Platform For Enzyme Immobilization Research Articles

Laccase Immobilized on Arginine-Functionalized Boron Nitride Nanosheets for Enhanced Atrazine Degradation.

Enzyme-mediated systems have been widely employed for the biotransformation of environmental contaminants. However, the catalytic performance of free enzymes is restricted by the rapid loss of their catalytic activity, stability, and reusability. In this work, we developed an enzyme immobilization platform by elaborately anchoring fungal laccase onto arginine-functionalized boron nitride nanosheets (BNNS-Arg@Lac). BNNS-Arg@Lac showcased ∼75% immobilization yield and enhanced stability against fluctuating pH values and temperatures, along with remarkable reusability across six consecutive cycles, outperforming free natural laccase (nlaccase). A model pollutant, atrazine, was selected for a proof-of-concept demonstration, given the substantial environmental and public health concerns in agriculture runoff. BNNS-Arg@Lac-catalyzed atrazine degradation rate was nearly twice that of nlaccase. Moreover, BNNS-Arg@Lac consistently demonstrated superior atrazine degradation in synthetic agricultural wastewater and various mediator systems compared to nlaccase. Comprehensive product analysis unraveled distinct degradation pathways for BNNS-Arg@Lac and nlaccase. Overall, this research provides a foundation for the future development of enzyme-nanomaterial hybrids for degrading environmental chemicals and may unlock new potential for green and efficient resource recovery and waste management strategies.

Designable immobilization of D-allulose 3-epimerase on bimetallic organic frameworks based on metal ion compatibility for enhanced D-allulose production

D-allulose, a low-calorie rare sugar catalyzed by D-allulose 3-epimerase (DAE), is highly sought after for its potential health benefits. However, poor reusability and stability of DAE limited its popularization in industrial applications. Although metal-organic frameworks (MOFs) offer a promising enzyme platform for enzyme immobilization, developing customized strategies for MOF immobilization of enzymes remains challenging. In this study, we introduce a designable strategy involving the construction of bimetal-organic frameworks (ZnCo-MOF) based on metal ions compatibility. The DAE@MOFs materials were prepared and characterized, and the immobilization of DAE and the enzymatic characteristics of the MOF-immobilized DAE were subsequently evaluated. Remarkably, DAE@ZnCo-MOF exhibited superior recyclability which could maintain 95 % relative activity after 8 consecutive cycles. The storage stability is significantly improved compared to the free form, with a relative activity of 116 % remaining after 30 days. Molecular docking was also employed to investigate the interaction between DAE and the components of MOFs synthesis. The results demonstrate that the DAE@ZnCo-MOF exhibited enhanced catalytic efficiency and increased stability. This study introduces a viable and adaptable MOF-based immobilization strategy for enzymes, which holds the potential to expand the implementation of enzyme biocatalysts in a multitude of disciplines.

Multivariate mesoporous MOFs with regulatable hydrophilic/hydrophobic surfaces as a versatile platform for enzyme immobilization

Zeolitic imidazole frameworks materials-8 (ZIF-8), a member of the metal-organic framework (MOFs) series, have been extensively used as a host matrix for enzyme immobilization. However, the microporous structure and hydrophobicity, as well as the protonation of the precursor 2-methylimidazole (2-MeIm) of ZIF-8, remain considerable challenges in maintaining the activity of immobilized enzymes. Here, novel multivariate mesoporous MOFs (mMOFs) with regulatable hydrophilic/hydrophobic surfaces were designed by the multivariate competitive strategy and pore modification engineering. 3-Methyl-1H-1,2,4-triazole (3-MTZ) and 5-methyltetrazole (5-MTA) were employed to partially replace 2-MeIm. These were then combined with zinc sulfate heptahydrate (soft templates) to yield mMOFs in a methanol solution. As a proof-of-concept application, we used mMOFs as carriers for enzyme immobilization and investigated the properties of the immobilized enzymes. Benefiting from their mesoporous structure, hydrophilic surface, and improved microenvironment, multivariate mMOFs exhibit a strong ability to stabilize enzyme conformation and increase enzyme activity compared with traditional ZIF-8. Our study offers an avenue for the controllable preparation of well-designed MOF structures, which will further broaden the application opportunities of MOF materials for enzyme immobilization.

