Preparation of combined cross-linked enzyme aggregates containing galactitol dehydrogenase and NADH oxidase for L-tagatose synthesis via in situ cofactor regeneration.

The combined cross-linked enzyme aggregates (combi-CLEAs) containing galactitol dehydrogenase (Gdh) and NADH oxidase (Nox) were prepared for L-tagatose synthesis. To prevent the excess consumption of cofactor, Nox in the combi-CLEAs was used to in situ regenerate NAD+. In the immobilization process, ammonia sulfate and glutaraldehyde were used as the precipitant and cross-linking reagent, respectively. The preparation conditions were optimized as follows: 60% ammonium sulfate, 1:1 (molar ratio) of Gdh to Nox, 20:1 (molar ratio) of protein to glutaraldehyde, and 6 h of cross-linking time at 35 °C. Under these conditions, the activity of the combi-CLEAs was 210 U g-1. The combi-CLEAs exhibited higher thermostability and preserved 51.5% of the original activity after eight cycles of reuses at 45 °C. The combi-CLEAs were utilized for the preparation of L-tagatose without by-products. Therefore, the combi-CLEAs have the industrial potential for the bioconversion of galactitol to L-tagatose.

Efficient production of the rare sugar l-gulose using a wheat-bran culture extract of Penicillium sp. KU-1.

We found that l-gulose, a rare sugar, was produced from d-sorbitol efficiently, using a wheat-bran culture extract of the fungus Penicillium sp. KU-1 isolated from soil. The culture extract showed enzyme activity for the oxidation of d-sorbitol to produce l-gulose; a high production yield of approximately 94% was achieved.

Efficient whole-cell biosynthesis of l-gulose by coupling mannitol-1-dehydrogenase with NADH oxidase.

L-Gulose is a rare aldohexose to serve as a building block for anticancer drug bleomycin and nucleoside-based antivirals. However, preparative inaccessibility and high cost have hindered its pharmaceutical application. Despite a regio- and stereo-selective enzymatic synthesis of l-gulose from d-sorbitol using a variant of NAD+-dependent mannitol-1-dehydrogenase from Apium graveolens (mMDH) was explored, low efficiency and productivity caused by NADH accumulation or insufficient amount of NAD+ limited the practical utility of this process. In this study, a stable and efficient NADH oxidase from Bacillus cereus (bcNOX) was found to be more compatible with mMDH to recycle NAD+ in E. coli cells for l-gulose biosynthesis. After a systematic optimization of the whole-cell system, efficient biosynthesis of l-gulose was achieved. Starting with 70 g/L of readily available and cheap d-sorbitol resulted in a volumetric productivity of 5.5 g/L/d. This whole-cell approach enables practical, efficient and environmentally friendly biosynthesis of l-gulose and exhibits the potential of becoming a biocatalytic strategy for various enzymatic oxidative transformations.

Using galactitol dehydrogenase coupled with water-forming NADH oxidase for efficient enzymatic synthesis of L-tagatose.

L-Tagatose, a promising building block in the production of many value-added chemicals, is generally produced by chemical routes with a low yield, which may not meet the increasing demands. Synthesis of l-tagatose by enzymatic oxidation of d-galactitol has not been applied on an industrial scale because of the high cofactor costs and the lack of efficient cofactor regeneration methods. In this work, an efficient and environmentally friendly enzymatic method containing a galactitol dehydrogenase for d-galactitol oxidation and a water-forming NADH oxidase for regeneration of NAD+ was first designed and used for l-tagatose production. Supplied with only 3 mM NAD+, subsequent reaction optimization facilitated the efficient transformation of 100 mM of d-galactitol into l-tagatose with a yield of 90.2 % after 12 h (obtained productivity: 7.61 mM.h-1). Compared with the current chemical and biocatalytic methods, the strategy developed avoids by-product formation and achieves the highest yield of l-tagatose with low costs. It is expected to become a cleaner and more promising route for industrial biosynthesis of l-tagatose.

Deciphering the Biosynthesis of TDP-ß-l-oleandrose in Avermectin.

