Engineering the thermostability of d-lyxose isomerase from Caldanaerobius polysaccharolyticus via multiple computer-aided rational design for efficient synthesis of d-mannose.

d-Mannose is an attractive functional sugar that exhibits many physiological benefits on human health. The demand for low-calorie sugars and sweeteners in foods are increasingly available on the market. Some sugar isomerases, such as d-lyxose isomerase (d-LIase), can achieve an isomerization reaction between d-mannose and d-fructose. However, the weak thermostability of d-LIase limits its efficient conversion from d-fructose to d-mannose. Nonetheless, few studies are available that have investigated the molecular modification of d-LIase to improve its thermal stability. In this study, computer-aided tools including FireProt, PROSS, and Consensus Finder were employed to jointly design d-LIase mutants with improved thermostability for the first time. Finally, the obtained five-point mutant M5 (N21G/E78P/V58Y/C119Y/K170P) showed high thermal stability and catalytic activity. The half-life of M5 at 65 °C was 10.22 fold, and the catalytic efficiency towards 600 g/L of d-fructose was 2.6 times to that of the wild type enzyme, respectively. Molecular dynamics simulation and intramolecular forces analysis revealed a thermostability mechanism of highly rigidity conformation, newly formed hydrogen bonds and π-cation interaction between and within protein domains, and redistributed surface electrostatic charges for the mutant M5. This research provided a promising d-LIase mutant for the industrial production of d-mannose from d-fructose.

Identification of a novel recombinant D-lyxose isomerase from Thermoprotei archaeon with high thermostable, weak-acid and nickel ion dependent properties.

Recently, production of D-mannose becomes a hotspot owing to it exhibiting many physiological functions on people's health and wide applications in food and pharmaceutical field. The use of biological enzymes to production of D-mannose is of particular receiving considerable concerns due to it possessing many merits over chemical synthesis and plant extraction strategies. D-Lyxose isomerase (D-LIase) plays a pivotal role in preparation of D-mannose from d-fructose through isomerization reaction. Thus, a novel putative D-LIase from thermophiles strain Thermoprotei archaeon which was expressed in E. coli BL21(DE3) was first identified and biochemically characterized. The recombinant D-LIase showed an optimal temperature of 80 and 85 °C and pH of 6.5. It was highly thermostable at 70 °C and 80 °C after incubating for 48 h and 33 h, respectively, with retaining over 50% of the initial activity. A lower concentration of Ni2+ (0.5 mM) could greatly increase the activity by 25-fold, which was rare reported in other D-LIases. It was a dimer structure with melting temperature of 88.3 °C. Under the optimal conditions, 15.8 g L-1 of D-mannose and 33.8 g L-1 of D-xylulose were produced from 80 g L-1 of d-fructose and D-lyxose, respectively. This work provided a promising candidate sugar isomerase T. archaeon D-LIase for the production of D-mannose and D-xylulose.

Characterization of a recombinant D-mannose-producing D-lyxose isomerase from Caldanaerobius polysaccharolyticus.

Recently, functional sugars, such as d-mannose, have attracted considerable attention due to their excellent physiological benefits for human health and wide applications in food and pharmaceutical industries. Therefore, d-mannose production using a sugar isomerase such as d-lyxose isomerase (d-LIase) has emerged as a research hotspot owing to its advantages over plant extraction and chemical synthesis methods. In this study, a putative d-LIase gene from Caldanaerobius polysaccharolyticus was cloned and expressed in Escherichia coli. Then, a biochemical characterization of the recombinant d-LIase was carried out and its potential use in d-mannose production also assessed. Results showed that d-LIase exhibited its maximum activity under these optimal conditions: temperature of 65 °C, a pH of 6.5, and the Mn2+ metal ion. The d-LIase was active at pH 6.0-8.0; it was also quite thermostable up to 60 °C and approximately 85 % of its maximum activity was retained after incubating for 4 h. Further, our Nano-DSC analysis determined that its melting temperature (Tm) was 70.74 °C. Using 100, 300, and 500 g L-1 of d-fructose as substrate, 25.6, 74.4, and 115 g L-1 of d-mannose were produced respectively, corresponding to a conversion rate of 25.6 %, 24.8 %, and 23.0 % under optimal conditions. Taken together, our results provide evidence for a promising candidate d-LIase for producing d-mannose directly from d-fructose.

