High-throughput assay of tyrosine phenol-lyase activity using a cascade of enzymatic reactions

Hang-Qin Zhu; Wen-Ye Hu; Xiao-Ling Tang; Ren-Chao Zheng; Yu-Guo Zheng

doi:10.1016/j.ab.2022.114547

High-throughput assay of tyrosine phenol-lyase activity using a cascade of enzymatic reactions

Anal Biochem. 2022 Mar 1:640:114547. doi: 10.1016/j.ab.2022.114547. Epub 2022 Jan 10.

Authors

Hang-Qin Zhu¹, Wen-Ye Hu¹, Xiao-Ling Tang¹, Ren-Chao Zheng², Yu-Guo Zheng¹

Affiliations

¹ Key Laboratory of Bioorganic Synthesis of Zhejiang Province, College of Biotechnology and Bioengineering, Zhejiang University of Technology, Hangzhou, 310014, People's Republic of China; Engineering Research Center of Bioconversion and Biopurification of Ministry of Education, Zhejiang University of Technology, Hangzhou, 310014, People's Republic of China.
² Key Laboratory of Bioorganic Synthesis of Zhejiang Province, College of Biotechnology and Bioengineering, Zhejiang University of Technology, Hangzhou, 310014, People's Republic of China; Engineering Research Center of Bioconversion and Biopurification of Ministry of Education, Zhejiang University of Technology, Hangzhou, 310014, People's Republic of China. Electronic address: zhengrc@zjut.edu.cn.

PMID: 35026146
DOI: 10.1016/j.ab.2022.114547

Abstract

Tyrosine phenol-lyase (TPL) exhibits great potential in industrial biosynthesis of l-tyrosine and its derivates. To uncover and screen TPLs with excellent catalytic properties, there is unmet demand for development of facile and reliable screening system for TPL. Here we presented a novel assay format for the detection of TPL activity based on catechol 2,3-dioxygenase (C23O)-catalyzed reaction. Catechol released from TPL-catalyzed cleavage of 3,4-dihydroxy-l-phenylalanine (l-DOPA) was further oxidized by C23O to form 2-hydroxymuconate semialdehyde, which could be readily detected by spectrophotometric measurements at 375 nm. The assay achieved a unique balance between the ease of operation and superiority of analytical performances including linearity, sensitivity and accuracy. In addition, this assay enabled real-time monitoring of TPL activity with high efficiency and reliability. As C23O is highly specific towards catechol, a non-natural product of microorganism, the assay was therefore accessible to both crude cell extracts and the whole-cell system without elaborate purification steps of enzymes, which could greatly expedite discovery and engineering of TPLs. This study provided fundamental principle for high-throughput screening of other enzymes consuming or producing catechol derivatives.

Keywords: Catechol 2,3-dioxygenase; Coupled enzymatic reactions; High-throughput screening; Real-time assay; Tyrosine phenol lyase.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Tyrosine Phenol-Lyase*

Substances

Tyrosine Phenol-Lyase