Enzyme Loading Efficiency Research Articles

Synthesis of Alginate Nanogels with Polyvalent 3D Transition Metal Cations: Applications in Urease Immobilization.

Biocompatible nanogels are highly in demand and have the potential to be used in various applications, e.g., for the encapsulation of sensitive biomacromolecules. In the present study, we have developed water-in-oil microemulsions of sodium alginate sol/hexane/Span 20 as a template for controlled synthesis of alginate nanogels, cross-linked with 3d transition metal cations (Mn2+, Fe3+, and Co2+). The results suggest that the stable template of 110 nm dimensions can be obtained by microemulsion technique using Span 20 at concentrations of 10mM and above, showing a zeta potential of −57.3 mV. A comparison of the effects of the cross-links on the morphology, surface charge, protein (urease enzyme) encapsulation properties, and stability of the resulting nanogels were studied. Alginate nanogels, cross-linked with Mn2+, Fe3+, or Co2+ did not show any gradation in the hydrodynamic diameter. The shape of alginate nanogels, cross-linked with Mn2+ or Co2+, were spherical; whereas, nanogels cross-linked with Fe3+ (Fe–alginate) were non-spherical and rice-shaped. The zeta potential, enzyme loading efficiency, and enzyme activity of Fe–alginate was the highest among all the nanogels studied. It was found that the morphology of particles influenced the percent immobilization, loading capacity, and loading efficiency of encapsulated enzymes. These particles are promising candidates for biosensing and efficient drug delivery due to their relatively high loading capacity, biocompatibility, easy fabrication, and easy handling.

Layer-by-Layer Engineered All-Liquid Microfluidic Chips for Enzyme Immobilization.

Enzyme immobilization in the confines of microfluidic chips, that promote enzyme activity and stability, has become a powerful strategy to enhance biocatalysis and biomass conversion. Here, based on a newly developed all-liquid microfluidic chip, fabricated by the interfacial assembly of nanoparticle surfactants (NPSs) in a biphasic system, a layer-by-layer assembly strategy to generate polysaccharide multilayers on the surface of a microchannel, greatly enhancing the mechanical properties of the microchannel and offering a biocompatible microenvironment for enzyme immobilization, is presented. Using horseradish peroxidase and glucose oxidase as model enzymes, all-liquid microfluidic enzymatic and cascade reactors have been constructed and the crucial role of polysaccharide multilayers on enhancing the enzyme loading and catalytic efficiency is demonstrated.

Synergistic role of carbon quantum dots in the activity and stability of Candida rugosa lipase encapsulated within metal–organic frameworks (ZIF-8)

Carbon quantum dots (CQDs) are particularly attractive in biosensing, bioimaging and drug delivery applications due to their luminescent properties, water dispersibility and favorable biocompatibility. In the study, by combining CQDs derived from edible mushroom Agaricus bisporus and zeolitic imidazolate frameworks (ZIFs) crystals, a new support material was prepared for encapsulation of Candida rugosa lipase. The study aimed to shed light on the activity, thermal and storage stability, and reusability of physically adsorbed or in-situ encapsulated enzyme. The enzyme loading efficiency of the in-situ encapsulation method was 3.11-fold higher (371 mg g−1). Also, catalytic activities were 119.8 U/g (CQDs@ZIF- 8@CRLenc) and 34.2 U/g (CQDs@ZIF-8@CRLads). The CQDs@ZIF-8@CRLenc with the biocatalytic activity was 3.5-folds higher than that of the CQDs@ZIF-8@CRLads. In the in-situ encapsulation, the CQDs and the enzyme were physically entrapped within three-dimensional structures of ZIF-8 that were constructed from tetrahedral zinc ions bridged by imidazolate. The structural, morphological, and spectrophotometric analyses were performed using TEM, SEM-EDX, FT-IR, XRD, UV–vis, fluorescence, and confocal microscopy. The effects of pH (4.0–9.0) and temperature (30–60 °C) on the enzymatic activity were studied and optimized. The coexistence of the CQDs and ZIF-8 crystals remarkably enhanced the activity, reusability, thermal resistance, and storage stability of the enzyme encapsulated within ZIF-8 crystals (the relative enzyme activity; after 6 re-uses: 73%, 2-hour-incubation at 60 °C: 70%, 5-week-storage at 4 °C: 70%). The CQDs from A. bisporus entrapped within ZIF-8 can synergistically enhance the catalytic performance and thermal/storage stability of immobilized C. rugosa lipase.

