Improved soluble expression and use of recombinant human renalase
| Autor: | Marvin A Rios, James R Hu, William B. Armiger, Lucy Q Lin, Jonathan S. Dordick, Clifford S. Morrison, Thomas R Beusse, Mattheos A. G. Koffas, Elena E. Paskaleva, David R. Dodds, Aidan H Gorby, Elaina M Blair |
|---|---|
| Jazyk: | angličtina |
| Rok vydání: | 2020 |
| Předmět: |
Protein Expression
Biochemistry Isomers Electricity Stereochemistry Enzyme Stability Electrochemistry Enzyme Inhibitors Enzyme Chemistry Renalase chemistry.chemical_classification 0303 health sciences Multidisciplinary Downstream processing biology Physics 030302 biochemistry & molecular biology Biochemical Cofactors Microbial electrosynthesis Recombinant Proteins Enzymes Chemistry Physical Sciences Medicine Research Article Science Materials Science Material Properties Static Electricity Oxidative phosphorylation Research and Analysis Methods Cofactor 03 medical and health sciences Industrial Microbiology Isomerism Protein Domains Electrostatics Gene Expression and Vector Techniques Escherichia coli Humans Molecular Biology Techniques Molecular Biology Monoamine Oxidase Molecular Biology Assays and Analysis Techniques 030306 microbiology Chemical Compounds Substrate (chemistry) Biology and Life Sciences Proteins Enzyme chemistry Solubility Mutation biology.protein Enzymology NAD+ kinase NADP |
| Zdroj: | PLoS ONE, Vol 15, Iss 11, p e0242109 (2020) PLoS ONE |
| ISSN: | 1932-6203 |
| Popis: | Electrochemical bioreactor systems have enjoyed significant attention in the past few decades, particularly because of their applications to biobatteries, artificial photosynthetic systems, and microbial electrosynthesis. A key opportunity with electrochemical bioreactors is the ability to employ cofactor regeneration strategies critical in oxidative and reductive enzymatic and cell-based biotransformations. Electrochemical cofactor regeneration presents several advantages over other current cofactor regeneration systems, such as chemoenzymatic multi-enzyme reactions, because there is no need for a sacrificial substrate and a recycling enzyme. Additionally, process monitoring is simpler and downstream processing is less costly. However, the direct electrochemical reduction of NAD(P)+on a cathode may produce adventitious side products, including isomers of NAD(P)H that can act as potent competitive inhibitors to NAD(P)H-requiring enzymes such as dehydrogenases. To overcome this limitation, we examined how nature addresses the adventitious formation of isomers of NAD(P)H. Specifically, renalases are enzymes that catalyze the oxidation of 1,2- and 1,6-NAD(P)H to NAD(P)+, yielding an effective recycling of unproductive NAD(P)H isomers. We designed several mutants of recombinant human renalase isoform 1 (rhRen1), expressed them inE.coliBL21(DE3) to enhance protein solubility, and evaluated the activity profiles of the renalase variants against NAD(P)H isomers. The potential for rhRen1 to be employed in engineering applications was then assessed in view of the enzyme’s stability upon immobilization. Finally, comparative modeling was performed to assess the underlying reasons for the enhanced solubility and activity of the mutant enzymes. |
| Databáze: | OpenAIRE |
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