基于恒温催化反应过程中吸光度实时测定的漆酶活性快速测定

Rapid Detection Method for Laccase Activity Based on Real-Time Absorbance Measurement at Constant Temperature

通过使用恒温反应分光光度系统,建立基于恒温催化反应过程中实时测定吸光度的漆酶活性快速测定方法。酶的催化反应与吸光度的检测在同一时空进行,实现了检测过程中酶催化反应温度的精确控制及催化反应时长的精确计量。通过考察与催化反应体系溶氧浓度相匹配的底物2,2’-连氮基-双-(3-乙基苯并二氢噻唑啉-6-磺酸)二铵盐[2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)diammonium salt,ABTS]和待测酶液的适宜浓度,以确保依据初始催化反应速率计算酶活性,最短仅需2 s,连续测定3个吸光度的变化,即可计算酶活性。

A rapid laccase activity determination technique was established using a spectrophotometer with accurate online temperature control.This is based on the mechanism of continuous real-time absorbance measurement during the enzymatic reaction conducted at a constant temperature.The enzymatic reaction and the absorbance measurement were conducted simultaneously at the same location,resulting in accurate temperature control and precise measurement of the catalytic reaction duration.The appropriate concentration ranges of substrate and enzyme solution under investigation,whose values must match the dissolved oxygen concentration in the catalytic system,were determined to ensure that the enzyme activity was calculated using the initial catalytic reaction rate.The minimum test duration to measure alterations in the three consecutive absorbances was 2 s,and the enzyme activity could be calculated.