Modified magnetite nanoparticle as biocatalytic support for magnetically stabilized fluidized bed reactors

Magnetically stabilized fluidized bed reactor (MSFBR) is a sustainable and cost-effective biotechnological process, which justifies the increasing search for biocatalytic superparamagnetic supports to enzyme immobilization. This work, investigates the effect of modified nanomagnetite, Fe3O4 (nM), for biocatalytic support on properties associated with catalytic behavior and potential use in MSFBR. The nM was either synthesized and then modified with 3-aminopropyltriethoxysilano (nM-APTES) or stabilized with oleic acid (nM-OA) and later modified with chitosan (nM-OA-Cs). A novel correlation study was carried out about the support dimension effect, enzymatic surface loading and retained activity. Both nM-based supports were morphologically, structurally and magnetically characterized by X-ray diffraction, thermogravimetry, Mössbauer and Fourier-transform Infrared spectroscopies, as well as vibration sample magnetometry, scanning (SEM) and transmission (TEM) electron microscopies. The nM-APTES and nM-OA samples disclosed crystal sizes of 8.07 and 8.69 nm, respectively. From TEM imagens, the average particles sizes were 10.9 nm for nM-APTES and 12.6 nm for nM-OA. The nM-OA-Cs displayed an average particles size of 918.6 μm. These magnetic supports displayed high saturated magnetization, 79.6 emu/g for nM-APTES and 72.7 emu/g for nM-OA. The amano lipase AK enzyme was immobilized on supports activated with glutaraldehyde. The enzyme loading density of nM-APTES-GA-Lip (122 mg/g) with 61% immobilization yield was higher than that of nM-OA-Cs-GA-Lip (46 mg/g) with 23% immobilization yield. The catalytic activity of nM-APTES-GA-Lip (85%) was higher than that of nM-OA-Cs-GA-Lip (46%), which reflected the correlation between enzyme loading efficiency on the magnetic nanosupports and their relative activity, essential to potential use in MSFBR.

Synthesis of chitosan-lysozyme microspheres, physicochemical characterization, enzymatic and antimicrobial activity

Chitosan microspheres (CMS) by the emulsion-chemical cross-linking method with and without lysozyme immobilization were synthesized and characterized. The technique conditions were adjusted, and spherical particles with approximate diameters of 3.74 ± 1.08 μm and 0. 29 ± 0.029 μm to CMS and chitosan-lysozyme microspheres (C-LMS), respectively, were obtained. The microspheres were characterized by scanning electron microscopy (FESEM), Spectroscopy Fourier Transform Spectroscopy (ATR-FTIR), X-ray diffraction (XRD), and zeta potential. Particle size was identified by laser light scattering (DLS) and the thermal properties by Differential Scanning Calorimetry (DSC) and Thermogravimetry (TGA) were determined. By the lysis of Micrococcus lysodeikticus, the activity of the microspheres was determined, and the results correlated with the amount of lysozyme used in the immobilization process and the enzyme loading efficiency was 67%. Finally, release tests pointed out the amount of enzyme immobilized on the microsphere surface. These results showed that chitosan microspheres could be used as material for lysozyme immobilization by cross-linking technique. The antimicrobial activity was tested by inhibition percent determination, and it evidenced both chitosan microspheres (CMS) and chitosan-lysozyme microspheres (C-LMS) positive antimicrobial activity to Staphylococcus aureus, Enterococcus faecalis and Pseudomonas aeruginosa.

