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›› 2010, Vol. 10 ›› Issue (3): 576-581.

• 生化工程专栏 • Previous Articles     Next Articles

Rapid Screening of a Nicotine Acid Hydroxylase Producing Strain and Its Catalytic Conditions

JI Chun-miao, LUO Hui CHANG Yan-hong, ZHOU Bei-hai   

  1. Department of Biological Science and Techonology, University of Science and Technology Beijing Department of Biological Science and Techonology, University of Science and Technology Beijing Department of Environmental Engineering , University of Science and Technology Beijing School of Civil and Environmental Engineering,University of Science and Technology Beijing
  • Received:2010-01-21 Revised:2010-04-19 Online:2010-06-20 Published:2010-06-20
  • Contact: LUO Hui

烟酸羟基化酶产生菌的快速筛选及催化条件

冀春苗 罗晖 常雁红 周北海   

  1. 北京科技大学生物科学与技术系 北京科技大学生物科学与技术系 北京科技大学土木与环境工程学院 北京科技大学土木与环境工程学院
  • 通讯作者: 罗晖

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Abstract: A rapid screening method for nicotine acid hydroxylase producing strain was established based on the ultraviolet development. The strain was cultured in 96-well microplate and a drop of broth was detected on the filter paper. The nicotine acid hydroxylase producing strain was screened fast and conveniently by the development of 6-hydroxynicotinic acid under ultraviolet illumination (300 nm). Study on the strain BKC4 with the highest activity showed that it was rod, Gram negative bacterium, and identified as Pseudomonas putida by the analysis of 16S rRNA sequence. The strain of BKC4 was used to convert nicotinic acid to 6-hydroxynicotinic acid. The experimental result showed that the optimal catalytic conditions of BKC4 were 10 g/L of nicotinic acid, temperature 37℃, pH 6.5, 200 r/min of rotation speed, and the enzyme is stable at pH 6.5 and temperature below 30℃.

Key words: nicotine acid hydroxylase, ultraviolet developmen, nicotinic acid, 6-hydroxynicotinic acid

摘要： 建立了一种基于紫外显影法快速筛选烟酸羟基化酶产生菌的方法，用96孔板培养菌后，将菌液点样到滤纸上，利用6-羟基烟酸在紫外光照射(300 nm)下显影的特性，可以快速、方便地筛选出烟酸羟基化酶产生菌. 对筛选出的最高酶活菌株BKC4进行了研究，菌株为杆状，革兰氏染色呈阴性，通过16S rRNA序列分析鉴定为恶臭假单胞菌(Pseudomonas putida). 利用BKC4菌株催化烟酸合成6-羟基烟酸，结果表明，BKC4菌株烟酸羟基化酶的最适催化条件为：底物浓度10 g/L，温度37℃, pH 6.5，摇床转速200 r/min，酶在低于30℃及pH 6.0~6.5范围内较稳定.

关键词: 烟酸羟基化酶, 紫外显影, 烟酸, 6-羟基烟酸

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