Substrate Specificity of a Mannose-6-Phosphate Isomerase from Bacillus subtilis and Its Application in the Production of l -Ribose

Abstract

The uncharacterized gene previously proposed as a mannose-6-phosphate isomerase from Bacillus subtilis was cloned and expressed in Escherichia coli. The maximal activity of the recombinant enzyme was observed at pH 7.5 and 40°C in the presence of 0.5 mM Co 2؉ . The isomerization activity was specific for aldose substrates possessing hydroxyl groups oriented in the same direction at the C-2 and C-3 positions, such as the D and L forms of ribose, lyxose, talose, mannose, and allose. The enzyme exhibited the highest activity for L-ribulose among all pentoses and hexoses. Thus, L-ribose, as a potential starting material for many L-nucleoside-based pharmaceutical compounds, was produced at 213 g/liter from 300-g/liter L-ribulose by mannose-6-phosphate isomerase at 40°C for 3 h, with a conversion yield of 71% and a volumetric productivity of 71 g liter ؊1 h ؊1 .L-Ribose is a potential starting material for the synthesis of many L-nucleoside-based pharmaceutical compounds, and it is not abundant in nature (5, 19). L-Ribose has been produced mainly by chemical synthesis from L-arabinose, L-xylose, Dglucose, D-galactose, D-ribose, or D-mannono-1,4-lactone (2,17,23). Biological L-ribose manufacture has been investigated using ribitol or L-ribulose. Recently, L-ribose was produced from ribitol by a recombinant Escherichia coli containing an NAD-dependent mannitol-1-dehydrogenase (MDH) with a 55% conversion yield when 100 g/liter ribitol was used in a 72-h fermentation (18). However, the volumetric productivity of L-ribose in the fermentation is 28-fold lower than that of the chemical method synthesized from L-arabinose (8). L-Ribulose has been biochemically converted from L-ribose using an Lribose isomerase from an Acinetobacter sp. (9), an L-arabinose isomerase mutant from Escherichia coli (4), a D-xylose isomerase mutant from Actinoplanes missouriensis (14), and a D-lyxose isomerase from Cohnella laeviribosi (3), indicating that L-ribose can be produced from L-ribulose by these enzymes. However, the enzymatic production of L-ribulose is slow, and the enzymatic production of L-ribose from L-ribulose has been not reported.Sugar phosphate isomerases, such as ribose-5-phosphate isomerase, glucose-6-phosphate isomerase, and galactose-6-phosphate isomerase, work as general aldose-ketose isomerases and are useful tools for producing rare sugars, because they convert the substrate sugar phosphates and the substrate sugars without phosphate to have a similar configuration (11,12,21,22). L-Ribose isomerase from an Acinetobacter sp. (9) and D-lyxose isomerase from C. laeviribosi (3) had activity with L-ribose, D-lyxose, and D-mannose. Thus, we can apply mannose-6-phosphate (EC 5.3.1.8) isomerase to the production of L-ribose, because there are no sugar phosphate isomerases relating to L-ribose and D-lyxose. The production of the expensive sugar L-ribose (bulk price, $1,000/kg) from the rare sugar L-ribulose by mannose-6-phosphate isomerase may prove to be a valuable industrial process, because we have produced Lribulose from the ch...

The Extracellular Wall-Bound β- N -Acetylglucosaminidase from Lactobacillus casei Is Involved in the Metabolism of the Human Milk Oligosaccharide Lacto- N -Triose

“…We analyzed if the manA gene was involved in the metabolism of that disaccharide. The deduced amino acid sequence of manA showed 53% and 48% identity with the sequences of the characterized type I mannose-6P isomerases (EC.5.3.1.8) from Streptococcus mutans (30) and Bacillus subtilis (31), respectively. The manA gene from L.…”

Metabolism Shapes the Cell

“…However, since the UDP-GalNAc used to synthesize teichoic acids is primarily generated from epimerization of UDP-GlcNAc (72), the result hints that the manA mutant is also likely compromised in its ability to synthesize UDP-GlcNAc. Consistent with this possibility, the manA mutant phenocopied cells inhibited for synthesis of lipid I by tunicamycin (71). No difference in GlcNAc levels was observed between the manA mutant and the wild type (71); however, the mutant also stopped growing shortly after the shift to LB, and neither intracellular GlcNAc levels nor PG synthesis were measured directly; thus, the possibility that PG synthesis was essentially paused in the manA mutant growing in LB was not ruled out.…”

“…Since LB does not contain mannose, these results are somewhat perplexing. The cell envelope of the manA mutant shows reduced levels of galactose and GalNAc (N-acetylglucosamine, a component of teichoic acid), so it was proposed that the defect in the manA mutant was due to problems with teichoic acid synthesis (71). However, since the UDP-GalNAc used to synthesize teichoic acids is primarily generated from epimerization of UDP-GlcNAc (72), the result hints that the manA mutant is also likely compromised in its ability to synthesize UDP-GlcNAc.…”

Substrate Specificity of the Bacillus licheniformis Lyxose Isomerase YdaE and Its Application in In Vitro Catalysis for Bioproduction of Lyxose and Glucose by Two-Step Isomerization

“…Mannose isomerases of Agrobacterium radiobacter and Mycobacterium smegmatis have been proposed for the catalysis of lyxose and mannose to xylulose and fructose, respectively, but their efficiency with lyxose is lower than that with mannose (14,15). Phosphomannose isomerase also carries out the conversion of lyxose to xylulose, but it shows low specificity for lyxose (37). Available information about several uncharacterized genes present in prokaryotes has generated interest in the characterization of the encoded proteins and their use in bioproduction.…”