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Catalysts 2016, 6(5), 67; doi:10.3390/catal6050067

Cloning, Expression and Characterization of a Novel Fructosyltransferase from Aspergillus oryzae ZZ-01 for the Synthesis of Sucrose 6-Acetate

School of Food and Biological Engineering, Zhengzhou University of Light Industry, 5 Dongfeng Rd, Zhengzhou 450002, China
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Author to whom correspondence should be addressed.
Academic Editor: David D. Boehr
Received: 26 March 2016 / Revised: 18 April 2016 / Accepted: 4 May 2016 / Published: 9 May 2016
(This article belongs to the Special Issue Enzyme Catalysis)
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Abstract

A 1521 bp gene encoding for a novel fructosyltransferase from Aspergillus oryzae ZZ-01 (AoFT) has been amplified by RACE and TAIL PCR, and functionally overexpressed in Escherichia coli BL 21-CodonPlus (DE3)-RIL. The recombinant A. oryzae ZZ-01 fructosyltransferases (r-AoFT) was purified to homogeneity after Ni-NTA affinity and Superdex-200 gel filtration chromatography. SDS-PAGE analysis of the purified r-AoFT revealed a single protein band with an apparent molecular mass of 60.0 kDa. The r-AoFT enzyme exhibited its optimal activity at 55 °C and pH 5.5, and maintained about 63% of its activity even after 60 min of treatment at 60 °C. The addition of Mg2+ led to an increase in the activity of r-AoFT, whereas Zn2+, Cu2+ and Ni2+ led to a reduction in its activity. Six site-directed mutants of r-AoFT (D39A, D164A, E216A, N38L, S99A and Y282A) were constructed and characterized biochemically. The N38L, S99A and Y282A mutants had lower Km and higher Vmax values than the wild-type enzyme, highlighting their higher binding affinity for the substrates. These results therefore suggest that r-AoFT could be used for the enzymatic synthesis of Suc6A from sucrose and glucose 6-acetate. View Full-Text
Keywords: fructosyltransferase; Aspergillus oryzae; gene cloning; site-directed mutagenesis; characterization fructosyltransferase; Aspergillus oryzae; gene cloning; site-directed mutagenesis; characterization
This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. (CC BY 4.0).

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Wei, T.; Huang, S.; Zang, J.; Jia, C.; Mao, D. Cloning, Expression and Characterization of a Novel Fructosyltransferase from Aspergillus oryzae ZZ-01 for the Synthesis of Sucrose 6-Acetate. Catalysts 2016, 6, 67.

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