Hengtao Tang,Zheng Zhou,Zhi Chen,Xin Ju,Liangzhi Li
Monosaccharides from biomass can be used as cost-effective raw materials to produce rare sugars which are valuable sweeteners. Enzymes play a pivotal role in such biomass biorefinery processes to develop novel synthetic routes, and improve the environmental friendliness of current routes. Here we propose a proof of concept biorefinery process to prepare useful rare sugars of D-xylulose and D-ribulose from D-xylose which was abundant in biomass. Ribose-5-phosphate isomerase B from Ochrobactrum sp. (OsRpiB) with a unique substrate scope was engineered to intensify the activity of transforming D-xylose to D-xylulose. A double mutant R94N/N137Q with 80% higher catalytic efficiency was identified, and kinetic and molecular dynamic studies were conducted to explore the mechanism underneath. The results revealed a correlation between enhancing stability and activity simultaneously. Then a one-pot enzyme cascade was constructed with OsRpiB and D-tagatose-3-epimerase from Pseudomonas cichorii and transformed D-xylulose further to D-ribulose. The conversions of D-xylose and D-xylulose in cascade reaction were 1.80-fold and 1.83-fold of that in separate reactions with the aid of process parameter adjustment. The presented work is the first reported enzyme cascade converting D-xylose to D-ribulose, and proves the promising role of monosaccharide isomerase platforms in the refinery of biomass.
Yuqiu Wu,Fanjin Meng,Jiaolong Fu,Yuyang Zhou,Huan Fan,Xin Ju,Liangzhi Li
D-xylose, a biomass derivative, is used as a substrate to generate D-xylulose and D-ribulose with high-added value through a cascade reaction. During this process, the thermal stability and catalytic efficiency of rate-limiting enzymes during cascade reactions are important issues that must be addressed in industrial applications. In this study, we prepared and validated PDA/SiO 2 microspheres as highly loaded immobilization carriers by oxidative self-polymerization of polydopamine (PDA) on silica (SiO 2 ) microspheres. D-xylose was successfully catalyzed by the co-immobilized D-5-phosphate ribose isomerase B (RpiB) and D-tagatose-3-epimerase (DTE), denoted PDA/SiO 2 -RpiB-DTE, to produce D-ribulose and D-xylulose. After immobilization, the K m of co-immobilized RpiB and DTE decreased, which suggested increased substrate affinity. The half-life of the co-immobilized enzymes was 3.5–8 fold higher than that of the free enzymes at 50–70 °C, and the inactivation rate constant was reduced, demonstrating that immobilization improved significantly the thermal stability. The conversion of D-xylose at 10 mM was 29.98% under optimal conditions, which contained 9.85% D-ribulose and 20.13% D-xylulose. PDA/SiO 2 -RpiB-DTE showed up to 89.10% initial enzyme activity after 3 recycle runs. The thermal stability and recovery aspects were improved by immobilization, which facilitates the use of biomass to produce high-value-added products and further exploitation.
Isomerization cascade is a promising strategy to prepare valuable rare sugars from biomass xylose. However current conversions are not satisfactory due to factors of enzyme efficiency and reaction equilibrium. An isomerase/isomerase cascade has been newly developed employing an isomerase of xylose and a tagatose-3-epimerase (DTE) to prepare xylulose and ribulose from xylose, with a total xylose conversion of 43.2 %. In this study, two microbial glucose isomerases (GI) were tested as alternatives to isomerize xylose for their high activity towards xylose and thermal stability, aiming to explore the adaption effect of enzyme in the isomerization type of cascade. GIs from Streptomyces rubiginosus (SrGI) and Piromyces sp. (PsGI) both showed higher xylose conversions than the previously used isomerase of xylose, ribose-5-phosphate isomerase. The optimal reaction conditions of GIs for xylose conversion were compared in the single transformation of xylose and the cascade reactions catalyzed by GI/DTE, and it revealed the importance of high reaction temperature to substrate conversion and enzyme deactivation. The activity profile and thermal stability of SrGI were better than PsGI, making it a potential biocatalyst in biomass sugar isomerization which was favorable at high temperatures. The conversion of xylose by the PsGI/DTE cascade was improved to 71.3 % by efforts of enzyme adaption and reaction condition adjusting. Additionally, the standstill of isomerization reaction of monosaccharide can be attributed to thermal dynamic equilibrium at low temperatures and enzyme deactivation at high temperatures based on the results of this study.
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