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

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.

Recent advancements in enzyme engineering via site-specific incorporation of unnatural amino acids.

With increased attention to excellent biocatalysts, evolving methods based on nature or unnatural amino acid (UAAs) mutagenesis have become an important part of enzyme engineering. The emergence of powerful method through expanding the genetic code allows to incorporate UAAs with unique chemical functionalities into proteins, endowing proteins with more structural and functional features. To date, over 200 diverse UAAs have been incorporated site-specifically into proteins via this methodology and many of them have been widely exploited in the field of enzyme engineering, making this genetic code expansion approach possible to be a promising tool for modulating the properties of enzymes. In this context, we focus on how this robust method to specifically incorporate UAAs into proteins and summarize their applications in enzyme engineering for tuning and expanding the functional properties of enzymes. Meanwhile, we aim to discuss how the benefits can be achieved by using the genetically encoded UAAs. We hope that this method will become an integral part of the field of enzyme engineering in the future.

Purification and Biochemical Characterization of a Tyrosine Phenol-lyase from Morganella morganii.

Tyrosine phenol-lyase (TPL) is a valuable and cost-effective biocatalyst for the biosynthesis of L-tyrosine and its derivatives, which are valuable intermediates in the pharmaceutical industry. A TPL from Morganella morganii (Mm-TPL) was overexpressed in Escherichia coli and characterized. Mm-TPL was determined as a homotetramer with molecular weight of 52 kDa per subunit. Its optimal temperature and pH for ß-elimination of L-tyrosine were 45 °C and pH 8.5, respectively. Mm-TPL manifested strict substrate specificity for the reverse reaction of ß-elimination and ortho- and meta-substituted phenols with small steric size were preferred substrates. The enzyme showed excellent catalytic performance for synthesis of L-tyrosine, 3-fluoro-L-tyrosine, and L-DOPA with a yield of 98.1%, 95.1%, and 87.2%, respectively. Furthermore, the fed-batch bioprocess displayed space-time yields of 9.6 g L-1 h-1 for L-tyrosine and 4.2 g L-1 h-1 for 3-fluoro-L-tyrosine with a yield of 67.4 g L-1 and 29.5 g L-1, respectively. These results demonstrated the great potential of Mm-TPL for industrial application.