http://scholars.ntou.edu.tw/handle/123456789/8614
Title: | Characterization of a recombinant D-allulose 3-epimerase from Agrobacterium sp ATCC 31749 and identification of an important interfacial residue | Authors: | Tseng, Wen-Chi Chen, Chao-Nan Hsu, Chung-Ting Lee, Hsu-Chieh Fang, Hong-Yi Wang, Ming-Jun Wu, Yi-Hung Fang, Tsuei-Yun |
Keywords: | D-Allulose 3-epimerase;Rare sugar;Agrobacterium;D-PSICOSE 3-EPIMERASE;L-RHAMNOSE ISOMERASE;D-GLUCOSE ISOMERASE;D-ALLOSE;ESCHERICHIA-COLI;ACTIVE-SITE;D-FRUCTOSE;MUTATION;PURIFICATION;TUMEFACIENS | Issue Date: | Jun-2018 | Publisher: | Biochemistry & Molecular Biology | Journal Volume: | 112 | Start page/Pages: | 767-774 | Source: | International Journal of Biological Macromolecules | Abstract: | 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 beta-D-1-thio-galactopyranoside induction at 20 degrees 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 degrees C, respectively. The half-life of the enzyme is 267 min at 55 degrees C in the presence of 0.1 mM Co2+ and the equilibrium ratio between D-allulose and D-fructose is 30:70 at 55 degrees 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 wilD-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. (C) 2018 Elsevier B.V. All rights reserved. |
URI: | http://scholars.ntou.edu.tw/handle/123456789/8614 | ISSN: | 0141-8130 | DOI: | 10.1016/j.ijbiomac.2018.02.036 |
Appears in Collections: | 食品科學系 |
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