Double-Network structure chemically programmed DNA Cryogel: Enzyme immobilization platform with enhanced catalytic activity for portable biosensing

Enzyme-based hydrogels have conferred multiple benefits, particularly for disease diagnosis and environmental monitoring due to their pronounced biocompatibility, ability to load cargo, and optical properties. However, traditional hydrogels suffer from some significant limitations, such as enzyme leakage and catalytic activity decreased, restricting the extent to which hydrogels can be beneficially used. Herein, chemically programmed DNA-DNA interactions were designed as a promising solution for enzyme immobilization that substantially maintains biological functionality with remarkable leakage reduction. Additionally, their responsiveness and mechanical properties are dramatically enhanced after adjusting the porous structure of hydrogels via freezing. As a proof-of-concept, a DNA cryogel with a hierarchical structure was developed with adjustable distance among cascade enzymes, increasing the local concentration of intermediates and reducing their mass transfer energy barriers for direct glucose monitoring and glutathione (GSH) sensing. Moreover, after being integrated with a smartphone system, a universal sensing platform (immunosensor) was fabricated for rapid detection of environmental chemicals (i.e., bisphenol A) without expensive instruments and sophisticated procedures. This strategy provided some novel insights on hydrogel- based bioassays, implying their great potentials for conducting onsite analysis against various targets.

Hollow Hierarchical Porous and Antihydrolytic Spherical Zeolitic Imidazolate Frameworks for Enzyme Encapsulation and Biocatalysis.

The creation of a new metal-organic framework (MOF) with a hollow hierarchical porous structure has gained significant attention in the realm of enzyme immobilization. The present work employed a novel, facile, and effective combinatorial technique to synthesize modified MOF (N-PVP/HZIF-8) with a hierarchically porous core-shell structure, allowing for the preservation of the structural integrity of the encapsulated enzyme molecules. Scanning electron microscopy, X-ray diffraction, X-ray photoelectron spectroscopy, confocal laser scanning microscopy, and other characterization tools were used to fully explore the changes of morphological structure and surface properties in different stages of the preparation of immobilization enzyme CRL-N-PVP/HZIF-8, thus showing the superiority of N-PVP/HZIF-8 as an enzyme immobilization platform and the logic of the immobilization process on the carrier. Additionally, the maximum enzyme loading was 216.3 mg mL-1, the relative activity of CRL-N-PVP/HZIF-8 increased by 15 times compared with the CRL@ZIF-8 immobilized in situ, and exhibited quite good thermal, chemical, and operational stability. With a maximal conversion of 88.8%, CRL-N-PVP/HZIF-8 demonstrated good catalytic performance in the biosynthesis of phytosterol esters as a proof of concept. It is anticipated that this work will offer fresh concepts from several perspectives for the creation of MOF-based immobilized enzymes for biotechnological uses.

Support Materials of Organic and Inorganic Origin as Platforms for Horseradish Peroxidase Immobilization: Comparison Study for High Stability and Activity Recovery.

In the presented study, a variety of hybrid and single nanomaterials of various origins were tested as novel platforms for horseradish peroxidase immobilization. A thorough characterization was performed to establish the suitability of the support materials for immobilization, as well as the activity and stability retention of the biocatalysts, which were analyzed and discussed. The physicochemical characterization of the obtained systems proved successful enzyme deposition on all the presented materials. The immobilization of horseradish peroxidase on all the tested supports occurred with an efficiency above 70%. However, for multi-walled carbon nanotubes and hybrids made of chitosan, magnetic nanoparticles, and selenium ions, it reached up to 90%. For these materials, the immobilization yield exceeded 80%, resulting in high amounts of immobilized enzymes. The produced system showed the same optimal pH and temperature conditions as free enzymes; however, over a wider range of conditions, the immobilized enzymes showed activity of over 50%. Finally, a reusability study and storage stability tests showed that horseradish peroxidase immobilized on a hybrid made of chitosan, magnetic nanoparticles, and selenium ions retained around 80% of its initial activity after 10 repeated catalytic cycles and after 20 days of storage. Of all the tested materials, the most favorable for immobilization was the above-mentioned chitosan-based hybrid material. The selenium additive present in the discussed material gives it supplementary properties that increase the immobilization yield of the enzyme and improve enzyme stability. The obtained results confirm the applicability of these nanomaterials as useful platforms for enzyme immobilization in the contemplation of the structural stability of an enzyme and the high catalytic activity of fabricated biocatalysts.

