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d-Allose is a low-calorie rare sugar with great application potential in the food and pharmaceutical industries. The production of d-allose has been accomplished using l-rhamnose isomerase (L-RI), but concomitantly increasing the enzyme's stability and activity remains challenging. Here, we rationally engineered an L-RI from Clostridium stercorarium to enhance its stability by comprehensive computation-aided redesign of its flexible regions, which were successively identified using molecular dynamics simulations. The resulting combinatorial mutant M2-4 exhibited a 5.7-fold increased half-life at 75 °C while also exhibiting improved catalytic efficiency. Especially, by combining structure modeling and multiple sequence alignment, we identified an α0 region that was universal in the L-RI family and likely acted as a "helix-breaker". Truncating this region is crucial for improving the thermostability of related enzymes. Our work provides a significantly stable biocatalyst with potential for the industrial production of d-allose.

Metal-organic frameworks (MOFs) are a class of porous materials with versatile properties. In this study, ZIF-8 was employed to establish a two-enzyme system by encapsulating permeabilized Bacillus subtilis cells coated with glucose isomerase. B. subtilis was constructed by introducing the shuttle plasmid PMA5 associated with the overexpression of trehalose synthase. Using this two-enzyme system, trehalose was produced by trehalose synthase and the byproduct glucose was converted to fructose with the help of glucose isomerase. The decrease in glucose production not only relieved the inhibition of the entire reaction chain but also increased the final yield of trehalose. The highest trehalose production rate reached 67.7% and remained above 50% after 20 batches. In addition, the toxicity of the ZIF-8 coating for B. subtilis was investigated by fluorescence microscopy and was found to be negligible. By simulating an extreme environment, the ZIF-8 coating was demonstrated to have a protective effect on the cells and enzymes. This study provides a theoretical basis for the application of MOFs in the immobilization of microorganisms and enzymes.

d-Tagatose is a rare monosaccharide that is used in products in the food industry as a low-calorie sweetener. To facilitate biological conversion of d-tagatose, the agarolytic enzyme complexes based on the principle of the cellulosome structure were constructed through dockerin-cohesin interaction with the scaffoldin. The construction of agarolytic complexes composed of l-arabinose isomerase caused efficient isomerization activity on the agar-derived sugars. In a trienzymatic complex, the chimeric β-agarase (cAgaB) and anhydro-galactosidase (cAhgA) from Zobellia galactanivorans could synergistically hydrolyze natural agar substrates and l-arabinose isomerase (LsAraA Doc) from Lactobacillus sakei 23K could convert d-galactose into d-tagatose. The trienzymatic complex increased the concentration of d-tagatose from the agar substrate to 4.2 g/L. Compared with the monomeric enzyme, the multimeric enzyme showed a 1.4-fold increase in tagatose production, good thermostability, and reusability. A residual activity of 75% remained, and 52% of conversion was noted after five recycles. These results indicated that the dockerin-fused chimeric enzymes on the scaffoldin successfully isomerized d-galactose into d-tagatose with synergistic activity. Thus, the results demonstrated the possibility of advancing efficient strategies for utilizing red algae as a biomass source to produce d-tagatose in the industrial food field that uses marine biomass as the feedstock.

The rare sugar l-ribulose is produced from the abundant sugar l-arabinose by enzymatic conversion. An l-arabinose isomerase (AI) from Geobacillus thermodenitrificans was efficiently expressed and encapsulated in Saccharomyces cerevisiae spores. Deletion of the yeast OSW2 gene, which causes a mild defect in the integrity of the spore wall, substantially improved the activity of encapsulated AI, without damaging its superior enzymatic properties of thermostability, pH tolerance,and resistance toward SDS and proteinase treatments. In a 10 mL reaction, 100 mg of dry AI encapsulated in spores produced 250 mg of l-ribulose from 1 g of l-arabinose, indicating a 25% conversion rate. Notably, the product of l-ribulose was directly purified from the reaction solution with an approximately 91% recovery using a Ca 2+ ion exchange column. Our results describe not only a facile approach for the production of l-ribulose but also a useful strategy for the enzymatic conversion of rare sugars in "Izumoring".

