylate and glycine as substrates respectively. Which showed that SpGLO displayed the highest affinity and activity with glycolate as substrate,and the Km of SpGLO for glycolate was 0. 41 mmol/L,and the Vm was 45. 92μmol/( min·mg). Oxalate could more strongly inhibit the glyoxylate-oxidizing activity of SpGLO,and the Ki values were 4. 61,2. 09 mmol/L,respectively. The purified SpGLO isozyme zymogram analysis was performed with a Caps-ammonium Clear Native-PAGE system,and two isozyme bands appeared after staining,indicating that there may be two GLO isozymes existed in spinach leaves. It provided a good foundation for future research on the biochemical difference between GLO isozyme in plant and the analysis of its physiological function.
引文
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