No abstract

Geobacillus thermodenitrificans, with a double-site mutation in L-arabinose isomerase, produced 95 g L-ribulose l(-1 ) from 500 g L-arabinose l(-1) under optimum conditions of pH 8, 70 degrees C, and 10 units enzyme ml(-1) with a conversion yield of 19% over 2 h. The half-lives of the mutated enzyme at 70 and 75 degrees C were 35 and 4.5 h, respectively.

Aims:  To characterize of a thermostable recombinant α‐l‐arabinofuranosidase from Caldicellulosiruptor saccharolyticus for the hydrolysis of arabino‐oligosaccharides to l‐arabinose. Methods and Results:  A recombinant α‐l‐arabinofuranosidase from C. saccharolyticus was purified by heat treatment and Hi‐Trap anion exchange chromatography with a specific activity of 28·2 U mg−1. The native enzyme was a 58‐kDa octamer with a molecular mass of 460 kDa, as measured by gel filtration. The catalytic residues and consensus sequences of the glycoside hydrolase 51 family of α‐l‐arabinofuranosidases were completely conserved in α‐l‐arabinofuranosidase from C. saccharolyticus. The maximum enzyme activity was observed at pH 5·5 and 80°C with a half‐life of 49 h at 75°C. Among aryl‐glycoside substrates, the enzyme displayed activity only for p‐nitrophenyl‐α‐l‐arabinofuranoside [maximum kcat/Km of 220 m(mol l−1)−1 s−1] and p‐nitrophenyl‐α‐l‐arabinopyranoside. This substrate specificity differs from those of other α‐l‐arabinofuranosidases. In a 1 mmol l−1 solution of each sugar, arabino‐oligosaccharides with 2–5 monomer units were completely hydrolysed to l‐arabinose within 13 h in the presence of 30 U ml−1 of enzyme at 75°C. Conclusions:  The novel substrate specificity and hydrolytic properties for arabino‐oligosaccharides of α‐l‐arabinofuranosidase from C. saccharolyticus demonstrate the potential in the commercial production of l‐arabinose in concert with endoarabinanase and/or xylanase. Significance and Impact of the Study:  The findings of this work contribute to the knowledge of hydrolytic properties for arabino‐oligosaccharides performed by thermostable α‐l‐arabinofuranosidase.

We purified recombinant glucose-6-phosphate isomerase from Pyrococcus furiosus using heat treatment and Hi-Trap anion-exchange chromatography with a final specific activity of 0.39 U mg(-1). The activity of the glucose-6-phosphate isomerase for L: -talose isomerization was optimal at pH 7.0, 95 degrees C, and 1.5 mM Co(2+). The half-lives of the enzyme at 65 degrees C, 75 degrees C, 85 degrees C, and 95 degrees C were 170, 41, 19, and 7.9 h, respectively. Glucose-6-phosphate isomerase catalyzed the interconversion between two different aldoses and ketose for all pentoses and hexoses via two isomerization reactions. This enzyme has a unique activity order as follows: aldose substrates with hydroxyl groups oriented in the same direction at C2, C3, and C4 > C2 and C4 > C2 and C3 > C3 and C4. L: -Talose and D: -ribulose exhibited the most preferred substrates among the aldoses and ketoses, respectively. L: -Talose was converted to L: -tagatose and L: -galactose by glucose-6-phosphate isomerase with 80% and 5% conversion yields after about 420 min, respectively, whereas D: -ribulose was converted to D: -ribose and D: -arabinose with 53% and 8% conversion yields after about 240 min, respectively.

A recombinant mannose-6-phosphate isomerase from Geobacillus thermodenitrificans (GTMpi) isomerizes aldose substrates possessing hydroxyl groups oriented in the same direction at the C2 and C3 positions such as the D- and L-forms of ribose, lyxose, talose, mannose, and allose. The activity of GTMpi for D-lyxose isomerization was optimal at pH 7.0, 70 degrees C and 1 mM Co(2+). Under these conditions, the k(cat) and K(m) values were 74,300 s(-1) and 390 mM for D-lyxose and 28,800 s(-1) and 470 mM for L-ribose, respectively. The half-lives of the enzyme at 60, 65, and 70 degrees C were 388, 73, and 27 h, respectively. GTMpi catalyzed the conversion of D-lyxose to D-xylulose with a 38% conversion yield after 3 h, and converted L-ribose to L-ribulose with a 29% conversion yield.