Characterization of a recombinant d-allulose 3-epimerase from Agrobacterium sp. ATCC 31749 and identification of an important interfacial residue
| Autor: | Wen-Chi Tseng, Ming-Jun Wang, Yi-Hung Wu, Hsu-Chieh Lee, Hong-Yi Fang, Chung-Ting Hsu, Chao-Nan Chen, Tsuei-Yun Fang |
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| Rok vydání: | 2018 |
| Předmět: |
0106 biological sciences
0301 basic medicine Models Molecular Agrobacterium Stereochemistry Fructose medicine.disease_cause 01 natural sciences Biochemistry Substrate Specificity 03 medical and health sciences chemistry.chemical_compound Residue (chemistry) Structural Biology 010608 biotechnology Enzyme Stability medicine Amino Acids Molecular Biology Escherichia coli chemistry.chemical_classification biology Temperature General Medicine Cobalt Hydrogen-Ion Concentration biology.organism_classification Rare sugar Enzyme assay Recombinant Proteins Molecular Weight Kinetics 030104 developmental biology Enzyme chemistry Agrobacterium tumefaciens Structural Homology Protein biology.protein Mutagenesis Site-Directed Specific activity Carbohydrate Epimerases |
| Zdroj: | International journal of biological macromolecules. 112 |
| ISSN: | 1879-0003 |
| Popis: | d -Allulose 3-epimerase (DAEase) catalyzes the epimerization between d -fructose and d -allulose. We had PCR-cloned and overexpressed the gene encoding Agrobacterium sp. ATCC 31749 DAEase (AsDAEase) in Escherichia coli. A high yield of active AsDAEase, 35,300 U/L or 1350 U/g of wet cells, was acquired with isopropyl β- d -1-thiogalactopyranoside induction at 20 °C for 20 h. Although only six residues including residue 234 located in tetrameric interface are different between AsDAEase and A. tumefaciens DAEase (AtDAEase), the specific activity of purified AsDAEase is much larger than that of AtDAEase. The optimal pHs and optimal temperatures of the purified recombinant AsDAEase are 7.5–8.0 and 55–60 °C, respectively. The half-life of the enzyme is 267 min at 55 °C in the presence of 0.1 mM Co2+, and the equilibrium ratio between d -allulose and d -fructose is 30:70 at 55 °C. Besides characterizing AsDAEase, mutation N234D was constructed to assess its influence on activity. The specific activity of the purified N234D AsDAEase is only 25.5% of wil d -type's activity, suggesting residue N234 is an important interfacial residue which substantially affects enzyme activity. The high specific activity and high expression yield of AsDAEase suggest its prospect to be applied in d -allulose production. |
| Databáze: | OpenAIRE |
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