Transformable Spinose Nanodrums with Self-Supplied H2 O2 for Photothermal and Cascade Catalytic Therapy of Tumor.

Advances in enzymes involve an efficient biocatalytic process, which has demonstrated great potential in biomedical applications. However, designing a functional carrier for enzymes equipped with satisfactory degradability and loading efficiency, remains a challenge. Here, based on transformable liquid metal (LM), a spinose nanodrum is designed as protein carrier to deliver enzyme for tumor treatment. With the assistance of spines and a special drum-like shape, it is found that the spiny LM can carry much more enzymes than spherical LM under the same condition. Benefiting from the satisfactory enzyme loading efficiency of spiny LM, a plasma amine oxidase immobilized spinose LM nanosystem enveloped with epigallocatechin gallate (EGCG)-Fe3+ (LMPE) is fabricated for photothermal and cascade catalytic tumor therapy. Activated by the acidic condition in the tumor microenvironment, the LMPE can oxidize spermine (Spm) and spermidine (Spd) to generate hydrogen peroxide (H2 O2 ) for Fenton catalytic reaction to produce the lethal hydroxyl radical (•OH) for tumor cell killing. Combined with remarkable photothermal performance of LM, LMPE exhibits significant inhibition of tumor in vivo.

Amine and aldehyde functionalized mesoporous silica on magnetic nanoparticles for enhanced lipase immobilization, biodiesel production, and facile separation

In this research, synthesis of fatty acid methyl ester (FAME) from olive oil as model feedstock was carried out in the presence of Rhizopus oryzae lipase (ROL) that was immobilized on the mesoporous silica magnetic nanoparticles (MNPs). MNPs were coated with mesoporous silica (MS) functionalized by amine and aldehyde groups used for immobilization of a lipase, used for biodiesel production, and easy magnetic separation of the immobilized lipase from the biodiesel product. MNPs were synthesized by using the co-precipitation method and coated with MS by sol-gel synthesis using cetyltrimethylammonium bromide (CTAB) as a template for mesoporous formation. ROL was immobilized on these supports via physical adsorption and covalent attachment. Enzyme loading efficiency on mesoporous was doubled compared to that on bare MNPs. Also, the covalent bonding led to an increase in enzyme loading from 67.8 to 82.4%. The physico-chemical properties of MNPs were determined by XRD, EDX, VSM, FESEM, BET, and FTIR and were correlated with their activities for biodiesel synthesis. The evaluations of hydrolytic activity and kinetic parameters revealed that MNPs@MS-AP-GA is a suitable nanomaterial for ROL immobilization. Transesterification of olive oil using ROL-MNPs@MS-AP-GA showed the highest biodiesel production of 84.6% among other nanobiocatalysts.

Synthesis and characterization of magnetic cross-linked enzyme aggregate and its evaluation of the alternating magnetic field (AMF) effects in the catalytic activity

A magnetic cross-linked enzyme aggregate (MCLEA) of cellulases and a non-magnetic analogue (CLEA) were synthesized and further investigated. The obtained MCLEA and CLEA showed a high enzyme loading efficiency of 90% and 88%, respectively. However, the MCLEA presented an activity 33% higher than the CLEA one, suggesting a catalytic enhancement effect as a result of magnetic nanoparticle presence in cross-linked cellulase aggregates structure. In addition, MCLEA shows an unusual behavior with highest enzymatic activity at low temperatures near to 37 °C. Lastly, a full factorial design (2x2) with two variables at two levels, frequency (203–420 kHz) and amplitude (3–6 kA m−1), was used in order to better investigate the effects of an alternating magnetic field (AMF) on MCLEA activity. AMF application decreased MCLEA activity, particularly in the highest values of frequency and amplitude, when compared to condition field free. In addition, frequency implied in more significant effects on MCLEA activity when compared to AMF amplitude.