Production of l-ribose from l-arabinose by co-expression of l-arabinose isomerase and d-lyxose isomerase in Escherichia coli.

l-Ribose is an important pharmaceutical intermediate that is used in the synthesis of numerous antiviral and anticancer drugs. However, it is a non-natural and expensive rare sugar. Recently, the enzymatic synthesis of l-ribose has attracted considerable attention owing to its considerable advantages over chemical approaches. In this work, a new strategy was developed for the production of l-ribose from the inexpensive starting material l-arabinose. The l-arabinose isomerase (l-AIase) gene from Alicyclobacillus hesperidum and the d-lyxose isomerase (d-LIase) gene from Thermoflavimicrobium dichotomicum were cloned and co-expressed in Escherichia coli, resulting in recombinant cells harboring the vector pCDFDuet-Alhe-LAI/Thdi-DLI. The co-expression system exhibited optimal activity at a temperature of 70 °C and pH 6.0, and the addition of Co2+ enhanced the catalytic activity by 27.8-fold. The system containing 50 g L-1 of recombinant cells were relatively stable up to 55 °C. The co-expression system (50 g L-1 of recombinant cells) afforded 20.9, 39.7, and 50.3 g L-1 of l-ribose from initial l-arabinose concentrations of 100, 300, and 500 g L-1, corresponding to conversion rate of 20.9%, 13.2%, and 10.0%, respectively. Overall, this study provides a viable approach for producing l-ribose from l-arabinose under slightly acidic conditions using a co-expression system harboring l-AIase and d-LIase genes.

Characterization of a D-lyxose isomerase from Bacillus velezensis and its application for the production of D-mannose and L-ribose.

D-Mannose and L-ribose are two important monosaccharides, which have attracted public attention recently because of their great application potentials in food, cosmetic and pharmaceutical industries. Sugar isomerases catalyze the sugar isomerization and therefore can be used as the biocatalysts for production of the high-value sugars from inexpensive sugars. L-arabinose isomerase catalyzes the conversion of L-arabinose to L-ribulose, while D-lyxose isomerase catalyzes L-ribulose and D-fructose to L-ribose and D-mannose, respectively. In this paper, a putative D-LI from Bacillus velezensis (BvLI) was identified, characterized and used to produce D-mannose and L-ribose from D-fructose and L-arabinose, respectively. The recombinant BvLI exhibited a maximum activity at 55 °C and pH 6.5, in the presence of 0.1 mM Co2+. Approximately 110.75 g/L D-mannose was obtained from 500 g/L D-fructose in 6 h by the recombinant BvLI, and approximately 105 g/L L-ribose was obtained from 500 g/L L-arabinose in 8 h by the successive biocatalysis of L-arabinose isomerase from Bacillus licheniformis (BlAI) and BvLI.

Production of d-mannose from d-glucose by co-expression of d-glucose isomerase and d-lyxose isomerase in Escherichia coli.

BACKGROUND: d-Mannose is not only the epimer of d-glucose at the C-2 position, but also the aldose isomer of d-fructose. Because of its physiological properties and health benefits, d-mannose has attracted public interest. It has been confirmed that d-mannose has broad applications in food, cosmetics, and pharmaceutical industries. According to the Izumoring strategy, d-glucose isomerase (d-GI) and d-lyxose isomerase (d-LI) play important roles in the conversions of d-fructose from d-glucose and of d-mannose from d-fructose respectively. In this study, a one-step enzyme process of d-mannose production from d-glucose has been constructed by co-expression of the d-GI from Acidothermus cellulolyticus and d-LI from Thermosediminibacter oceani in Escherichia coli BL21(DE3) cells. RESULTS: The co-expression system exhibits maximum activity at pH 6.5 and 65 °C with Co2+ supplement. It is relatively thermostable at less than 65 °C. When the reaction reaches equilibrium, the ratio of d-glucose, d-fructose, and d-mannose is approximately 34 : 49.6 : 16.4. By using this co-expression system, about 60.0 g L-1 d-mannose is obtained from 400 g L-1 d-glucose in 8 h. CONCLUSION: This co-expression of d-GI and d-LI system provides a novel and efficient approach for d-mannose production. © 2018 Society of Chemical Industry.

D-lyxose isomerase and its application for functional sugar production.

Functional sugars have attracted attention because of their wide application prospects in the food, cosmetics, and pharmaceutical industries in recent decades. Compared with complex chemical synthesis, enzymatic methods of creating functional sugars, characterized by high specificity, moderate reaction conditions, and sustainability, are favored. D-lyxose isomerase (D-LI, EC 5.3.1.15), an important aldose-ketose isomerase, catalyzes the reverse isomerization reaction between D-xylulose and D-lyxose, as well as D-fructose and D-mannose. D-LI has drawn researchers' attention due to its broad substrate specificity and high potential for enzymatic production of some functional sugars such as D-xylulose, D-mannose, and D-ribose. In this article, an overview of recent advances in the biochemical properties of various D-LIs is explored in detail. Structural analysis, active site identification, and catalytic mechanisms are also provided. Additionally, the applications of D-LIs for functional sugar production, including D-lyxose, D-mannose, and L-ribose, are reviewed in detail in this paper.