One-pot multi-step transformation of D-allose from D-fructose using a co-immobilized biocatalytic system

[摘要] In this report, Shinella zoogloeoides NN6 was discovered to produce two rare sugar producingenzymes, d-allulose 3-epimerase (DAE) and l-rhamnose isomerase (LRhI), when cultured with l-rhamnose as the sole carbon source. Sodium dodecylsulfate-polyacrylamide gel electrophoresis of purified DAE and LRhI revealed that the molecularmasses of the monomeric subunits are 37 and 43kDa, respectively, whereas gel filtration analysisshowed that purified DAE and LRhI are 148 and162 kDa, respectively, indicating that both enzymesform tetramers. The activity of DAE was the highest at 80°C in acetate buffer (pH 6.5) with Co2+,whereas LRhI exhibited maximum activity at 60°Cin glycine–NaOH buffer (pH 9.0) with Mn2+. Aco-immobilized biocatalyst was constructed usingDAE (3.2 U) and LRhI (40 U). Activity profile analysis of this co-immobilized biocatalyst revealed thatDAE activity was highest at 80°C in acetate buffer(pH 5.5), whereas the highest activity for LRhI wasobserved at 55°C in sodium phosphate buffer (pH7.0). d-Allose was produced from 2% (w/w) d-fructose via d-allulose at 60°C and pH 9.0 in a one-potreaction, providing a mixture of d-glucose, d-fructose, d-allulose and d-allose at a ratio of1.3:62.7:23.6:12.4. This is the first report describingone-pot d-allose production using LRhI and DAEexpressed in a single microorganism.

[学科分类] 微生物学和免疫学

[关键词] d-allose;d-allulose;co-immobilization;enzymatic reaction;one-pot multi-step production