Cloning, expression, and characterization of xylose reductase with higher activity from Candida tropicalis.
Xylose reductase (XR) is a key enzyme in xylose metabolism because it catalyzes the reduction of xylose to xylitol. In order to study the characteristics of XR from Candida tropicalis SCTCC 300249, its XR gene (xyll) was cloned and expressed in Escherichia coli BL21 (DE3). The fusion protein was purified effectively by Ni2+-chelating chromatography, and the kinetics of the recombinant XR was investigated. The Km values of the C. tropicalis XR for NADPH and NADH were 45.5 microM and 161.9 microM, respectively, which demonstrated that this XR had dual coenzyme specificity. Moreover, this XR showed the highest catalytic efficiency (kcat =1.44 x 10(4) min(-1)) for xylose among the characterized aldose reductases. Batch fermentation was performed with Saccharomyces serivisiae W303-lA:pYES2XR, and resulted in 7.63 g/L cell mass, 93.67 g/L xylitol, and 2.34 g/L x h xylitol productivity. This XR coupled with its dual coenzyme specificity, high activity, and catalytic efficiency proved its utility in in vitro xylitol production.
Zhang
Feiwei
F
College of Life Sciences, Sichuan University, Chengdu 610064, P. R. China.
Qiao
Dairong
D
Xu
Hui
H
Liao
Chong
C
Li
Shilin
S
Cao
Yi
Y
D000449:Aldehyde Reductase / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D041022:Candida tropicalis / Q000201:enzymology* / Q000235:genetics*; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D003067:Coenzymes / Q000378:metabolism / Q000494:pharmacology; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008958:Models, Molecular; D009243:NAD / Q000378:metabolism / Q000494:pharmacology; D009249:NADP / Q000378:metabolism / Q000494:pharmacology; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D013379:Substrate Specificity; D013696:Temperature; D014993:Xylitol / Q000378:metabolism
D003067:Coenzymes; D011993:Recombinant Fusion Proteins; D009243:NAD; D009249:NADP; D000449:Aldehyde Reductase; D014993:Xylitol