Avermectin (AVM) refers to eight macrolides containing a common l-oleandrosyl disaccharide chain indispensable to their antiparasitic bioactivities. We delineated the biosynthetic pathway of TDP-ß-l-oleandrose (1), the sugar donor of AVM, by characterizing AveBVIII, AveBV, and AveBVII as TDP-sugar 3-ketoreductase, 5-epimerase, and 3-O-methyltransferase, respectively. On the basis of this pathway, we successfully reconstituted the biosynthesis of 1 in Escherichia coli. Our work completes the biosynthetic pathway of AVM and lays a solid foundation for further studies.

Characteristics of a fructose 6-phosphate 4-epimerase from Caldilinea aerophila DSM 14535 and its application for biosynthesis of tagatose.

Tagatose is a rare hexoketose with potential health benefits. Here, an enzyme, GatZ subunit ofd-tagatose-1,6-bisphosphate aldolase, was characterized. GatZ is involved in a multi-enzyme cascade reaction system that can produce tagatose from maltodextrin. It showed maximum activity at 70 °C and a pH 8.0, and required supplementation with 5 mM Mg2+ to achieve the highest catalytic activity. The Km and Vmax values of GatZ using fructose 6-phosphate as substrate were 5.66 mM and 0.0329 mmol/L min, respectively. An in vitro multi-enzyme system containing GatZ was constructed, and 1.75 g/L tagatose was produced from 5 g/L maltodextrin after 10 h. This biosystem could potentially enrich the application of C4 epimerases in rare sugar bioproduction.

Stabilization of immobilized l-arabinose isomerase for the production of d-tagatose from d-galactose.

The aim of this work was to develop a stable immobilized enzyme biocatalyst for the isomerization of d-galactose to d-tagatose at high temperature. l-Arabinose isomerase from the hyperthermophilic bacterium Thermotoga maritima (TMAI) was produced as a (His)6 -tagged protein, immobilized on a copper-chelate epoxy support and subjected to several postimmobilization treatments aimed at increasing its operational and structural stability. Treatment with glutaraldehyde and ethylenediamine resulted in a more than twofold increase in the operational stability and in all enzyme subunits linked, directly or indirectly, to the support via covalent bonds. A postimmobilization treatment of the immobilized derivatives with mercaptoethanol for the removal of any remaining copper ions, determined a further increase of the operational biocatalytic activity. Immobilized derivatives subjected to both treatments were used for the bioconversion of 18 g/L d-galactose to d-tagatose at 80°C in a packed bed reactor in three repeated cycles and showed a better operational stability compared with the literature data. This study shows that a postimmobilization stabilization treatment with glutaraldehyde and ethylenediamine can stabilize the multi-subunit structure of an enzyme immobilized on a metal-chelate epoxy support with an increase of its operational stability, results that are not easily achievable with the sole immobilization on epoxy or metal chelate-epoxy supports in the case of complex multimeric enzymes with geometric incongruence with the support.

Biochemical characterization of recombinant L-fucose isomerase from Caldanaerobius polysaccharolyticus for L-fuculose production.

L-fuculose is a rare sugar that is useful for the agriculture and medicine industries. L-fucose isomerase (E.C.5.3.1.25), which is an aldose-ketose isomerase, plays a significant role in producing rare sugars. A recommended L-fucose isomerase gene was cloned from Caldanaerobius polysaccharolyticus and purified with a single band of 65 kDa using nickel-affinity chromatography, with a specific activity of 108.23 U mg-1. The native molecular mass existed with 214 kDa was a trimer. The purified enzyme showed a maximum activity in 1 mM Mn2+ at 55 °C and pH 6.5 with a melting temperature (Tm) of 80.3 °C in the presence of one molecule per monomer. L-fucose isomerase from C. polysaccharolyticus (Capo-LfIase) exhibited the highest activity of L-fucose with Km, kcat and Kcat/km values of 94.2 mM, 23854 min-1 and 253.3 min-1 mM-1, respectively. Capo-LfIase showed more than 50% thermostability after 20 h of incubation at 45, 55, 65, 75 and 85 °C. The 9 putative active site residues of the L-fucose substrate were described using a homology model, and the results showed that Tyr440, Met185, Trp499 and Asn527 are the candidates of metal-binding residues, while Ser393, Glu337, Glu302, His528 and Asp361 would be involved in substrate binding. The conversion rate of L-fuculose from L-fucose was almost 28.2%, with 80 g L-1 L-fucose, and no byproduct was found. To the best of our knowledge, Capo-LfIase produces high yield of L-fuculose from L-fucose by enzymatic methods.

