以丁香醛嗪为显色底物,自动比色法测定发酵样品中重组真菌漆酶活性。

K A Holm, D M Nielsen, J Eriksen
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引用次数: 9

摘要

建立了一种自动Cobas - Fara法测定重组嗜热分枝杆菌漆酶(rMtL)活性的方法。所使用的显色底物为丁香醛dazine。在好氧条件下,rMtL催化丁香醛嗪氧化生成四甲氧基偶氮二亚甲基醌。在530 nm处动态测量紫色作为酶活性的表达,rMtL是一种非常敏感的氧化还原酶,因此必须仔细控制许多因素,以获得可靠的分析试验。为了稳定rMtL,在酶稀释培养基中加入PEG 6000。此外,Triton X-I00加入酶孵育液中。对分析条件和工艺条件进行了优化,使该方法具有良好的精密度、灵敏度和分析速度。Michaelis-Menten常数K(m)为22muM。定量限为0.010 Uml(-1),定量限为0.0002 Uml(-1),酶稀释曲线分析范围为0.01 ~ 0.044 Uml(-1),重复性为1.9%,重现性为3.1%。对该方法的稳健性检验表明,影响rMtL分析的主要因素为:孵育温度、Triton X-I00的浓度、孵育缓冲液的摩尔浓度和pH,最后是丁香醛嗪的浓度。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
Automated colorimetric determination of recombinant fungal laccase activity in fermentation sarples using syringaldazine as chromogenic substrate.

An automated Cobas Fara method was developed determining the activity of recombinant M. thermophila laccase (rMtL). The chromogenic substrate used was syringaldazine. Under aerobic conditions, rMtL catalyses the oxidation of syringaldazine forming tetrametoxy-azo bis methylene quinone. The developed violet colour was measured kinetically at 530 nm as an expression of the enzyme activity, rMtL is a very sensitive oxidoreductase, therefore many factors had to be carefully controlled in order to get a robust analytical assay. In order to stabilize rMtL, PEG 6000 was added to the enzyme dilution medium. Furthermore, Triton X-I00 was included in the enzyme incubation solution.The analytical as well as technical conditions have been optimized, resulting in a method with good precision, sensitivity and speed of analysis. The Michaelis-Menten constant, K(m), was determined to be 22muM syringaldazine. LOQ was determined to be 0.010 Uml(-1), LOD to be 0.0002 Uml(-1) The analytical range of the enzyme dilution curve was from 0.01 to 0.044 Uml(-1) The repeatability was 1.9%, the reproducibility 3.1%. Testing the robustness of the method showed that the most sensible factors in the rMtL analysis in decreasing range were: incubation temperature, concentration of Triton X-I00, molarity and pH of the incubation buffer, and finally the concentration of syringaldazine.

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