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Tytuł:
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Continuous Spectrophotometric Assay for High-Throughput Screening of Predominant d-Allulose 3-Epimerases.
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Autorzy:
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Li C; Key Laboratory of Industrial Fermentation Microbiology of the Ministry of Education; Tianjin Key Laboratory of Industrial Microbiology, College of Biotechnology, Tianjin University of Science and Technology; National Engineering Laboratory for Industrial Enzymes, Tianjin 300457, P. R. China.
Zhang W; Key Laboratory of Industrial Fermentation Microbiology of the Ministry of Education; Tianjin Key Laboratory of Industrial Microbiology, College of Biotechnology, Tianjin University of Science and Technology; National Engineering Laboratory for Industrial Enzymes, Tianjin 300457, P. R. China.
Wei C; Key Laboratory of Industrial Fermentation Microbiology of the Ministry of Education; Tianjin Key Laboratory of Industrial Microbiology, College of Biotechnology, Tianjin University of Science and Technology; National Engineering Laboratory for Industrial Enzymes, Tianjin 300457, P. R. China.
Gao X; Key Laboratory of Industrial Fermentation Microbiology of the Ministry of Education; Tianjin Key Laboratory of Industrial Microbiology, College of Biotechnology, Tianjin University of Science and Technology; National Engineering Laboratory for Industrial Enzymes, Tianjin 300457, P. R. China.
Mao S; Key Laboratory of Industrial Fermentation Microbiology of the Ministry of Education; Tianjin Key Laboratory of Industrial Microbiology, College of Biotechnology, Tianjin University of Science and Technology; National Engineering Laboratory for Industrial Enzymes, Tianjin 300457, P. R. China.
Lu F; Key Laboratory of Industrial Fermentation Microbiology of the Ministry of Education; Tianjin Key Laboratory of Industrial Microbiology, College of Biotechnology, Tianjin University of Science and Technology; National Engineering Laboratory for Industrial Enzymes, Tianjin 300457, P. R. China.
Qin HM; Key Laboratory of Industrial Fermentation Microbiology of the Ministry of Education; Tianjin Key Laboratory of Industrial Microbiology, College of Biotechnology, Tianjin University of Science and Technology; National Engineering Laboratory for Industrial Enzymes, Tianjin 300457, P. R. China.
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Źródło:
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Journal of agricultural and food chemistry [J Agric Food Chem] 2021 Oct 06; Vol. 69 (39), pp. 11637-11645. Date of Electronic Publication: 2021 Sep 26.
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Typ publikacji:
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Journal Article
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Język:
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English
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Imprint Name(s):
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Original Publication: Washington, American Chemical Society.
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MeSH Terms:
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High-Throughput Screening Assays*
Racemases and Epimerases*
Carbohydrate Epimerases/metabolism ; Fructose ; Hydrogen-Ion Concentration ; Kinetics
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Contributed Indexing:
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Keywords: continuous spectrophotometric assay; d-allulose; d-allulose 3-epimerase; molecular dynamics simulation
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Substance Nomenclature:
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23140-52-5 (psicose)
30237-26-4 (Fructose)
EC 5.1.- (Racemases and Epimerases)
EC 5.1.3.- (Carbohydrate Epimerases)
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Entry Date(s):
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Date Created: 20210927 Date Completed: 20211007 Latest Revision: 20211007
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Update Code:
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20240105
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DOI:
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10.1021/acs.jafc.1c04716
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PMID:
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34569239
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d-Allulose is an attractive noncaloric sugar substitute with numerous health benefits, which can be biosynthesized by d-allulose 3-epimerases (DAEases). However, enzyme instability under harsh industrial reaction conditions hampered its practical applications. Here, we developed a continuous spectrophotometric assay (CSA) for the efficient analysis of d-allulose in a mixture. Furthermore, a high-throughput screening strategy for DAEases was developed using CSA by coupling DAEase with a NADH-dependent ribitol dehydrogenase, enabling high-throughput screening of DAEase variants with desired properties. The variant M15S/P40N/S209N exhibited a half-life of 22 h at 60 °C and an 8.7 °C increase of the T 50 60 value, with a 1.2-fold increase of activity. Structural modeling and molecular dynamics simulations indicated that the improvement of thermostability and activity was due to some new hydrogen bonds between chains at the dimer interface and between the residue and the substrate d-fructose. This work offers a robust tool and theoretical basis for the improvement of DAEases, which will benefit the enzymatic biosynthesis of d-allulose and promote its industrial application.