Enzyme immobilization on porous chitosan hydrogel capsules formed by anionic surfactant gelation.

Sodium dodecyl sulfate (SDS)-chitosan hydrogels have been employed for adsorption of anionic dyes and metallic substances. Two mutant forms of Thermoanaerobacter ethanolicus alcohol dehydrogenase (TeSADH) were used as model enzymes to develop a novel enzyme immobilization technique employing newly formulated porous chitosan hydrogels. The enzyme immobilized on chitosan hydrogel capsules formed by 5g/l SDS gelation and subsequent treatment with 0.05M NaOH was 28-35% higher in NADPH production than that formed by 20g/l SDS gelation only under the same conditions. A 48-h asymmetric biphasic reduction of acetophenone with immobilized TeSADH enzyme at 50 °C showed 68% increase in (R)-1-phenylethanol production than the free enzyme. Compared to the free enzyme which denatured and lost its activity at 80 °C, the immobilized enzyme retained about 25% of its initial activity after 2-h incubation. In contrast to the conventional chitosan hydrogel which suffers thermal and operational stability, the newly formulated porous chitosan hydrogel capsules have excellent enzyme loading efficiency and stable at harsh temperatures. Especially, this newly developed enzyme immobilization method would be applicable for food processing.

The Carboxylated Multi-walled Carbon Nanotubes/l-Asparaginase Doped Calcium-Alginate Beads: Structural and Biocatalytic Characterization

The calcium-alginate/multi-walled carbon nanotube hybrid beads (Ca-ALG/MWCNT-COOH) as a novel kind of matrix were fabricated and characterized in detailed. l-Asparaginase (l-ASNase), which is important chemotherapeutic enzyme-drug in leukemia, was immobilized on the Ca-ALG/MWCNT-COOH hybrid beads. To the best of our knowledge, this is the first study using Ca-ALG/MWCNT-COOH hybrid beads for l-ASNase immobilization. Our characterization investigations displayed that the hybridization between ALG and MWCNT-COOH caused significant changes on the surface morphology and structure. ALG of 0.5% (w/v), CaCl2 of 0.2 M concentration, enzyme of 187.5 U and bead size of 2 mm was found to be best with respect to enzyme loading efficiency. The enzyme was loaded a high yield (97.0%) on these hybrid beads. Remarkably, the tolerance of immobilized enzyme developed towards temperature and pH changes. The maximum activity for the free enzyme was observed at 35 °C, pH 7.5, whereas the immobilized enzyme showed maximum activity at 45 °C pH 8.5. After immobilization, storage stability of enzyme improved and retained more than 70% of its initial activity after 4 weeks at ~ 30 °C as compared with free enzyme which showed only 20% of residual activity. After immobilization, Km value decreased 1.27-fold compared to free counterpart, indicating increased the affinity between the substrate and enzyme. Moreover, immobilized enzyme maintained more than 36% of its original activity even after consecutive 14 reuse. As result, it is worthy of noting that this kind of hybrid materials may become a promising support material for the immobilization of commercial enzymes in areas such as industrial and medical.

Stabilization of phytase on multi-walled carbon nanotubes via covalent immobilization.

This chapter provides one of the covalent methods to immobilize phytase, the most important feed enzyme, on Multi-walled carbon nanotube in order to improve enzyme characteristics. Various techniques such as scanning electron microscopy, FTIR and CD spectroscopy, along with zeta potential measurement were performed to confirm immobilization. Catalytic activity measurement of phytase was explained in details and enzyme loading efficiency was measured. The results indicated a 38% decrease in enzyme activity after immobilization which could because of structural changes or wrong enzyme orientation after immobilization. Also, optimum temperature and pH of free and immobilized enzyme were performed. The remaining activity of free and immobilized enzymes was also investigated after treating with higher temperatures using in the pelleting process. Immobilized phytase showed 10-fold resistance against 90°C temperature in comparison with free phytase. Finally, the effect of NaCl on the catalytic activity of free and immobilized phytase was examined and showed interesting results. We found that the catalytic activity of immobilized could increase by about 80% in the presence of 0.2M NaCl.

