{"title":"Utilizing repurposed myoglobin to develop biosensors of vitamin C","authors":"Sriyani Liyanage , Sriparna Ray","doi":"10.1016/j.envc.2025.101319","DOIUrl":null,"url":null,"abstract":"<div><div>The iron-based metalloprotein, Myoglobin (Mb) does not possess any catalytic property. Its essential function is oxygen storage in vertebrates. By modifying the essential amino acids present near the active site of the protein, it is possible to generate peroxidase activity in Mb through genetic engineering. Such “repurposed” activity can be incorporated by altering the essential amino acid with an unnatural amino acid (UAA). In addition, it can be easily expressed and purified, thereby increasing its ease of application in catalysis and biosensing. Here, we report a genetic modification of the leucine residue at the 29th position with dihydroxy-L-phenylalanine (DOPA), which is an UAA, followed by the substitution of H64 by glycine. This double mutation enabled the L29-DOPA/H64G mutant to efficiently catalyze the oxidation of thioanisole with H<sub>2</sub>O<sub>2</sub>, a common method to test peroxidase activity. In fact, unusual products arising from radical mechanism – biphenyl, diphenyl sulfide, etc., were obtained along with mono-oxidized sulfoxide product. Such a uniquely “repurposed” moiety could be utilized to design biosensors. This enabled the achievement of the 11th principle of Green Chemistry, which expresses the need of monitoring different substances on a real-time basis. The genetically modified and easily procured L29DOPA H64G Mb could be immobilized onto a surface-modified electrode and this could easily detect the electrocatalytic oxidation of vitamin C. Thus, the Au/L-Cys/L29DOPAH64G mutant Mb electrode was modified into a biosensor for ascorbic acid, which provides a convenient way to monitor organic food items containing vitamin C. The L29DOPA/H64G Mb immobilized electrode could detect as low as 5 mmol/L of vitamin C, using scan rates from 50 to 200 mVs<sup>−1</sup>.</div></div>","PeriodicalId":34794,"journal":{"name":"Environmental Challenges","volume":"21 ","pages":"Article 101319"},"PeriodicalIF":0.0000,"publicationDate":"2025-09-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"Environmental Challenges","FirstCategoryId":"1085","ListUrlMain":"https://www.sciencedirect.com/science/article/pii/S2667010025002380","RegionNum":0,"RegionCategory":null,"ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q2","JCRName":"Environmental Science","Score":null,"Total":0}
引用次数: 0
Abstract
The iron-based metalloprotein, Myoglobin (Mb) does not possess any catalytic property. Its essential function is oxygen storage in vertebrates. By modifying the essential amino acids present near the active site of the protein, it is possible to generate peroxidase activity in Mb through genetic engineering. Such “repurposed” activity can be incorporated by altering the essential amino acid with an unnatural amino acid (UAA). In addition, it can be easily expressed and purified, thereby increasing its ease of application in catalysis and biosensing. Here, we report a genetic modification of the leucine residue at the 29th position with dihydroxy-L-phenylalanine (DOPA), which is an UAA, followed by the substitution of H64 by glycine. This double mutation enabled the L29-DOPA/H64G mutant to efficiently catalyze the oxidation of thioanisole with H2O2, a common method to test peroxidase activity. In fact, unusual products arising from radical mechanism – biphenyl, diphenyl sulfide, etc., were obtained along with mono-oxidized sulfoxide product. Such a uniquely “repurposed” moiety could be utilized to design biosensors. This enabled the achievement of the 11th principle of Green Chemistry, which expresses the need of monitoring different substances on a real-time basis. The genetically modified and easily procured L29DOPA H64G Mb could be immobilized onto a surface-modified electrode and this could easily detect the electrocatalytic oxidation of vitamin C. Thus, the Au/L-Cys/L29DOPAH64G mutant Mb electrode was modified into a biosensor for ascorbic acid, which provides a convenient way to monitor organic food items containing vitamin C. The L29DOPA/H64G Mb immobilized electrode could detect as low as 5 mmol/L of vitamin C, using scan rates from 50 to 200 mVs−1.