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

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.