High-throughput screening and identification of lignin peroxidase based on spore surface display of Bacillus subtilis.

Background: Lignin peroxidase is closely related to agriculture and food as it improves the quality of feedstuffs, facilitates the degradation of lignin in agricultural wastes, and degrades azo dyes that have similar complex structures to lignin. However, the current status of homologous or heterologous expression of lignin peroxidase is unsatisfactory and needs to be modified with the help of immobilization and directed evolution to maximize its potential. Directed evolution technology is an effective strategy for designing and improving enzyme characteristics, and Bacillus subtilis spore surface display technology is an efficient method for preparing immobilized enzymes.

Results: A colorimetric dye decolorization assay using Congo red as a substrate was developed and optimized for high-throughput screening of spore surface display in a 96-well plate. After two rounds of screening, a superior mutant strain was selected from 2700 mutants. Its highest catalytic activity was 196.36%. The amino acid substitution sites were identified as N120D and I242T.

Conclusion: The mechanism for the enhanced catalytic activity was explained using protein modeling and functional analysis software. This study provides insights into the rational design of lignin peroxidase and its application in food and agriculture. © 2024 Society of Chemical Industry.