Biochemical Characterization of Heat-Tolerant Recombinant L-Arabinose Isomerase from Enterococcus faecium DBFIQ E36 Strain with Feasible Applications in D-Tagatose Production.

D-Tagatose is a ketohexose, which presents unique properties as a low-calorie functional sweetener possessing a sweet flavor profile similar to D-sucrose and having no aftertaste. Considered a generally recognized as safe (GRAS) substance by FAO/WHO, D-tagatose can be used as an intermediate for the synthesis of other optically active compounds as well as an additive in detergent, cosmetic, and pharmaceutical formulations. This study reports important features for L-arabinose isomerase (EC 5.3.1.4) (L-AI) use in industry. We describe arabinose (araA) gene virulence analysis, gene isolation, sequencing, cloning, and heterologous overexpression of L-AI from the food-grade GRAS bacterium Enterococcus faecium DBFIQ E36 in Escherichia coli and assess biochemical properties of this recombinant enzyme. Recombinant L-AI (rL-AI) was one-step purified to homogeneity by Ni2+-agarose resin affinity chromatography and biochemical characterization revealed low identity with both thermophilic and mesophilic L-AIs but high degree of conservation in residues involved in substrate recognition. Optimal conditions for rL-AI activity were 50 °C, pH 5.5, and 0.3 mM Mn2+, exhibiting a low cofactor concentration requirement and an acidic optimum pH. Half-life at 45 °C and 50 °C were 1427 h and 11 h, respectively, and 21.5 h and 39.5 h at pH 4.5 and 5.6, respectively, showing the high stability of the enzyme in the presence of a metallic cofactor. Bioconversion yield for D-tagatose biosynthesis was 45% at 50 °C after 48 h. These properties highlight the technological potential of E. faecium rL-AI as biocatalyst for D-tagatose production.

Cloning and expression of d-glucoside 3-dehydrogenase from Rhizobium sp. S10 in Escherichia coli and its application for d-gulose production.

The novel isolated Rhizobium sp. S10 was identified as d-glucoside 3-dehydrogenase (G3DH) producing microbe. Therefore, the gene encoding for G3DH from Rhizobium sp. S10 was cloned and overexpressed in Escherichia coli strain JM109 as a soluble enzyme complex. The recombinant G3DH (rG3DH) was purified with relatively high specific activity of 38.54 U/mg compared to the previously characterized and cloned G3DHs. The purified rG3DH showed the highest activity at pH 7.0, 40 °C toward cellobiose. It can also oxidize a broad range of mono-disaccharides including saccharide derivatives. The glycosides oxidizing activity combined with chemical reaction, could produce d-gulose from lactitol via 3-ketolactitol.

One-Step Immobilization and Stabilization of a Recombinant Enterococcus faecium DBFIQ E36 L-Arabinose Isomerase for D-Tagatose Synthesis.

A recombinant L-arabinose isomerase from Enterococcus faecium DBFIQ E36 was immobilized onto multifunctional epoxide supports by chemical adsorption and onto a chelate-activated support via polyhistidine-tag, located on the N-terminal (N-His-L-AI) or on the C-terminal (C-His-L-AI) sequence, followed by covalent bonding between the enzyme and the support. The results were compared to reversible L-AI immobilization by adsorption onto charged agarose supports with improved stability. All the derivatives presented immobilization yields of above 75%. The ionic interaction established between agarose gels containing monoaminoethyl-N-aminoethyl structures (MANAE) and the enzyme was the most suitable strategy for L-AI immobilization in comparison to the chelate-activated agarose. In addition, the immobilized biocatalysts by ionic interaction in MANAE showed to be the most stable, retaining up to 100% of enzyme activity for 60 min at 60 °C and with Km values of 28 and 218 mM for MANAE-N-His-L-AI and MANAE-C-His-L-AI, respectively.

Tagatose as a Potential Nutraceutical: Production, Properties, Biological Roles, and Applications.

