摘要
通过RP-HPLC同时检测酶水解反应前后底物5-吲哚甲基海因及产物D-氨甲酰-N-色氨酸的含量变化,不仅可以检测海因酶的活性,还能分析酶反应过程的转化效率。采用Merck公司的PurospherSTARRP-C18e色谱柱(4.6mm×250.0mm,5μm),以乙腈-20mmoL/L磷酸二氢钾缓冲液(体积比25∶75)为流动相,磷酸调pH值为3.0,流速为1.0mL/min,柱温为30℃,检测波长为220nm,于10min内很好地实现了各组分的分离。采用外标法进行定量分析,D-色氨酸、5-吲哚甲基海因和D-氨甲酰-N-色氨酸的线性范围分别是0.10～1.00mg/mL(r=0.9991)、0.20～2.00mg/mL(r=0.9989)、0.20～2.00mg/mL(r=0.9989);酶反应体系中5-吲哚甲基海因和D-氨甲酰-N-色氨酸的加标回收率分别为99.3%～100.2%和98.0%～99.3%,精密度分别为0.51%～0.87%和0.43%～0.94%。试验结果表明该法快速简便、准确可靠。
The HPLC method was used to determine the concentrations of substrate 5-（3-Indolylmethyl）hydantoin,intermediate product N- carbamyl-D-Tryptophan and product D-Tryptophan in the reaction solution. The sample was determined using a Purospher STAR RP-C18e column （4.6 mm×250.0 mm ,5 μm particle size）at the temperature of 30 ℃ with the mobile phase of acetonitrile-20mmol/L Sodium acetate （25：75 by volume） which adjusted to pH 3.0 with phosphoric acid at a flow rate of 1.0 mL/min. The detection wavelength was set at 220 nm.Under these conditions ,the components in the reaction system were separated completely within 10 min. A good linear relationship was obtained by employing external standard method for quantitative analysis. The linear calibration ranges for the three target analytes were 0.10-1.00 mg/mL（r=0.999 1 ） ,0.20-2.00 mg/mL（r= 0.998 9 ）and 0.20-2.00 mg/mL（r=0.998 9 ）, respectively. The recoveries of the analytes from spiking experiments were 99.3%-102.0% and 98.0%- 99.3% ,respectively ,with the relative standard deviation being 0.51%N0.87% and 0.43%-0.94% ,respectively. The results showed that the method was rapid,eonvenient and accurate.
出处
《现代农业科技》
2010年第7期17-19,共3页
Modern Agricultural Science and Technology
基金
新一代生物催化和生物转化的科学基础(2009CB724700)