Chemical characterization of Levan and optimization of immobilized Bacillus tequilensis levansucrase onto 𝜅-Carrageenan –CMC Gel Beads.

B acillus tequilens was a good levansucrase producer (222.2 U/mL) with levan yield (130 g/L). The levan yield was characterized by FT-IR and the results recorded that the product was mainly fructose. Levansucrase produced by Bacillus tequilens was immobilized by covalent binding on κ-carrageenan and carboxy methyl cellulose gel beads activated by two-step method; the gel beads were soaked in polyethyleneimine followed by glutaraldehyde. Then22 full-factorial central composite experiment designs were employed to optimize the conditions for the maximum enzyme loading efficiency to reach (14.01852 U/of enzyme/g gel beads).The free enzyme showed optimum pH at7 while immobilization process increased the tolerance of enzyme at both alkaline range pH3 and acidic range pH10. The apparent Km after immobilization was 2.85 mg/mL compared to 2.5 mg/mL for free enzyme. Maximum velocity Vmax was 71.4 mg.min−1 for free enzyme while it was 62.4 mg.min−1 for immobilized formula of enzyme. An inhibition of enzyme activity was recognized with all tested metal ion as well as EDETA for either free or immobilized formula of levansucrase.

Facile Synthesis of S‐Substituted L‐Cysteines with Nano‐sized Immobilized O‐Acetylserine Sulfhydrylase

AbstractMany S‐substituted l‐cysteines are useful pharmaceutical intermediates but require a simple synthesis method. Here we developed enzymatic synthesis of several S‐aryl‐l‐cysteines and S‐benzyl‐l‐cysteine directly from O‐acetylserine (OAS) with immobilized O‐acetylserine sulfhydrylase CysM. Novel iron‐oxide magnetic nanoparticles (MNPs) with cobalt‐nitrilotriacetic acid function were prepared with a diameter of 80 nm in 75 % yield. Direct immobilization of His‐tagged CysM in the cell‐free extract on the MNPs via affinity attachment afforded stable nanobiocatalyst with 97 % enzyme loading efficiency and 93 % free enzyme activity. The immobilized enzyme catalyzed the biotransformation of benzylthiol and OAS to give S‐benzyl‐l‐cysteine in 88 % yield. The nanobiocatalyst also demonstrated high recyclability, retaining 95 % productivity in the fifth cycle. The immobilized CysM accepts various arylthiols to react with OAS, giving rise to a new synthesis route of several S‐substituted l‐cysteines in 60–96 % yield.

Covalent immobilization of phytase on the multi-walled carbon nanotubes via diimide-activated amidation: structural and stability study

Phytase is the most widely used feed enzyme in the world. This enzyme inactivates under pelleting conditions such as temperatures above 80 °C. The present study reported covalent attachment of phytase onto functionalized multi-walled carbon nanotubes (F-MWNT). The optimum enzyme loading and immobilization efficiency were 110 µg/mg of functionalized multi-walled carbon nanotubes and 62%, respectively. Characterization, structural changes and immobilization of phytase were studied by scanning electron microscopy (SEM), circular dichroism (CD), zeta potential measurement and Fourier transform infrared (FTIR) spectroscopy. Immobilized phytase exhibited improved stability towards temperature than the free phytase. The free phytase retain 3% and 27% of relative activity at 90 and 80 °C, respectively after 2 min of incubations. While immobilized phytase retained about 33% and 51% at the same condition. Also the results showed that the presence of small quantities of NaCl (<1 M) in the reaction media increased enzyme activity of immobilized phytase up to 78% but free phytase activity was not significantly changed until 1 M NaCl which increased by 30%. Higher concentrations of 1 M NaCl drastically reduced both free and immobilized phytase activities.

