A halotolerant aldose reductase from Debaryomyces nepalensis: gene isolation, overexpression and biochemical characterization

Abstract

Aldose reductase (AR) catalyzes the conversion of aldoses to their corresponding polyols in yeasts and filamentous fungi. ARs have the potential to be exploited for the enzymatic production of xylitol, thus the identification and characterization of ARs from novel strains have gained interest. In this study, we chose the novel yeast Debaryomyces nepalensis as a source for an AR gene. For the first time, here we isolated the AR gene from D. nepalensis (DnAR) that encodes a protein of 320 amino acids with a predicted molecular weight of 36.7 kDa using the RACE technique. It was heterologously expressed in Escherichia coli as a His-tagged fusion protein and purified. The enzyme showed strict NADPH dependence and broad substrate specificity with high catalytic efficiency for arabinose, xylose and 3-nitro benzaldehyde.Remarkably, it was active and stable in the presence of high concentrations of salts (KCl/NaCl), thus exhibiting halotolerance. It showed 75% and 45% activity at 0.5 and 1 M concentration of salts respectively. Enzyme half-lifetime at 1 M KCl and 1 M NaCl was found to be 30 h and 16.5 h respectively. Furthermore, to explore the structural basis of its halotolerance, we built a homology model of DnAR.Surprisingly, we found that the existence of a uniform negative electrostatic potential over the protein surface, which is one of the known mechanisms governing protein halotolerance. Therefore, DnAR could be exploited as a biocatalyst to develop an enzyme based bioprocess for xylitol production from lignocelluloses. Moreover, this is the first report providing the genetic sequence and biochemical characteristics of a halotolerant aldose reductase.

Crystal structure of yeast xylose reductase in complex with a novel NADP‐DTT adduct provides insights into substrate recognition and catalysis

“…The C4 sugars d ‐erythrose and l ‐threose were assayed for enzyme activity. The kinetic parameters were determined at constant NADPH (0.3 m m as determined previously and different d ‐erythrose/ l ‐threose concentrations (0.1–60 m m ). The obtained data were used to calculate the kinetic constants by fitting for the Michaelis–Menten equation using the software graphpad prism 5 (GraphPad Software, La Jolla, CA, USA).…”

“…Protein expression, purification, and oligomeric state DnXR (GenBank: KT239024) in the vector pET28a(+) was overexpressed and purified as described previously [37]. The plasmid construct contains a 21-residue-long N-terminal expression and purification tag including a His 6 region.…”

“…D. nepalensis, a nonpathogenic saccharomycetes yeast can utilize both hexose and pentose sugars to produce polyols [36]. DnXR, a key metabolic enzyme in the D-xylose utilization pathway belongs to the AKR2 family (AKR2B10) and has previously been shown to have strict dependence on NADPH and displays activity for a range of aldehydes [37]. In this study, an ordered conformation of the safety belt loop was observed in the absence of the cosubstrate and provides a bonafide depiction of the apo form of an AR.…”

In Vitro Inhibitory Activity and Molecular Docking Study of Selected Natural Phenolic Compounds as AR and SDH Inhibitors**

“…In the present study, r h AR and r h SDH were expressed as a SUMO fusion protein containing 6xHis Tag. The r h AR and r h SDH were purified using Ni‐NTA affinity chromatography [60–65] . The target proteins were eluted using 250 mM imidazole [66,67] .…”

“…The rhAR and rhSDH were purified using Ni-NTA affinity chromatography. [60][61][62][63][64][65] The target proteins were eluted using 250 mM imidazole. [66,67] rhAR and rhSDH enzymes were obtained with 16.67 and 6.54 purification fold and with a specific activity of 0.717 and 0.333 EU/mg protein, respectively.…”