Nutraceuticals are gaining importance owing to their potential applications in numerous sectors including food and feed industries. Among the emerging nutraceuticals, d-tagatose occupies a significant niche because of its low calorific value, antidiabetic property and growth promoting effects on beneficial gut bacteria. As d-tagatose is present in minute quantities in naturally occurring food substances, it is produced mainly by chemical or biological means. Recently, attempts were made for bio-production of d-tagatose using l-arabinose isomerase enzyme to overcome the challenges of chemical process of production. Applications of d-tagatose for maintaining health and wellbeing are increasing due to growing consumer awareness and apprehension against modern therapeutic agents. This review outlines the current status on d-tagatose, particularly its production, properties, biological role, applications, and the future perspectives.

Engineering the l-Arabinose Isomerase from Enterococcus Faecium for d-Tagatose Synthesis.

l-Arabinose isomerase (EC 5.3.1.4) (l-AI) from Enterococcus faecium DBFIQ E36 was overproduced in Escherichia coli by designing a codon-optimized synthetic araA gene. Using this optimized gene, two N- and C-terminal His-tagged-l-AI proteins were produced. The cloning of the two chimeric genes into regulated expression vectors resulted in the production of high amounts of recombinant N-His-l-AI and C-His-l-AI in soluble and active forms. Both His-tagged enzymes were purified in a single step through metal-affinity chromatography and showed different kinetic and structural characteristics. Analytical ultracentrifugation revealed that C-His-l-AI was preferentially hexameric in solution, whereas N-His-l-AI was mainly monomeric. The specific activity of the N-His-l-AI at acidic pH was higher than that of C-His-l-AI and showed a maximum bioconversion yield of 26% at 50 °C for d-tagatose biosynthesis, with Km and Vmax parameters of 252 mM and 0.092 U mg-1, respectively. However, C-His-l-AI was more active and stable at alkaline pH than N-His-l-AI. N-His-l-AI follows a Michaelis-Menten kinetic, whereas C-His-l-AI fitted to a sigmoidal saturation curve.

Enhancements in sucrose biosynthesis capacity affect shoot branching in Arabidopsis.

We previously demonstrated that transgenic tobacco plants expressing cyanobacterial fructose-1,6-/sedoheptulose-1,7-bisphosphatase in the cytosol increased the number of lateral shoots and leaves at elevated CO2 levels. These findings suggest that alterations in carbon partitioning affect the development of shoot branching. In order to elucidate the underlying mechanisms at the molecular level, we generated transgenic Arabidopsis plants overexpressing cyanobacterial fructose-1,6-bisphosphatase-II in the cytosol (AcF). At elevated CO2 levels, the number of lateral shoots was significantly increased in AcF plants. Sucrose and hexose levels were also higher in AcF plants than in wild-type plants. The expression levels of MAX1, MAX4, YUCCA8, YUCCA9, and BRC1, which are involved in auxin or strigolactone biosynthesis and responses, were lower in AcF plants than in wild-type plants. These results suggest that alterations in sugar partitioning affect hormone metabolism and responses, resulting in enhanced shoot branching.

Rational Design of Bacillus coagulans NL01 l-Arabinose Isomerase and Use of Its F279I Variant in d-Tagatose Production.

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.

Biocatalytic production of D-tagatose: A potential rare sugar with versatile applications.

D-tagatose is a naturally existing rare monosaccharide having prebiotic properties. Minimal absorption, low metabolizing energy, and unique clinical properties are the characteristics of D-tagatose. D-tagatose gained international attention by matching the purpose of alternate sweeteners that is much needed for the control of diabetes among world population. Recent efforts in understanding tagatose bioconversion have generated essential information regarding its production and application. This article reviews the evolution of D-tagatose as an important rare sugar by appreciable improvements in production results and its significant applications resulted of its unique physical, chemical, biological, and clinical properties thus considering it an appropriate product for requisite improvements in technical viability. Based on current knowledge and technology projections, the commercialization of D-tagatose rare sugar as food additive is close to reality.

A single and two step isomerization process for d-tagatose and l-ribose bioproduction using l-arabinose isomerase and d-lyxose isomerase.

l-ribose and d-tagatose are biochemically synthesized using sugar isomerases. The l-arabinose isomerase gene from Shigella flexneri (Sf-AI) was cloned and expressed in Escherichia coli BL-21. Sf-AI was applied for the bioproduction of d-tagatose from d-galactose. l-ribose synthesis was performed by two step isomerization using Sf-AI and d-lyxose/ribose isomerase from Cohnella laevoribosii. The overall 22.3% and 25% conversion rate were observed for d-tagatose and l-ribose production from d-galactose and l-arabinose respectively. In the present manuscript, synthesis of rare sugars from naturally available sugars is discussed along with the biochemical characterization of Sf-AI and its efficiency.

