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2,6-Dimethoxyphenol-Based Assay for Quantitation of Polysaccharide Monooxygenase in Multienzyme Cocktails
Moscow University Chemistry Bulletin Pub Date : 2020-05-25 , DOI: 10.3103/s0027131420020157
V. D. Telitsin , M. V. Semenova , D. O. Osipov , A. V. Gusakov , A. P. Sinitsyn

Abstract

A rapid method for quantitation of lytic polysaccharide monooxygenase (LPMO) in multienzyme cocktails produced by mutant strains of filamentous fungi is developed. In this method, 2,6-dimethoxyphenol and hydrogen peroxide are used as cosubstrates in a nonspecific LPMO-catalyzed reaction leading to the formation of a colored product. With this method, we determine the LPMO content in the multienzyme cocktails produced by Penicillium verruculosum recombinant strains that homologously express LPMO. The results agree closely with the findings obtained using a method based on the fine chromatographic fractionation of the cocktails followed by the qualitative and quantitative evaluation of their content by electrophoresis and mass-spectrometry and protein quantitation assays in fractions collected from chromatographic separation.


中文翻译:

基于2,6-二甲氧基苯酚的定量方法测定多酶鸡尾酒中的多糖单加氧酶

摘要

建立了一种快速定量丝状真菌突变株产生的多酶混合物中溶解性多糖单加氧酶(LPMO)的方法。在这种方法中,2,6-二甲氧基苯酚和过氧化氢被用作非特异性LPMO催化反应的共底物,导致形成有色产物。通过这种方法,我们确定了由青霉产生的多酶鸡尾酒中的LPMO含量。同源表达LPMO的重组菌株。结果与使用基于鸡尾酒的精细色谱分馏,然后通过电泳和质谱法以及从色谱分离中收集的馏分中的蛋白质定量测定对它们的含量进行定性和定量评估的方法所获得的发现非常吻合。
更新日期:2020-05-25
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