Advanced applications in enzyme-induced electrospun nanofibers.

Electrospun nanofibers, renowned for their high specific surface area, robust mechanical properties, and versatile chemical functionalities, offer a promising platform for enzyme immobilization. Over the past decade, significant strides have been made in developing enzyme-induced electrospun nanofibers (EIEN). This review systematically summarizes the advanced applications of EIEN which are fabricated using both non-specific immobilization methods including interfacial adsorption (direct adsorption, cross-linking, and covalent binding) and encapsulation, and specific immobilization techniques (coordination and affinity immobilization). Future research should prioritize optimizing immobilization techniques to achieve a balance between enzyme activity, stability, and cost-effectiveness, thereby facilitating the industrialization of EIEN. We elucidate the rationale behind various immobilization methods and their applications, such as wastewater treatment, biosensors, and biomedicine. We aim to provide guidelines for developing suitable EIEN immobilization techniques tailored to specific future applications.

Graphene oxide dispersed mesoporous ZIF-8-encapsulated laccase for removal of toluidine blue with multiple enhanced stability.

The extensive use and discharge of toluidine blue have caused serious problems to the water environment. As a green biocatalyst, laccase has the ability to decolorize the dyes, but it is limited by poor reusability and low stability. Metal-organic frameworks (MOFs) are a good platform for enzyme immobilization. However, due to the weak dispersion of MOFs, the enzyme activity is inevitably inhibited. Herein, we proposed to use graphene oxide (GO) as the dispersion medium of mesoporous ZIF-8 to construct MZIF-8/GO bi-carrier for laccase (FL) immobilization. On account of the narrower bandgap energy of FL@MZIF-8/GO (4.07eV) than that of FL@MZIF-8 (4.69eV), electron transport was enhanced which later increased the catalytic activity of the immobilized enzyme. Meanwhile, the improved hydrophilicity characterized by contact angle and full infiltration time further promoted the efficiency of the enzymatic reaction. Benefiting from such regulatory effects of GO, the composite showed excellent storage stability and reusability, as well as multifaceted enhancements including pH, thermal, and solvent adaptation. On the basis of the characterized synergistic effect of adsorption and degradation, FL@MZIF-8/GO was successfully applied to the degradation of toluidine blue (TB) with a removal rate of 94.8%. Even in actual treated wastewater, the highest removal rate still reached more than 80%. Based on the inner mechanism analysis and the universality study, this material is expected to be widely used in the degradation of pollutants in real water under complex environmental conditions.

Cation-assisted stabilization of carbonic anhydrase one-step in situ loaded in diatom-inspired silica nanospheres for potential applications in CO2 capture and utilization

Carbonic anhydrase (CA) is a powerful green catalyst for CO2 capture and utilization due to its ultrafast kinetics and biobased nature. For industrial utilization, immobilization of CA is needed to increase enzyme stability and recovery. Diatom-inspired silica nanoparticle provides a green platform for the efficient immobilization of enzymes in a fast and facile manner. Herein, we describe a simple and effective method of salt addition during diatom-inspired silicification for the development of an immobilized and highly stabilized catalyst with bovine CA (bCA). Silica synthesis was facilitated by the silica-forming R5 peptide fused to the bCA; bCA-R5 was immobilized in situ with an excellent immobilization yield. The thermal stability of bCA-R5 was improved via salt supplementation, which was controlled by the cation-assisted increase in silica synthesis with a high packing density. The salt effect was dependent on both the pH and the enzyme’s electrostatic nature, suggesting the interplay among all the reaction components. The thermal stability of immobilized bCA-R5 was improved 11-fold in a biological buffer and 18-fold in 4.2 M MDEA (an organic solvent used as a CO2 absorbent) via the cation-assisted method. The suggested strategy is useful for developing CO2-capturing nanomaterials and can be widely applicable to immobilizing and stabilizing various proteins, maximally exploiting the potential of diatom-inspired silicification.

Computational Insights into the Selecting Mechanism of α-Amylase Immobilized on Cellulose Nanocrystals: Unveiling the Potential of α-Amylases Immobilized for Efficient Poultry Feed Hydrolysis.