Pathway Construction in Corynebacterium glutamicum and Strain Engineering To Produce Rare Sugars from Glycerol.

Rare sugars are valuable natural products widely used in pharmaceutical and food industries. In this study, we expected to synthesize rare ketoses from abundant glycerol using dihydroxyacetone phosphate (DHAP)-dependent aldolases. First, a new glycerol assimilation pathway was constructed to synthesize DHAP. The enzymes which convert glycerol to 3-hydroxypropionaldehyde and l-glyceraldehyde were selected, and their corresponding aldehyde synthesis pathways were constructed in vivo. Four aldol pathways based on different aldolases and phosphorylase were gathered. Next, three pathways were assembled and the resulting strains synthesized 5-deoxypsicose, 5-deoxysorbose, and 5-deoxyfructose from glucose and glycerol and produce l-fructose, l-tagatose, l-sorbose, and l-psicose with glycerol as the only carbon source. To achieve higher product titer and yield, the recombinant strains were further engineered and fermentation conditions were optimized. Fed-batch culture of engineered strains obtained 38.1 g/L 5-deoxypsicose with a yield of 0.91 ± 0.04 mol product per mol of glycerol and synthesized 20.8 g/L l-fructose, 10.3 g/L l-tagatose, 1.2 g/L l-sorbose, and 0.95 g/L l-psicose.

Increased Production of Food-Grade d-Tagatose from d-Galactose by Permeabilized and Immobilized Cells of Corynebacterium glutamicum, a GRAS Host, Expressing d-Galactose Isomerase from Geobacillus thermodenitrificans.

The generally recognized as safe microorganism Corynebacterium glutamicum expressing Geobacillus thermodenitrificans d-galactose isomerase (d-GaI) was an efficient host for the production of d-tagatose, a functional sweetener. The d-tagatose production at 500 g/L d-galactose by the host was 1.4-fold higher than that by Escherichia coli expressing d-GaI. The d-tagatose-producing activity of permeabilized C. glutamicum (PCG) cells treated with 1% (w/v) Triton X-100 was 2.1-fold higher than that of untreated cells. Permeabilized and immobilized C. glutamicum (PICG) cells in 3% (w/v) alginate showed a 3.1-fold longer half-life at 50 °C and 3.1-fold higher total d-tagatose concentration in repeated batch reactions than PCG cells. PICG cells, which produced 165 g/L d-tagatose after 3 h, with a conversion of 55% (w/w) and a productivity of 55 g/L/h, showed significantly higher d-tagatose productivity than that reported for other cells. Thus, d-tagatose production by PICG cells may be an economical process to produce food-grade d-tagatose.

Characterization of the Two Methylation Steps Involved in the Biosynthesis of Mycinose in Tylosin.

The S-adenosyl-l-methionine-dependent O-methyltransferases TylE and TylF catalyze the last two methylation reactions in the tylosin biosynthetic pathway of Streptomyces fradiae. It has long been known that the TylE-catalyzed C2‴-O-methylation of the 6-deoxy-d-allose bound to demethylmacrocin or demethyllactenocin precedes the TylF-catalyzed C3‴-O-methylation of the d-javose (C2‴-O-methylated 6-deoxy-d-allose) attached to macrocin or lactenocin. This study reveals the unexpected substrate promiscuity of TylE and TylF responsible for the biosynthesis of d-mycinose (C3‴-O-methylated d-javose) in tylosin through the identification of a new minor intermediate 2‴-O-demethyldesmycosin (2; 3‴-methyl-demethyllactenocin), which lacks a 2‴-O-methyl group on the mycinose moiety of desmycosin, along with 2‴-O-demethyltylosin (1; 3‴-methyl-demethylmacrocin) that was previously detected from the S. fradiae mutant containing a mutation in the tylE gene. These results unveil the unique substrate flexibility of TylE and TylF and demonstrate their potential for the engineered biosynthesis of novel glycosylated macrolide derivatives.