Folate-Conjugated Superoxide Dismutase Adsorbed Over Antioxidant Mimicking Nanomatrix Frameworks for Treatment of Rheumatoid Arthritis

Rheumatoid arthritis (RA) is an autoimmune disease occurring in larger population, characterized by synovial inflammation followed by destruction of joint. Major concerned factor for cause of RA has been related to oxidative stress due to environmental toxicity and immune imbalance. Reactive oxygen species generated from macrophages communes series of oxidation at cellular and genetic level and leads to generation of inflammatory cytokines for provoking inflammation in RA. Superoxide dismutase and catalase are supposed to be potential antioxidant enzymes to scavange free radicals. However, many clinical studies report reduced potency of enzyme due to lack of its targeting efficacy. Therefore, the aim of the work involves development of nanomatrix mimicking as catalase over which folate-linked superoxide dismutase was adsorbed for a macrophage targeting. The developed formulation was optimized, characterized for morphological studies, enzyme loading efficiency, protein activity, and in vitro drug release. Quantification of interleukin 6 was performed by indirect enzyme linked immunosborent assay over macrophage cell lines to determine in vitro treatment efficacy. It is concluded that the prepared system can act as enzyme reservoir to deliver acid labile enzymes in controlled form to efficiently treat RA.

Nitrogen-doped hollow carbon nanospheres for high-energy-density biofuel cells and self-powered sensing of microRNA-21 and microRNA-141

High-energy-density enzymatic biofuel cells (EBFCs) are urgently needed not only as green energy conversion devices but also as energy generators for medical devices. Low enzyme loading efficiency and insufficient direct electron transfer (DET) between enzymes and electrodes greatly hamper the development of EBFCs. Herein, we fabricated nitrogen-doped hollow carbon nanospheres with large pores (pNHCSs) via a green microwave-assisted hydrothermal method (MWHM). Due to their three-dimensional structure and the incorporation of nitrogen, the pNHCS-functionalized electrode not only efficiently enriched the load of enzyme (ca. 8.65 × 10−10molcm−2) but also significantly facilitated DET rate (ca. 10.73s−1). Thus, the pNHCS-based EBFCs exhibited an outstanding maximum power output (Pmax), which achieved to 325 ± 0.6µWcm−2. Then, based on a dual-fuel-driven EBFC, we developed an innovative self-powered biosensor to sense two kinds of cancer-related microRNAs, miR-21 and miR-141, at the same time. The detection limits were as low as 0.1fM for miR-21 and 4.0fM for miR-141 (S/N = 3). This material has excellent potential in the development of superior green bio-batteries as well as economical and portable self-powered biomedical sensors.

An electrospun chitosan-based nanofibrous membrane reactor immobilized by the non-glycosylated rPGA for hydrolysis of pectin-rich biomass

Abstract Most native polygalacturonases are glycosylated. Owing to the influence of attached glycans on the function of proteins, an immobilized non-glycosylated polygalacturonase is fabricated for the degradation of pectin-rich biomass pectin into fermentable sugars. Recombinant non-glycosylated and glycosylated PGAs expressed by E. coli and P. pastoris, respectively, were prepared. As application model, recombinant polygalacturonases in immobilized enzyme membrane reactors were tested. The electrospun chitosan-based nanofibrous membrane was selected as the support matrix. The maximum enzyme loading efficiency and enzyme binding efficiency of non-glycosylated polygalacturonase and glycosylated polygalacturonase were 89.12 mg/g and 84.13% and 34.16 mg/g and 87.18%, respectively, suggesting that the glycosylation of polygalacturonases had a serious effect on its immobilization. The membranes were characterized by scanning electron microscope, Fourier transform infrared spectroscopy and X-ray diffraction. Furthermore, the glycosylation can significantly enhance the stability of recombinant polygalacturonases. Meanwhile, the glycosylation influenced their specific activity and degradation pattern. Most interesting of all, some d -galacturonic acid monomers occurred in the hydrolysate after the degradation of pectin by the non-glycosylated polygalacturonase, and the yield of reducing sugar could be 31.98 mg after 60min, with a conversion rate of 63.96%. Thus, the constructed reactor has potential in using pectin-rich biomass as the feedstock.

