{"title":"Expression and purification of bacterial dye-decolorizing peroxidases using Escherichia coli.","authors":"Hegne Pupart, Priit Eek","doi":"10.1016/bs.mie.2025.03.004","DOIUrl":null,"url":null,"abstract":"<p><p>Dye-decolorizing peroxidases (DyPs) are heme-dependent enzymes that utilize peroxide as a co-substrate. These enzymes catalyze the oxidation of various compounds, inculding dyes and lignin, and their industrial potential has been widely recognized. Studying these enzymes necessitates the production of an active form suitable for detailed investigation. However, bacterial recombinant expression of DyPs frequently results in heme-deficient proteins. Our previous research demonstrated that codon-optimized genes significantly enhance both protein yields and heme content per monomer. In this chapter, we detail our strategy for expressing and purifying DyPs in Escherichia coli to obtain active enzymes for further analysis. We also outline a method to determine the concentration of the active enzyme.</p>","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"716 ","pages":"143-156"},"PeriodicalIF":0.0000,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"Methods in enzymology","FirstCategoryId":"99","ListUrlMain":"https://doi.org/10.1016/bs.mie.2025.03.004","RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"2025/4/16 0:00:00","PubModel":"Epub","JCR":"Q3","JCRName":"Biochemistry, Genetics and Molecular Biology","Score":null,"Total":0}
引用次数: 0
Abstract
Dye-decolorizing peroxidases (DyPs) are heme-dependent enzymes that utilize peroxide as a co-substrate. These enzymes catalyze the oxidation of various compounds, inculding dyes and lignin, and their industrial potential has been widely recognized. Studying these enzymes necessitates the production of an active form suitable for detailed investigation. However, bacterial recombinant expression of DyPs frequently results in heme-deficient proteins. Our previous research demonstrated that codon-optimized genes significantly enhance both protein yields and heme content per monomer. In this chapter, we detail our strategy for expressing and purifying DyPs in Escherichia coli to obtain active enzymes for further analysis. We also outline a method to determine the concentration of the active enzyme.
期刊介绍：
The critically acclaimed laboratory standard for almost 50 years, Methods in Enzymology is one of the most highly respected publications in the field of biochemistry. Each volume is eagerly awaited, frequently consulted, and praised by researchers and reviewers alike. Now with over 500 volumes the series contains much material still relevant today and is truly an essential publication for researchers in all fields of life sciences, including microbiology, biochemistry, cancer research and genetics-just to name a few. Five of the 2013 Nobel Laureates have edited or contributed to volumes of MIE.
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