The selection of an appropriate amylase for hydrolysis poultry feed is crucial for achieving improved digestibility and high-quality feed. Cellulose nanocrystals (CNCs), which are known for their high surface area, provide an excellent platform for enzyme immobilization. Immobilization greatly enhances the operational stability of α-amylases and the efficiency of starch bioconversion in poultry feeds. In this study, we immobilized two metagenome-derived α-amylases, PersiAmy2 and PersiAmy3, on CNCs and employed computational methods to characterize and compare the degradation efficiencies of these enzymes for poultry feed hydrolysis. Experimental in vitro bioconversion assessments were performed to validate the computational outcomes. Molecular docking studies revealed the superior hydrolysis performance of PersiAmy3, which displayed stronger electrostatic interactions with CNCs. Experimental characterization demonstrated the improved performance of both α-amylases after immobilization at high temperatures (80 °C). A similar trend was observed under alkaline conditions, with α-amylase activity reaching 88% within a pH range of 8.0 to 9.0. Both immobilized α-amylases exhibited halotolerance at NaCl concentrations up to 3 M and retained over 50% of their initial activity after 13 use cycles. Notably, PersiAmy3 displayed more remarkable improvements than PersiAmy2 following immobilization, including a significant increase in activity from 65 to 80.73% at 80 °C, an increase in activity to 156.48% at a high salinity of 3 M NaCl, and a longer half-life, indicating greater thermal stability within the range of 60 to 80 °C. These findings were substantiated by the in vitro hydrolysis of poultry feed, where PersiAmy3 generated 53.53 g/L reducing sugars. This comprehensive comparison underscores the utility of computational methods as a faster and more efficient approach for selecting optimal enzymes for poultry feed hydrolysis, thereby providing valuable insights into enhancing feed digestibility and quality.

Biomimetic Metal-Pyrimidine Nanoflowers: Enzyme Immobilization Platforms with Boosted Activity.

For the enzyme immobilization platform, enhancing enzyme activity retention while improving enzyme stability remains a challenge for sensitive sensing analysis. Herein, an in situ biomimetic immobilized enzyme carrier (metal-pyrimidine nanoflowers, MPNFs) synthesized by the coordination of DNA base derivative (2-aminopyrimidine) with Zn2+ in the aqueous phase at room temperature is developed. The biocompatibility of 2-aminopyrimidine and the hydrophilicity and green synthetic conditions of MPNFs allows the immobilized enzymes to retain above 91.2% catalytic activity. On this basis, a cascade catalytic platform is constructed by simultaneously immobilizing acetylcholinesterase (AChE), choline oxidase (CHO), and horseradish peroxidase (HRP) in MPNFs (AChE/CHO/HRP@MPNFs) for organophosphorus pesticides (OPs) colorimetric biosensing detection. The assay could specifically detect parathion-methyl within 13min with a wider linear range (0.1-1000.0nm) and a lower limit of detection (LOD) (0.032nm). The remarkable stability of the immobilized enzymes is also achieved under harsh environments, room temperature storage, and recycling. Furthermore, a portable and cost-effective biosensing platform is developed by integrating AChE/CHO/HRP@MPNFs with a smartphone-assisted paper device for the on-site detection of OPs. Overall, the high catalytic activity retention and the enhanced detection performance demonstrate that MPNF is a robust carrier in enzyme immobilization and holds great promise in biosensing and other field applications.

Aniline Formaldehyde Cross-Linked Polyaniline Magnetic Nanocomposite with Core-Shell Structure As A New Platform for Enzyme Immobilization

Objectives: The industrial application of an enzyme is governed by its thermodynamic properties and stability, which can be effectively improved by immobilizing enzymes onto suitable supports. The present work is done by immobilizing a-amylase onto aniline formaldehyde condensate cross-linked polyaniline magnetic nanocomposite. Methods: Physiochemical characterization of synthesized Fe3O4-CLPANI nanocomposite was done by FT-IR, XRD, TG, and SEM images. Biochemical characterization related to immobilization efficiency, the kinetic parameters Vmax and the Km value, thermal stability, storage stability and reusability of immobilized enzymes were determined. Findings: Both physiochemical and biochemical characterization led to the conclusion that Fe3O4–CLPANI immobilized amylase indicated better thermal stability than that of free enzyme and it tailored to the requirement as an industrial catalyst. Novelty: To the best of our knowledge, no literature is available on the immobilization of the enzyme to Fe3O4–CLPANI composites with core-shell structure. Keywords: Nanocomposite; Amylase; Immobilization; Thermal stability; Thermal deactivation a

Enzyme@bismuth-ellagic acid: a versatile platform for enzyme immobilization with enhanced acid-base stability

Enzyme@bismuth-ellagic acid: a versatile platform for enzyme immobilization with enhanced acid-base stability

Enhanced Activity of Enzyme Immobilized on Hydrophobic ZIF-8 Modified by Ni2+ Ions.

