Ribose-5-phosphate isomerase from Clostridium thermocellum converted D-psicose to D-allose, which may be useful as a pharmaceutical compound, with no by-product. The 12 active-site residues, which were obtained by molecular modeling on the basis of the solved three-dimensional structure of the enzyme, were substituted individually with Ala. Among the 12 Ala-substituted mutants, only the R132A mutant exhibited an increase in D-psicose isomerization activity. The R132E mutant showed the highest activity when the residue at position 132 was substituted with Ala, Gln, Ile, Lys, Glu, or Asp. The maximal activity of the wild-type and R132E mutant enzymes for D-psicose was observed at pH 7.5 and 80°C. The half-lives of the wild-type enzyme at 60°C, 65°C, 70°C, 75°C, and 80°C were 11, 7.0, 4.2, 1.5, and 0.6 h, respectively, whereas those of the R132E mutant enzymes were 13, 8.2, 5.1, 3.1, and 0.9 h, respectively. The specific activity and catalytic efficiency (k(cat)/K(m)) of the R132E mutant for D-psicose were 1.4- and 1.5-fold higher than those of the wild-type enzyme, respectively. When the same amount of enzyme was used, the conversion yield of D-psicose to D-allose was 32% for the R132E mutant enzyme and 25% for the wild-type enzyme after 80 min.
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.
An uncharacterized gene from Thermus thermophilus, thought to encode a mannose-6-phosphate isomerase, was cloned and expressed in Escherichia coli. The maximal activity of the recombinant enzyme for L-ribulose isomerization was observed at pH 7.0 and 75°C in the presence of 0.5 mM Cu 2؉ . Among all of the pentoses and hexoses evaluated, the enzyme exhibited the highest activity for the conversion of L-ribulose to L-ribose, a potential starting material for many L-nucleoside-based pharmaceutical compounds. The active-site residues, predicted according to a homology-based model, were separately replaced with Ala. The residue at position 142 was correlated with an increase in L-ribulose isomerization activity. The R142N mutant showed the highest activity among mutants modified with Ala, Glu, Tyr, Lys, Asn, or Gln. The specific activity and catalytic efficiency (k cat /K m ) for L-ribulose using the R142N mutant were 1.4-and 1.6-fold higher than those of the wild-type enzyme, respectively. The k cat /K m of the R142N mutant was 3.8-fold higher than that of Geobacillus thermodenitrificans mannose-6-phosphate isomerase, which exhibited the highest activity to date for the previously reported k cat /K m . The R142N mutant enzyme produced 213 g/liter L-ribose from 300 g/liter L-ribulose for 2 h, with a volumetric productivity of 107 g liter ؊1 h ؊1 , which was 1.5-fold higher than that of the wild-type enzyme.Optically pure carbohydrates are important precursors for pharmaceutical, food, and agrochemical products (22). Among carbohydrates, L-enantiomers have been widely used as antiviral nucleoside analogue drugs in the treatment of severe viral diseases due to their potent biological activities and lower toxicity than the corresponding D-nucleosides (3). L-Ribose, a pentose sugar, can be used as a precursor for the synthesis of antiviral drugs, such as L-nucleoside derivatives (2,5,14). LRibose can be synthesized by chemical methods from L-arabinose (1, 6, 9), L-xylose (13), D-glucose (15), D-galactose (19), D-ribose (26), or D-mannono-1,4-lactone (20). However, chemical synthesis has several disadvantages, including multiple steps, by-product formation, and chemical waste production.Recently, the enzymatic production of L-ribose has been investigated using L-arabinose (7) or L-ribulose (25). L-Ribose has been produced primarily from the cheap sugar L-arabinose because L-ribulose is an expensive sugar. An L-arabinose isomerase mutant of Escherichia coli (4) and a D-xylose isomerase mutant of Actinoplanes missouriensis (17) converted Larabinose to L-ribose by a two-step isomerization reaction with low productivity. A recombinant E. coli strain containing Larabinose isomerase and L-ribose isomerase (7) and purified L-arabinose isomerase and mannose-6-phosphate isomerase from Geobacillus thermodenitrificans (24) were used to produce L-ribose from L-arabinose via L-ribulose with high productivity.However, a rate-limiting step in the two enzyme systems is the conversion of L-ribulose to L-ribose using L-ribose isomerase (7...
L-Ribose is an important precursor for antiviral agents, and thus its high-level production is urgently demanded. For this aim, immobilized recombinant Escherichia coli cells expressing the L-arabinose isomerase and variant mannose-6-phosphate isomerase genes from Geobacillus thermodenitrificans were developed. The immobilized cells produced 99 g/l L-ribose from 300 g/l L-arabinose in 3 h at pH 7.5 and 60 °C in the presence of 1 mM Co(2+), with a conversion yield of 33 % (w/w) and a productivity of 33 g/l/h. The immobilized cells in the packed-bed bioreactor at a dilution rate of 0.2 h(-1) produced an average of 100 g/l L-ribose with a conversion yield of 33 % and a productivity of 5.0 g/l/h for the first 12 days, and the operational half-life in the bioreactor was 28 days. Our study is first verification for L-ribose production by long-term operation and feasible for cost-effective commercialization. The immobilized cells in the present study also showed the highest conversion yield among processes from L-arabinose as the substrate.
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