Joo H, Arisawa A, Lin Z, Arnold F H
Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, CA 91125, USA.
Chem Biol. 1999 Oct;6(10):699-706. doi: 10.1016/s1074-5521(00)80017-4.
Oxygenases catalyze the hydroxylation of a wide variety of organic substrates. An ability to alter oxygenase substrate specificities and improve their activities and stabilities using recombinant DNA techniques would expand their use in processes such as chemical synthesis and bioremediation. Discovery and directed evolution of oxygenases require efficient screens that are sensitive to the activities of interest and can be applied to large numbers of crude enzyme samples.
Horseradish peroxidase (HRP) couples the phenolic products of hydroxylation of aromatic substrates to generate colored and/or fluorescent compounds that are easily detected spectroscopically in high-throughput screening. Coexpression of the coupling enzyme with a functional mono- or dioxygenase creates a pathway for the conversion of aromatic substrates into fluorescent compounds in vivo. We used this approach for detecting the products of the toluene-dioxygenase-catalyzed hydroxylation of chlorobenzene and to screen large mutant libraries of Pseudomonas putida cytochrome P450cam by fluorescence digital imaging. Colors generated by the HRP coupling reaction are sensitive to the site of oxygenase-catalyzed hydroxylation, allowing the screen to be used to identify catalysts with new or altered regiospecificities.
The coupled oxygenase-peroxidase reaction system is well suited for screening oxygenase libraries to identify mutants with desired features, including higher activity or stability and altered reaction specificity. This approach should also be useful for screening expressed DNA libraries and combinatorial chemical libraries for hydroxylation catalysts and for optimizing oxygenase reaction conditions.
加氧酶催化多种有机底物的羟基化反应。利用重组DNA技术改变加氧酶底物特异性并提高其活性和稳定性的能力，将扩大其在化学合成和生物修复等过程中的应用。加氧酶的发现和定向进化需要高效的筛选方法，这些方法要对感兴趣的活性敏感，并且能够应用于大量粗酶样品。
辣根过氧化物酶（HRP）将芳香族底物羟基化产生的酚类产物偶联，生成可在高通量筛选中通过光谱轻松检测的有色和/或荧光化合物。偶联酶与功能性单加氧酶或双加氧酶的共表达在体内创造了一条将芳香族底物转化为荧光化合物的途径。我们使用这种方法检测氯苯经甲苯双加氧酶催化羟基化的产物，并通过荧光数字成像筛选恶臭假单胞菌细胞色素P450cam的大型突变文库。HRP偶联反应产生的颜色对加氧酶催化羟基化的位点敏感，使得该筛选方法可用于鉴定具有新的或改变的区域特异性的催化剂。
加氧酶-过氧化物酶偶联反应系统非常适合筛选加氧酶文库，以鉴定具有所需特性的突变体，包括更高的活性或稳定性以及改变的反应特异性。这种方法对于筛选表达的DNA文库和组合化学文库中的羟基化催化剂以及优化加氧酶反应条件也应该是有用的。
