基于微孔板的高通量筛选6-羟基烟酸转化菌方法的建立  

Microplate for high throughput screening of 6-hydroxynicotinic acid transforming strains

作　　者：杨瑶 袁生[1] 戴亦军 尚广东 

机构地区：[1]南京师范大学生命科学学院,南京市微生物技术工程研究中心,南京师范大学生命科学学院微生物工程重点实验室,南京210046

出　　处：《微生物学报》2008年第1期112-115,共4页Acta Microbiologica Sinica

基　　金：国家"十五"科技攻关项目(2004BA308A22-12);江苏省高校自然科学重大基础研究项目(06KJA21016);江苏省"六大人才高峰"项目(06-C-014)~~

摘　　要：建立了一种基于96孔板-酶标仪的双波长紫外分光光度法高通量筛选6-羟基烟酸转化菌的方法。实验以251nm为测定波长、231nm为参比波长测定转化样品的6-羟基烟酸含量,6-羟基烟酸与ΔA251-231在0.5～11μg/mL浓度范围内有良好的线性关系,服从朗伯-比尔定律,平均回收率为99.11%～100.81%。利用96孔板-酶标仪,每天筛选量可达到2000～5000个反应,达到高通量筛选要求。Recently, new insecticides containing 3-chloropyridylmethyl group as a versatile building block have been developed, among which imidachloprid is a promising one. The synthesis of imidachloprid can use 6-Hydroxynicotinic acid, the first intermediate of the bacterial degradation of nicotinic acid, as a starting material. It is difficult to hydroxylate nicotinic acid at C6 position in a chemical synthesis. However, biotransformation can produce 6-hydroxynicotinic acid with industrial application possibility. Therefore, methods for large scale screening of 6-hydroxynicotinic acid-producing microorganisms are urgent. A high through-put screening method for 6-hydroxynicotinic acid transforming strains was established by determining 6-hydroxynicotinic acid based on 96-well Microplate-Multiskan Spectrum. The determination wavelength and the reference wavelength of 6-hydroxynicotinic acid were 251nm and 231nm respectively. Beer＇s law is obeyed in the range of 0.5-111xg/mL （R2=0.9999） for measuring 6-hydroxynicotinic acid. The average recovery rate was 99.11%-100.81%. The results showed that there was no apparent difference between our microplate method and previous HPLC method in the detecting 6-hydroxynicotinic acid formation. The microplate method is simple, convenient and accurate. It has the potential for large scale （about 2000 - 5000 reactions/d） screening of 6-hydroxynicotinic acid-producing microorganisms.

关 键 词：微孔板 高通量筛选 双波长紫外分光光度测定 烟酸 6-羟基烟酸 

分 类 号：Q93[生物学—微生物学]