The development of efficient enzyme immobilization to promote their recyclability and activity is highly desirable. Zeolitic imidazolate framework-8 (ZIF-8) has been proved to be an effective platform for enzyme immobilization due to its easy preparation and biocompatibility. However, the intrinsic hydrophobic characteristic hinders its further development in this filed. Herein, a facile synthesis approach was developed to immobilize pepsin (PEP) on the ZIF-8 carrier by using Ni2+ ions as anchor (ZIF-8@PEP-Ni). By contrast, the direct coating of PEP on the surface of ZIF-8 (ZIF-8@PEP) generated significant conformational changes. Electrochemical oxygen evolution reaction (OER) was employed to study the catalytic activity of immobilized PEP. The ZIF-8@PEP-Ni composite attains remarkable OER performance with an ultralow overpotential of only 127 mV at 10 mA cm-2 , which is much lower than the 690 and 919 mV overpotential values of ZIF-8@PEP and PEP, respectively.

Enhanced Activity of Enzyme Immobilized on Hydrophobic ZIF‐8 Modified by Ni2+ Ions

AbstractThe development of efficient enzyme immobilization to promote their recyclability and activity is highly desirable. Zeolitic imidazolate framework‐8 (ZIF‐8) has been proved to be an effective platform for enzyme immobilization due to its easy preparation and biocompatibility. However, the intrinsic hydrophobic characteristic hinders its further development in this filed. Herein, a facile synthesis approach was developed to immobilize pepsin (PEP) on the ZIF‐8 carrier by using Ni2+ ions as anchor (ZIF‐8@PEP‐Ni). By contrast, the direct coating of PEP on the surface of ZIF‐8 (ZIF‐8@PEP) generated significant conformational changes. Electrochemical oxygen evolution reaction (OER) was employed to study the catalytic activity of immobilized PEP. The ZIF‐8@PEP‐Ni composite attains remarkable OER performance with an ultralow overpotential of only 127 mV at 10 mA cm−2, which is much lower than the 690 and 919 mV overpotential values of ZIF‐8@PEP and PEP, respectively.

Expanding the "Library" of Metal-Organic Frameworks for Enzyme Biomineralization.

Metal-organic frameworks (MOFs) are advanced platforms for enzyme immobilization. Enzymes can be entrapped via either diffusion (into pre-formed MOFs) or co-crystallization. Enzyme co-crystallization with specific metals/ligands in the aqueous phase, also known as biomineralization, minimizes the enzyme loss compared to organic phase co-crystallization, removes the size limitation on enzymes and substrates, and can potentially broaden the application of enzyme@MOF composites. However, not all enzymes are stable/functional in the presence of excess metal ions and/or ligands currently available for co-crystallization. Furthermore, most current biomineralization-based MOFs have limited (acid) pH stability, making it necessary to explore other metal-ligand combinations that can also immobilize enzymes. Here, we report our discovery on the combination of five metal ions and two ligands that can form biocomposites with two model enzymes differing in size and hydrophobicity in the aqueous phase under ambient conditions. Surprisingly, most of the formed composites are single- or multiphase crystals, even though the reaction phase is aqueous, with the rest as amorphous powders. All 20 enzyme@MOF composites showed good to excellent reusability and were stable under weakly acidic pH values. The stability under weakly basic conditions depended upon the selection of enzyme and metal-ligand combinations, yet for both enzymes, 3-4 MOFs offered decent stability under basic conditions. This work initiates the expansion of the current "library" of metal-ligand selection for encapsulating/biomineralizing large enzymes/enzyme clusters, leading to customized encapsulation of enzymes according to enzyme stability, functionality, and optimal pH.

