Search

Abstract: Xylitol was used as a raw material for production of l-xylose and l-lyxose using Alcaligenes 701B strain and immobilized l-rhamnose isomerase enzyme. Alcaligenes 701B converted xylitol to l-xylulose with a yield of 34% in the bioreactor. l-Xylulose was converted to l-xylose and l-lyxose using immobilized l-rhamnose isomerase enzyme. The final equilibrium between l-xylulose, l-xylose and l-lyxose was 53:26:21. The enzyme assays indicated that Alcaligenes 701B strain has an NAD-dependent xylitol dehydrogenase enzyme responsible for l-xylulose production. Furthermore, NAD(P)H-dependent l-xylulose reductase enzyme was active during conversion of xylitol to l-xylulose. The highest l-xylulose production rate corresponded with the highest growth rate. The Alcaligenes 701B strain used d-xylose for biomass growth, but xylitol was used only for l-xylulose production during conversion phase. [Copyright &y& Elsevier]

Abstract: l-Rhamnose isomerase (l-RhI) from Pseudomonas stutzeri LL172 can convert d-psicose to d-allose. Partially purified recombinant l-RhI from Escherichia coli was immobilized on BCW-2510 Chitopearl beads and utilized to produce d-allose. Total 20,000 units of immobilized enzyme converted d-psicose to d-allose without remarkable decrease in the enzyme activity over 17 days. When 50% d-psicose (w/w) was applied to a column with a flow rate of 0.8ml/min at 42°C, approximately 30% d-psicose was isomerized to d-allose for 17 days. However, by reducing the flow rate to 0.4ml/min after 17 days, d-allose was transformed at the same rate for 13 days. The total of 27l reaction mixture was separated by Simulated-Moving-Bed Chromatograph system. Approximately 2.2l/d of 50% (w/w) reaction mixture was separated continuously. After separation, d-allose and d-psicose fractions were 3l of approximately 10% (w/w) with 95% purity and 10l of approximately 8% (w/w) with 95% purity per day, respectively. The separated d-allose solution was concentrated up to about 50% and crystallized gradually by being kept at room temperature. Crystals of d-allose were separated from the syrup by filtration and 1.65kg crystals of 100% purity were obtained. The d-allose crystal yield from the d-psicose substrate was approximately 10%. [Copyright &y& Elsevier]

Mass production of a rare aldohexose d-allose from d-psicose was achieved in a batch reaction by crude recombinant l-rhamnose isomerase (l-RhI) cross-linked with glutaraldehyde. The d-psicose substrate was, in turn, mass produced from a naturally abundant ketohexose d-fructose by immobilized recombinant d-tagatose 3-epimerase (d-TE). At an equilibrium state, 25% of d-psicose was isomerized to d-allose, that is, 25 g of d-allose was obtained from 100 g of d-psicose. The d-allose product was easily separated and crystallized from the reaction mixture that contains 25%d-allose, 8%d-altrose and 67%d-psicose using ethanol. Empirically, approximately 338 g, that is, 90% of a theoretical overall yield for the purification of pure d-allose crystals was produced from 1.5 kg of d-psicose within 30 d using a constructed bioreactor. The cross-linked enzyme had an operative half-life of two months after repeated usages. [Copyright &y& Elsevier]

Escherichia coli strain JM 109 harboring 6× His-tag l-rhamnose isomerase (l-RhI) from Pseudomonas stutzeri allowed a 20-fold increase in the volumetric yield of soluble enzyme compared to the value for the intrinsic yield. Detailed studies on the substrate specificity of the purified His-tagged protein revealed that it catalyzed previously unknown common and rare aldo/ketotetrose, aldo/ketopentose, and aldo/ketohexose substrates in both d- and l-forms, for instance, erythrose, threose, xylose, lyxose, ribose, glucose, mannose, galactose, altrose, tagatose, sorbose, psicose, and fructose. Using a high enzyme–substrate ratio in extended reactions, the enzyme-catalyzed interconversion reactions from which two different products from one substrate were formed: l-lyxose, l-glucose, l-tagatose and d-allose were isomerized to l-xylulose and l-xylose, l-fructose and l-mannose, l-galactose and l-talose, and d-psicose and d-altrose, in that order. Kinetic studies, however, showed that l-rhamnose with Km and Vmax values of 11 mM and 240 U/mg, respectively, was the most preferred substrate, followed by l-mannose, l-lyxose, d-ribose, and d-allose. Based on the observed catalytic mode of action, these new findings reflected a hitherto undetected interrelation between l-RhI and d-xylose isomerase (d-XI). [Copyright &y& Elsevier]

Wild-type strain of Pseudomonas cichorii ST-24 was unable to grow on D-psicose and inductively produced D-tagatose 3-epimerase (D-TE) with D-tagatose as an inducer. We have isolated a constitutive mutant, designated strain Ka75, which had acquired a new ability to grow on a mineral salts medium containing D-psicose as a sole carbon source. The D-psicose-metabolizing mutant synthesized a high level of D-TE. When grown on the culture medium supplemented with Mn2+, the mutant strain produced around 250-fold higher activity than did the parent strain. Enzymatic properties of the constitutive enzyme were similar to those of the wild-type. Using the immobilized D-TE and recombinant L-rhamnose isomerase (L-RhI) from Escherichia coli strain JM109, a two-step enzymatic reaction was performed for massproduction of a rare aldo-hexose monosaccharide, L-galactose, from a common one, L-sorbose. In the first step, L-sorbose was epimerized to L-tagatose in a yield of 28%. The L-tagatose obtained was utilized as a starting material for L-galactose preparation by the immobilized L-RhI. At equilibrium, approximately 30% L-tagatose was isomerized to L-galactose. Finally, 7.5 g of L-galactose was obtained from 100 g of L-sorbose, viz an overall yield of 7.5%. The product obtained was purified and identified to be L-galactose by specific optical rotation and high performance liquid chromatography (HPLC) analysis, and was ultimately confirmed by 13C nuclear magnetic resonance (13C NMR) and IR spectra. [Copyright &y& Elsevier]