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Metformin is one of the most regularly prescribed Type II diabetes drugs in the world, and its use is likely to expand as diabetes diagnoses rise globally. This drug and its main degradation byproduct, guanylurea, are not fully metabolized by humans and cannot be removed through conventional water treatment processes. These compounds have been detected in coastal waters around the world and are currently considered emerging pollutants. The goal of this research was to examine the catalytic mechanism and substrate specificity of Guanylurea Hydrolase (GuuH), a recently discovered enzyme that converts guanylurea to ammonia and guanidine. Bioinformatic analyses were conducted to predict the active site and three-dimensional structure of GuuH. Site-directed mutagenesis was performed to construct mutants in amino acids predicted to be part of the enzyme's catalytic triad and substrate binding site. The mutants created were K138R, N141K, E211D, E211Q, and E211N. The wild-type and mutant enzymes were purified using His-tag affinity chromatography. Enzyme activity was assessed by measuring ammonia released using Berthelot assays. The results showed that the K138R mutant had similar specific activity compared to the wild-type GuuH when reacting with guanylurea, while E211N and E221D showed low specific activity under the same conditions. All of the enzymes had no detectable activity when reacting with biuret, which suggests they have low affinity for this substrate. Future work will focus on kinetic analyses of the wild-type and K138R enzymes and additional mutagenesis to identify the amino acids that determine the substrate specificity to the enzyme. Understanding GuuH's catalytic activity and substrate specificity is essential to using this enzyme in the development of biotechnological applications for water treatment.
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This content will become publicly available on September 16, 2026
Effects of biochar adsorption on extracellular enzymes activity: measurement and interpretation
Abstract Extracellular enzymes play a key role in microbe‐mediated organic matter decomposition in soils, and the efficiency of these enzymes in substrate decomposition depends on their mobility and specific activity in soils. In this work, we explored the influence of biochar adsorption on extracellular enzyme activity across a spectrum of environmental conditions, from simple to complex. Batch adsorption results showed that biochar adsorption of two hydrolytic enzymes—α‐amylase and amyloglucosidase (AMG)—similarly decreases with pH and follows the Langmuir isotherm, suggesting electrostatic interaction between them. Activity of AMG and alkaline phosphatase (ALP), which belong to carbon and phosphorus cycling enzymes, was measured using a novel calorimetric method. The technique demonstrated advantages over conventional enzyme assays, such as in situ real‐time measurement of reaction rate and the ability to identify potential interferences. The technique enabled the measurement of specific activity of biochar‐adsorbed AMG, which ranged from 10% to 90% of that of free AMG. The effect of substrate adsorption on activity measurement was demonstrated through the examination of two substrates for ALP, which suggested the use of effective substrate concentration (instead of nominal concentration) in calculating enzyme activity kinetics. Soil column experiments showed that biochar amendment can affect the activity of AMG in starch hydrolysis through changing the mobility of AMG (and accessibility to substrate) and its specific activity. Results from this work improve our understanding of the effects of biochar adsorption on enzyme activity and suggest the need to appropriately interpret enzyme activity data and account for confounding processes.
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- Award ID(s):
- 2120547
- PAR ID:
- 10644370
- Publisher / Repository:
- Wiley Blackwell (John Wiley & Sons)
- Date Published:
- Journal Name:
- Soil Science Society of America Journal
- Volume:
- 89
- Issue:
- 5
- ISSN:
- 0361-5995
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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