Abstract
A thermostable uronate dehydrogenase Tb-UDH from Thermobispora bispora was over-expressed in Escherichia coli using the T7 polymerase expression system. The Tb-UDH was purified by metal affinity chromatography, and gave a single band on SDS-PAGE. The maximum activity on glucuronic acid was found at 60°C and pH 7.0. The purified enzyme retained over 58% of its activity after holding a pH ranging from 7.0 to 7.5 for 1h at 60°C. The Km and Vmax values of the purified Tb-UDH for Glucuronic acid (GluUA) were 0.165mM and 117.7Umg-1, respectively, those for galacturonic acid (GalUA) were 0.115mM and 104.2Umg-1, respectively, and those for NAD+ were 0.120mM and 133.3Umg-1, respectively; the turnover number (kcat) with GluUA as a substrate was higher than that with GalUA; however, the Michaelis constant (Km) for GalUA was lower than that for GluUA. After 60min of incubation at 50°C, Tb-UDH exhibited a conversion ratio for glucuronic acid to the glucaric acid of 84% on chemical reagent and 81.3% on hydrolysates from breech xylans formed by xylanase and α-glucuronidase. This work shows that biocatalytic routes have great potential for the conversion of hemicellulose substrate into value-added products derived from renewable biomass. TOC GRAPHIC: (A) The structure of the xylan is described and the site of action of the xylan degrading enzyme is indicated. (B) The effect of substrate concentration on recombinant Tb-UDH activity when galacturonic acid was used as substrate. (C) SDS-PAGE analysis of E. coli BL21 (DE3) harboring pET-20b(+) and pET-20b-Tb-UDH. (D) Oxidative conversion of glucuronic acid from a beechwood xylan to glucaric acid.
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