A method for high-throughput screening hydrolase of lignin β-aryl ether linkage from directed evolution by glutathione (GSH) assay

Abstract

Background

Lignin’s valorization plays a critical role in refining the bioresource. Considering that the β-aryl ether linkage (β-O-4 bond) accounts for 50–70% of lignin chemical linkage between aromatic rings, the hydrolase of lignin β-aryl ether linkage, especially the β-etherase, provided a promising way for the lignin depolymerization and valorization. As a result, it is essential to develop the effective high-throughput methods for screening the mutant library of β-etherase from directed evolution.

Results

Based on the enzymatic mechanism of β-O-4 bond’s cleavage by β-etherase, the LigF was selected as the model to study high-throughput method by GSH assay for screening the mutant library of β-etherase from directed evolution. After the primary study with purified LigF and cell lysate, the GSH assay was used to screen mutant library of β-etherase. The study on screening the mutant library with about 600 colonies indicated that the selected transformants all have one or two mutated sites in the gene sequence of LigF, and the activities from GSH assay of most selected transformants were the same as their activities from HPLC assay.

Conclusions

The results from the high-throughput screening of mutant library demonstrated that GSH assay could be applied to screen β-etherase mutant from directed evolution.

Keywords

Lignin valorization / β-etherase / Directed evolution / High-throughput screening

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Funding

National Key Research and Development Program of China(No. 2016YFD0400601)