Engineering of Covalent Organic Framework‐Based Advanced Platforms for Enzyme Immobilization: Strategies, Research Progress, and Prospects

AbstractDriven by green and sustainable methodologies for chemical manufacturing, enzymes are forecast to have much to prospect in the coming years due to their high activity and selectivity. However, the fragile active spatial‐conformation of an enzyme is predominantly maintained by weak intermolecular interactions, showing quite sensitive to thermal, pH, and chemical chemoreception. Thus, the low activity and recyclability result from structural instability can hardly meet the prototype of green chemistry in commercial process. Covalent organic frameworks (COFs) are stable extended porous network materials assembled by organic linkers in a moderate way. Due to their pre‐designable structures, an enzyme possesses the access to be immobilized to the interior or surface of COF skeletons via covalent or noncovalent processes, which can afford protection to enzymes in harsh conditions. Moreover, with the improved stability and cyclicity, the formed COF/enzyme biocomposites can facilitate broader horizon. Herein, this review mainly canvasses the advances in the emerging field of COF/enzyme biocomposites, sketching the factors that are critical in building COF/enzyme systems and the applications of registered enzyme host platforms reported in recent years.

Green and Scalable Fabrication of High-Performance Biocatalysts Using Covalent Organic Frameworks as Enzyme Carriers.

Enzyme immobilization is essential to the commercial viability of various critical large-scale biocatalytic processes. However, challenges remain for the immobilization systems, such as difficulties in loading large enzymes, enzyme leaching, and limitations for large-scale fabrication. Herein, we describe a green and scalable strategy to prepare high-performance biocatalysts through in situ assembly of enzymes with covalent organic frameworks (COFs) under ambient conditions (aqueous solution and room temperature). The obtained biocatalysts have exceptional reusability and stability and serve as efficient biocatalysts for important industrial reactions that cannot be efficiently catalyzed by free enzymes or traditional enzyme immobilization systems. Notably, this versatile enzyme immobilization platform is applicable to various COFs and enzymes. The reactions in an aqueous solution occurred within a short timeframe (ca. 10-30 min) and could be scaled up readily (ca. 2.3 g per reaction).

Green and Scalable Fabrication of High‐Performance Biocatalysts Using Covalent Organic Frameworks as Enzyme Carriers

AbstractEnzyme immobilization is essential to the commercial viability of various critical large‐scale biocatalytic processes. However, challenges remain for the immobilization systems, such as difficulties in loading large enzymes, enzyme leaching, and limitations for large‐scale fabrication. Herein, we describe a green and scalable strategy to prepare high‐performance biocatalysts through in situ assembly of enzymes with covalent organic frameworks (COFs) under ambient conditions (aqueous solution and room temperature). The obtained biocatalysts have exceptional reusability and stability and serve as efficient biocatalysts for important industrial reactions that cannot be efficiently catalyzed by free enzymes or traditional enzyme immobilization systems. Notably, this versatile enzyme immobilization platform is applicable to various COFs and enzymes. The reactions in an aqueous solution occurred within a short timeframe (ca. 10–30 min) and could be scaled up readily (ca. 2.3 g per reaction).

Mn-doped bimetallic synergistic catalysis boosts for enzymatic phosphorylation of N-Acetylglucosamine/ N-Acetylgalactosamine and their derivatives

Metal-organic frameworks (MOFs), as advanced enzyme immobilization platforms for improving biocatalysis and protein biophysics, are rarely investigated as solid supports in the enzymatic synthesis of carbohydrate and derivatives, which can be attributed to the complex biochemical reaction mechanisms and the adverse interactions between the high polarity of substrate sugars, glycoenzymes and traditional MOFs. Here, we introduced divalent metal ion Mn2+ into MOF to prepare bimetallic MOF microreactor that encapsulated N-acetylhexosamine 1-Kinase (NahK), a critical anomeric kinase involved in the enzymatic synthesis of sugar nucleotide. The introduced Mn ions not only adjusted the microstructure of MOFs, but also participated in the enzymatic catalysis as cofactor, thus facilitated the N-acetylglucosamine/ N-acetylgalactosamine (GlcNAc/GalNAc) phosphorylation. The Mn-doped NahK@Zn-metal organic material (MOM), integrated with high catalytic activity, high stability, and high recoverability, solved the issues of immobilization related to glucokinase activity. These features significantly improved the operability and reduced the processing cost, assuring industrial application prospects for sugar nucleotides synthesis.