Cloning, expression, and characterization of xylose reductase with higher activity from Candida tropicalis

Zhang, Feiwei; Qiao, Dan; Xu, Hui; Liao, Chong; Li, Shilin; Cao, Yi

doi:10.1007/s12275-008-0225-9

Cited by 21 publications

(12 citation statements)

References 26 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Many factors could cause the ineYcient xylose fermentation, of which redox imbalance and low enzyme activity in the xylose metabolic pathway are the most likely [7]. In the present study, the XR activity of K. marxianus (1.295 U/mg) was lower than some XRs reported [37,38], but higher than XR in Debaryomyces hansenii UFV-170 [26,27]. XDH from K. marxianus NBRC1777 preferred NAD + as the coenzyme and the activity was very low with NADP + (data not shown).…”

Section: Characterization Of Recombinant Kmxrcontrasting

confidence: 62%

“…The activity of KmXR was determined according to a previously described method [36,38] using a spectrophotometer to monitor the change in A 340 upon oxidation of NAD(P)H. Unless indicated otherwise, the KmXR assay mixture (1.0 ml) for the reaction contained 100 mM phosphate buVer (pH 7.0), 200 M NAD(P)H, 200 mM xylose, and enzyme solution (0.1 ml). This reaction mixture was allowed to stand for 1 min to eliminate the endogenous oxidation of NADPH, and the reaction was started by the addition of 0.1 ml of substrate.…”

Section: Xr Activity Assaymentioning

confidence: 99%

“…D-Xylose is one of the main hydrolysis products of lignocellulosic biomass and is the second most abundant fermentable material [6,19,33,38]. In contrast to the eYcient glucose fermentation in yeast, xylose fermentation is challenging because only a few ethanol-producing microorganisms can readily ferment xylose even though many microorganisms utilize xylose as a carbon source [19].…”

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

Identification of a xylose reductase gene in the xylose metabolic pathway of Kluyveromyces marxianus NBRC1777

Zhang¹,

Zhang²,

Wang³

et al. 2011

J Ind Microbiol Biotechnol

View full text Add to dashboard Cite

Kluyveromyces marxianus is thermotolerant yeast that is able to utilize a wider range of substrates and has greater thermal tolerance than most other yeast species. K. marxianus can assimilate xylose, but its ability to produce ethanol from xylose in oxygen-limited environments is poor. In the present study, the K. marxianus xylose reductase (KmXR) gene (Kmxyl1) was cloned and the recombinant enzyme was characterized to clarify the factors that limit xylose fermentation in K. marxianus NBRC1777. KmXR is a key enzyme in the xylose metabolism of K. marxianus, which was verified by disruption of the Kmxyl1 gene. The Km of the recombinant KmXR for NADPH is 65.67 μM and KmXR activity is 1.295 U/mg, which is lower than those of most reported yeast XRs, and the enzyme has no activity with coenzyme NADH. This result demonstrates that the XR from K. marxianus is highly coenzyme specific; combined with the extremely low XDH activity of K. marxianus with NADP+, the limitation of xylose fermentation is due to a redox imbalance under anaerobic conditions and low KmXR activity.

show abstract

Section: Characterization Of Recombinant Kmxrcontrasting

confidence: 62%

Section: Xr Activity Assaymentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Identification of a xylose reductase gene in the xylose metabolic pathway of Kluyveromyces marxianus NBRC1777

Zhang¹,

Zhang²,

Wang³

et al. 2011

J Ind Microbiol Biotechnol

View full text Add to dashboard Cite

show abstract

“…It is another feature that separate it from typical xylose reductases (usually XRs are highly specic to xylose). [22][23][24] Contrastingly, AR from C. tenuis showed maximum catalytic efficiency with D-erythrose.…”

Section: Discussionmentioning

confidence: 99%

“…23 Conversely, DnAR was susceptible to Cu 2+ toxicity as like enzymes from C. tropicalis and C. parapsilosis. 23,32 Cu 2+ is known to exert its effect by induction of site specic oxidation in human aldose reductase 25 and probably the same mechanism might be applicable for DnAR.…”

mentioning

confidence: 98%

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

2017

View full text Add to dashboard Cite

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.

show abstract

Cloning, expression, and characterization of a novel xylose reductase from Rhizopus oryzae

Zhang

Jiang

Zheng

et al. 2015

J. Basic Microbiol.

View full text Add to dashboard Cite

Rhizopus oryzae is valuable as a producer of organic acids via lignocellulose catalysis. R. oryzae metabolizes xylose, which is one component of lignocellulose hydrolysate. In this study, a novel NADPH-dependent xylose reductase gene from R. oryzae AS 3.819 (Roxr) was cloned and expressed in Pichia pastoris GS115. Homology alignment suggested that the 320-residue protein contained domains and active sites belonging to the aldo/keto reductase family. SDS-PAGE demonstrated that the recombinant xylose reductase has a molecular weight of approximately 37 kDa. The optimal catalytic pH and temperature of the purified recombinant protein were 5.8 and 50 °C, respectively. The recombinant protein was stable from pH 4.4 to 6.5 and at temperatures below 42 °C. The recombinant enzyme has bias for D-xylose and L-arabinose as substrates and NADPH as its coenzyme. Real-time quantitative reverse transcription PCR tests suggested that native Roxr expression is regulated by a carbon catabolite repression mechanism. Site-directed mutagenesis at two possible key sites involved in coenzyme binding, Thr(226) → Glu(226) and Val(274) → Asn(274), were performed, respectively. The coenzyme specificity constants of the resulted RoXR(T226E) and RoXR(V274N) for NADH increased 18.2-fold and 2.4-fold, which suggested possibility to improve the NADH preference of this enzyme through genetic modification.

show abstract

Cloning, expression, and characterization of xylose reductase with higher activity from Candida tropicalis

Cited by 21 publications

References 26 publications

Identification of a xylose reductase gene in the xylose metabolic pathway of Kluyveromyces marxianus NBRC1777

Identification of a xylose reductase gene in the xylose metabolic pathway of Kluyveromyces marxianus NBRC1777

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

Cloning, expression, and characterization of a novel xylose reductase from Rhizopus oryzae

Contact Info

Product

Resources

About