Facile fabrication of 3D porous hybrid sphere by co-immobilization of multi-enzyme directly from cell lysates as an efficient and recyclable biocatalyst for asymmetric reduction with coenzyme regeneration in situ

Ni2+-agarose bead-wrapped multi-enzyme/inorganic hybrid sphere composed of the immobilized enzymes as organic component and NaH2PO4 and NaCl as inorganic component was developed by co-immobilizing extracellular His-tagged 3-quinuclidinone reductases and glucose dehydrogenase without pre-purification. The resulting biocatalysts has 3D porous architectures as confirmed by SEM and FESEM, and it enabled the continuous biotransformation of 3-quinuclidone to (R)-3-quinuclidinol with cofactor regeneration in situ. The 3D porous biocatalysts were formed via three steps: First, immobilization of the His-tagged enzymes directly from the cell lysates supernatant. Next, formation of enzyme aggregates, ribbons and gels. Finally, the enzymes, the formed aggregates/ribbons/gels and salt were incorporated to the foam and then covered the Ni2+-agarose bead. The technique made the immobilization of these enzymes effective such that specific enzyme loading of 60.8mg/g support and enzyme loading efficiency of 92.3% were achieved. As a direct consequence, the biocatalyst catalyzed the conversion of 3-quinuclidinone (204g/L) to (R)-3-quinuclidinol in 100% yield and 100% ee at 4.5h, and the recyclability of the biocatalyst was excellent, retaining>95% conversion yield and 100% ee even after the fifteenth runs. Overall, our strategy is demonstrated to be a promising method for developing efficient and robust biocatalyst for asymmetric synthesis.

Enzyme Immobilized on Nanoporous Carbon Derived from Metal-Organic Framework: A New Support for Biodiesel Synthesis.

In this study, nanoporous carbon (NPC) derived from metal-organic framework was used as support for the immobilization of Burkholderia cepacia lipase. The decorated aluminum oxide within the mesoporous NPC improved the enzyme loading efficiency as well as the catalytic ability for the transesterification of soybean oil, thus making it a promising green and sustainable catalytic system for industrial application.

Coupled Immobilized Amine Dehydrogenase and Glucose Dehydrogenase for Asymmetric Synthesis of Amines by Reductive Amination with Cofactor Recycling

AbstractThe amine dehydrogenase (AmDH) engineered from the phenylalanine dehydrogenase of Rhodococcus sp. M4 was directly immobilized on magnetic nanoparticles (MNP) from the cell‐free extract containing his‐tagged AmDH through affinity attachment to give AmDH‐MNPs with high yield, enzyme loading efficiency, and specific enzyme loading. AmDH‐MNPs showed higher activity and productivity than the free enzyme for the asymmetric reductive amination of 4‐phenyl‐2‐butanone 1 a and phenylacetone 1 b, producing the corresponding amines (R)‐2 a,b in 99 % ee and 99 % yield, and with recycling of NADH for up to 3956 times. AmDH‐MNPs were easily recycled, retaining 91 % of the original productivity in the third cycle of the reductive amination of 1 a. Coupling of immobilized AmDH and immobilized glucose dehydrogenase (GDH) for the asymmetric reductive amination of 1 a gave (R)‐2 a in 99 % ee and 74 % yield, with a total turnover number (TTN) of 2940 for NADH recycling. Both immobilized enzymes showed good recyclability, retaining 81 % productivity in the third reaction cycle. The developed method with coupled immobilized AmDH and immobilized GDH for the asymmetric reductive amination of ketones is useful for the synthesis of enantiopure amines, superior to the use of coupled isolated enzymes with enhanced catalytic performance and reduced enzyme cost through catalyst recycling.