Cloning, purification, and characterization of xylose isomerase from Thermotoga naphthophila RKU‐10

Abstract

A 1.3 kb xyl-A gene encoding xylose isomerase from a hyperthermophilic eubacterium Thermotoga naphthophila RKU-10 (TnapXI) was cloned and over-expressed in Escherichia coli to produce the enzyme in mesophilic conditions that work at high temperature. The enzyme was concentrated by lyophilization and purified by heat treatment, fractional precipitation, and UNOsphere Q anion-exchange column chromatography to homogeneity level. The apparent molecular mass was estimated by SDS-PAGE to be 49.5 kDa. The active enzyme showed a clear zone on Native-PAGE when stained with 2, 3, 5-triphenyltetrazolium chloride. The optimum temperature and pH for D-glucose to D-fructose isomerization were 98 °C and 7.0, respectively. Xylose isomerase retains 85% of its activity at 50 °C (t1/2 1732 min) for 4 h and 32.5% at 90 °C (t1/2 58 min) for 2 h. It retains 90-95% of its activity at pH 6.5-7.5 for 30 min. The enzyme was highly activated (350%) with the addition of 0.5 mM Co(2+) and to a lesser extent about 180 and 80% with the addition of 5 and 10 mM Mn(2+) and Mg(2+) , respectively but it was inhibited (54-90%) in the presence of 0.5-10 mM Ca(2+) with respect to apo-enzyme. D-glucose isomerization product was also analyzed by Thin Layer Chromatography (Rf 0.65). The enzyme was very stable at neutral pH and sufficiently high temperature and required only a trace amount of Co(2+) for its optimal activity and stability. Overall, 52.2% conversion of D-glucose to D-fructose was achieved by TnapXI. Thus, it has a great potential for industrial applications.

Kinetics and thermodynamics for aldo/keto conversion of D‐xylose and D‐glucose catalyzed by xylose isomerase from Thermotoga naphthophilaRKU‐10

“…All genetic manipulations were performed by conventional molecular biological techniques 16‐18 . Recombinant TnapXI was concentrated by lyophilization and purified to homogeneity by heat treatment at 85 °C for 15 min, 70% ammonium sulphate precipitation, and anion exchange chromatography 19 …”

“…[16][17][18] Recombinant TnapXI was concentrated by lyophilization and purified to homogeneity by heat treatment at 85°C for 15 min, 70% ammonium sulphate precipitation, and anion exchange chromatography. 19 Enzyme assays Thirty micro-liters of TnapXI (2 mg.ml −1 ) were incubated with 20 μL MOPS buffer (100 mmol L -1 ) containing 400 mmol L -1 either of D-xylose or D-glucose and 0.5 mmol L -1 CoCl 2 in a 96 well microplate for 10 min. 20 Assays were performed at 95°C and pH 7.0.…”

Fermentation of Biodegradable Organic Waste by the Family Thermotogaceae

“…On the other hand, Thermotogaceae showed the presence of genes involved in monosaccharides conversion to glucose inducing an alternative flux to EMP and ED, enabling Thermotogaceae to use alternative sugar substrate sources [168][169][170]. Examples of these are uronate isomerase (E.C.…”

A Novel Glucose Isomerase fromCaldicellulosiruptor besciiwith Great Potentials in the Production of High-Fructose Corn Syrup

“…The other thermophilic GIs were reported to obtain high bioconversion rate at high temperature. Such as the maximal bioconversion rate of 54.7% was accomplished by GICA from C. algeriensis at 85°C for 3 h [13], which 52.2% was achieved by TnapXI from T. naphthophila RKU-10 [21].…”