d-Allose, a rare sugar, is an ideal table-sugar substitute and has many advantageous physiological functions. l-Rhamnose isomerase (l-RI) is an important d-allose-producing enzyme, but it exhibits comparatively low catalytic activity on d-allulose. In this study, an array of hydrophobic residues located within β1-α1-loop were solely or collectively replaced with polar amino acids by site-directed mutagenesis. A group of mutants was designed to weaken the hydrophobic environment and strengthen the catalytic behavior on d-allulose. Compared with that of the wild-type enzyme, the relative activities of the V48N/G59N/I63N and V48N/G59N/I63N/F335S mutants toward d-allulose were increased by 105.6 and 134.1%, respectively. Another group of mutants was designed to enhance thermostability. Finally, the t 1/2 values of mutant S81A were increased by 7.7 and 1.1 h at 70 and 80 °C, respectively. These results revealed that site-directed mutagenesis is efficient for improving thermostability and catalytic behavior toward d-allulose.

d-Tagatose is a prospective functional sweetener that can be produced by l-arabinose isomerase (AI) from d-galactose. To improve the activity of AI toward d-galactose, the AI of Bacillus coagulans was rationally designed on the basis of molecular modeling and docking. After alanine scanning and site-saturation mutagenesis, variant F279I that exhibited improved activity toward d-galactose was obtained. The optimal temperature and pH of F279I were determined to be 50 °C and 8.0, respectively. This variant possessed 1.4-fold catalytic efficiency compared with the wild-type (WT) enzyme. The recombinant Escherichia coli overexpressing F279I also showed obvious advantages over the WT in biotransformation. Under optimal conditions, 67.5 and 88.4 g L -1 d-tagatose could be produced from 150 and 250 g L -1 d-galactose, respectively, in 15 h. The biocatalyst constructed in this study presents a promising alternative for large-scale d-tagatose production.

The functional sweetener, d-tagatose, is commonly transformed from galactose by l-arabinose isomerase. To make use of a much cheaper starting material, lactose, hydrolization, and isomerization are required to take place collaboratively. Therefore, a single-step method involving β-d-galactosidase was explored for d-tagatose production. The two vital genes, β-d-galactosidase gene (lacZ) and l-arabinose isomerase mutant gene (araA') were extracted separately from Escherichia coli strains and incorporated into E. coli simultaneously. This gave us E. coli-ZY, a recombinant producing strain capable of coexpressing the two key enzymes. The resulted cells exhibited maximum d-tagatose producing activity at 34 °C and pH 6.5 and in the presence of borate, 10 mM Fe(2+), and 1 mM Mn(2+). Further monitoring showed that the recombinant cells could hydrolyze more than 95% lactose and convert 43% d-galactose into d-tagatose. This research has verified the feasibility of single-step d-tagatose fermentation, thereby laying down the foundation for industrial usage of lactose.

The L-arabinose isomerase (L-AI) and the D-xylose isomerase (D-XI) encoding genes from Lactobacillus reuteri (DSMZ 17509) were cloned and overexpressed in Escherichia coli BL21 (DE3). The proteins were purified to homogeneity by one-step affinity chromatography and characterized biochemically. L-AI displayed maximum activity at 65 °C and pH 6.0, whereas D-XI showed maximum activity at 65 °C and pH 5.0. Both enzymes require divalent metal ions. The genes were also ligated into the inducible lactobacillal expression vectors pSIP409 and pSIP609, the latter containing a food grade auxotrophy marker instead of an antibiotic resistance marker, and the L-AI- and D-XI-encoding sequences/genes were coexpressed in the food grade host Lactobacillus plantarum . The recombinant enzymes were tested for applications in carbohydrate conversion reactions of industrial relevance. The purified L-AI converted D-galactose to D-tagatose with a maximum conversion rate of 35%, and the D-XI isomerized D-glucose to D-fructose with a maximum conversion rate of 48% at 60 °C.

L-rhamnose isomerase (EC 5.3.1.14, L-RhI) catalyzes the reversible aldose-ketose isomerization between L-rhamnose and L-rhamnulose. In this study, the L-Rhi gene encoding L-Rhi was PCR-cloned from Thermoanaerobacterium saccharolyticum NTOU1 and then expressed in Escherichia coli. A high yield of the active L-RhI, 9780 U/g of wet cells, was obtained in the presence of 0.2 mM IPTG induction. L-RhI was purified sequentially using heat treatment, nucleic acid precipitation, and anion-exchange chromatography. The purified L-RhI showed an apparent optimal pH of 7 and an optimal temperature at 75 °C. The enzyme was stable at pH values ranging from 5 to 9, and the activity was fully retained after a 2 h incubation at 40-70 °C. L-RhI from T. saccharolyticum NTOU1 is the most thermostable L-RhI to date, and it has a high specific activity (163 U/mg) and an acceptable purity after heat treatment, suggesting that this enzyme has the potential to be used in rare sugar production.