公开(公告)号：US20030207345A1

公开(公告)日：2003-11-06

申请号：US10453104

申请日：2003-06-02

Applicant: California Institute of Technology

Inventor： Frances H. Arnold ,  Hyun Joo

IPC: C12Q001/26

CPC classification number: C12N9/0071 ,  C07K2319/02 ,  C12N9/0065 ,  C12N9/0069 ,  C12N9/0077 ,  C12N15/102 ,  C12N15/1034 ,  C12N15/67 ,  C12N15/815 ,  C12Q1/26 ,  C12Q1/28 ,  G01N2333/795 ,  G01N2333/80 ,  G01N2333/90245

Abstract: A method for detecting the presence of an oxygenated compound which is produced when a substrate is reacted with an oxygenase for the substrate. The method involves reacting a coupling enzyme with the oxygenated compound to form a polymeric oxygenated compound which is fluorescent or luminescent. Measurement of the fluorescence or luminescence of the polymeric oxygenated compound provides indirect detection of the oxygenated compound produced by reaction of the oxygenase with the substrate. The method is carried out in a whole cell environment wherein the cell is transformed to express both the oxygenase being screened and the coupling enzyme. The method can be used to measure the activity of monooxygenases and dioxygenases on aromatic substrates. The method is amenable to large scale screening of enzyme mutants to isolate those with maximum oxygenase activity.

Abstract translation: 用于检测当底物与用于底物的加氧酶反应时产生的含氧化合物的存在的方法。 该方法包括使偶联酶与含氧化合物反应以形成荧光或发光的聚合的氧化化合物。 聚合物氧化化合物的荧光或发光的测量提供了通过加氧酶与底物的反应产生的氧合化合物的间接检测。 该方法在整个细胞环境中进行，其中转化细胞以表达被筛选的加氧酶和偶联酶。 该方法可用于测量单加氧酶和双加氧酶在芳香族底物上的活性。 该方法适用于大规模筛选酶突变体以分离具有最大加氧酶活性的那些。