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

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 Mg 2+ led to an increase in the activity of r-AoFT, whereas Zn 2+ , Cu 2+ and Ni 2+ 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 K m and higher V max 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.

Purification of Aspergillus tamarii mycelial fructosyltransferase (m‐FTase), optimized FOS production, and evaluation of its anticancer potential

“…Two FTases, namely AcFT1 and AcFT2, from A. aculeatus were 70 kDa and 100 kDa proteins (Virgen‐Ortíz et al., 2016). Molecular weight of A. oryzae FTase was observed to be 50 kDa (T. Wei et al., 2016). Recently, A. oryzae S719 FTase (95 kDa) has been purified using Sephacryl S‐200 HR and DEAE‐Sepharose matrices (Han et al., 2020).…”

“…Deglycosylated (Endo‐H treated) extracellular recombinant Aureobasidium melanogenum 11−1 FTase purified using nickel‐NTA column had molecular weight of 82.4 kDa (Aung et al., 2019). Some other reports also suggested that most of the fungal FTases are reported to have molecular weights in the range of 50–90 kDa (Aung et al., 2019; T. Wei et al., 2016).…”

Research Progress and Prospects for Fructosyltransferases

“…The 3D model structure of A. oryzae ZZ‐01 fructosyltransferase was modeled based on the crystal structure of Schwanniomyces occidentalis fructofuranosidase (PDB: 3u75.1A). The model also includes two domains, i.e., an N ‐terminal five‐blade β ‐propeller fold harboring the active site, and a C ‐terminal β ‐sandwich domain 69.…”

“…Additionally, metal ions play an important role in the folding of proteins 13. Structures of enzymes can be altered by metal ions, which can lead to denaturation 69. Fructosyltransferase can be also activated or inhibited by certain metal ions.…”

Characterization of a Novel Nicotine Hydroxylase from Pseudomonas sp. ZZ-5 That Catalyzes the Conversion of 6-Hydroxy-3-Succinoylpyridine into 2,5-Dihydroxypyridine

“…Any further increase or decrease in the HSP concentration resulted in a decrease in 2,5-DHP production. Thus, increasing the HSP concentration (from 0.25 to 1 mM) facilitated contact between the enzyme and the substrate in the reaction system [27]. The Michaelis-Menten equation was applied for catalytic kinetic analysis.…”