High Activity Recovery Research Articles

Magnetic nickel nanostructure as cellulase immobilization surface for the hydrolysis of lignocellulosic biomass

In this research, a magnetic reusable nickel nanoparticle (NiNPs) supporting materials were prepared for cellulase enzyme immobilization. The immobilized cellulase showed high activity recovery, large & fast immobilization capacity and improved pH & temperature tolerance. The excellent stability and reusability enabled the immobilized cellulase to retain 84% of its initial activity after ten cycles. At 2 mg/mL enzyme concentration, highest 93% immobilization efficiency was achieved within two hours of immobilization. When the treatment temperature reached 40 °C and pH 5, the immobilized cellulase exhibited highest residual activity. The immobilized cellulase could be separated from the solution by a magnetic force. This study introduced a novel supporting material for cellulase immobilization, and the immobilized cellulase poses a great potential in the hydrolysis of lignocellulosic biomass which can used as an easily applicable and sustainable pre-treatment step for advanced biofuel production.

Efficient Synthesis of (S)-1-Boc-3-aminopiperidine in a Continuous Flow System Using ω-Transaminase-Immobilized Amino-Ethylenediamine-Modified Epoxide Supports

We developed a continuous flow system using ω-transaminase covalently immobilized on an epoxy resin modified with ethylenediamine (EDA) to synthesize (S)-1-Boc-3-aminopiperidine, which is a key intermediate for several novel inhibitors. A second functional group (−NH3+) was introduced to rapidly adsorb ω-transaminase, which was further rigidified by multipoint covalent attachment to the modified resin with high binding efficiency (>95%) and activity recovery (75%). The resulting immobilized ω-transaminase was stable with 90% residual activity after 15 reuse cycles. We synthesized (S)-1-Boc-3-aminopiperidine using this continuous flow system, and 95% conversion was obtained within 10 min of residence. The system was continuously operated for 24 h and achieved a space–time yield of 930.73 g·L–1·day–1, indicating outstanding reaction efficiency. This efficient continuous bioprocess has potential for large-scale production of (S)-1-Boc-3-aminopiperidine.

Synthesis of Novel Magnetic Carbon Microtube-Based Solid Acid and Its Catalytic Activities for Biodiesel Synthesis

Novel magnetic carbon microtube-based solid acid was synthesized via the carbonization of FeCl3-doped willow catkin and benzenediazoniumsulfonate acid-based sulfonation. The biomass willow catkin provided the special microtube structure and the high surface area of 215 m2/g for the novel solid acid. The microtube structure was well conserved during the mild carbonization (400 °C) and sulfonation process. The large number of acidic sites (2.3 mmol/g) on the microtube surface was quite accessible to reactants. Its magnetic properties offered a simple separation process. The novel solid acid showed very high activity for biodiesel synthesis, using cooking oils as raw material, which gave a total yield of 99% and free fatty acid conversion of 98.7% under mild conditions (70 °C). The facile synthetic process, high activity, high stability, and high recovery simplicity were the main properties of the novel magnetic solid acid, which is one of the best choices for biodiesel synthesis.

Mannich-mediated synthesis of a recyclable magnetic kraft lignin-coated copper nanostructure as an efficient catalyst for treatment of environmental contaminants in aqueous media

Recently, the fabrication of heterogeneous catalysts using biopolymers has been considered due to some advantages such as biodegradability, easy handling, high efficiency and so on. In this work, 5-amino-1H-tetrazole modified kraft lignin/Fe3O4 supported highly catalytic active copper complex (Fe3O4@LigA/Cu) has been synthesized as a novel catalyst. The support was prepared by the Mannich reaction of 5-amino-1H-tetrazole and kraft lignin. The prepared catalyst was characterized by different techniques including Fourier transform infrared (FT-IR), X-ray diffraction (XRD), thermogravimetric analysis (TGA), field emission scanning electron microscopy (FE-SEM), Energy-dispersive X-ray spectrometry (EDX), elemental mapping, Brunauer-Emmett-Teller (BET) and vibrating sample magnetometry (VSM). The Fe3O4@LigA/Cu catalyst was used in the catalytic reduction of organic pollutants such as 4-nitrophenol (4-NP), methylene blue (MB), and congo red (CR) using sodium borohydride (NaBH4) as the reducing agent and the catalytic efficiency was studied by UV–visible spectroscopy. Fe3O4@LigA/Cu catalyst showed high yield (˃ 99%) in the reduction of 4-NP in four min. Moreover, Fe3O4@LigA/Cu catalyst showed good recyclability and reusability. This work provides a simple method for the preparation of lignin based catalysts with high catalytic activity and easy recovery to degrade organic pollutants from aqueous solution.

Fabrication of ω‐Transaminase@Metal‐Organic Framework Biocomposites for Efficiently Synthesizing Benzylamines and Pyridylmethylamines

AbstractIn this study, ten ω‐transaminases (ω‐TAs) have been investigated to efficiently catalyze the synthesis of twenty‐four functionalized benzylamines and pyridylmethylamines. We optimized the reactions, screened suitable amino donors and compared ω‐transaminases activities for all aromatic aldehyde substrates. Under the optimized conditions, eighteen aromatic amines have been obtained with 60.4%–96.6% conversions and isolated only via simple extraction and recrystallization with 18.5%–81% yields on a preparative scale. Furthermore, we first immobilized the Bm‐STA onto the MOFs via the physical adsorption to overcome the limitation of free enzyme and improve their industrial applications. The obtained Bm‐STA/UiO‐66‐NH2 composites exhibited not only high enzymes loading (80.4 mg g−1) and enzyme activity recovery (95.8%), but also the better reusability, storage stability, pH stability and the tolerance to acetone and DMF.magnified image

Bioaccessibility, safety, and antidiabetic effect of phenolic-rich extract from fermented Psidium guajava Linn. leaves

• Phenolic-rich extract (PE-fgl) was obtained from fermented guava leaves. • The high bioaccessibility of PE-fgl was observed during gastrointestinal digestion. • PE-fgl improved blood glucose levels and antioxidant capacity in diabetic mice. • No toxicity evidence was noted by acute or subchronic toxicity assessment. Fermentation results in the release and biotransformation of phenolic compounds in guava leaves. This study aimed to evaluate the bioaccessibility, safety and antidiabetic effect of phenolic-rich extract from fermented guava leaves (PE-fgl) using Monascus anka and Bacillus sp. BS2. The total phenolics and flavonoids were 51.18% and 16.60% bioaccessible after gastrointestinal digestion, respectively. High bioaccessibility was observed, particularly for ellagic acid, isoquercitrin, quercetin-3-O-β-D-xylopyranoside, quercetin-3-O-α-L-arabinofuranoside and avicularin, resulted in high recovery of α-glucosidase inhibition activity and antioxidant capacity. Moreover, PE-fgl was practically nontoxic with a median lethal dose (LD 50 ) greater than 5000 mg/kg and was without evidence of toxicity following subchronic administration up to the dose limit of 2000 mg/kg. Furthermore, PE-fgl significantly improved serum glucose and lipid levels as well as antioxidant capacity in diabetic mice. Liver, kidney and pancreas damage was also significantly alleviated. These results suggested that PE-fgl could be a nontoxic candidate for diabetes treatment.

Entrapment of the Fastest Known Carbonic Anhydrase with Biomimetic Silica and Its Application for CO2 Sequestration.

Capturing and storing CO2 is of prime importance. The rate of CO2 sequestration is often limited by the hydration of CO2, which can be greatly accelerated by using carbonic anhydrase (CA, EC 4.2.1.1) as a catalyst. In order to improve the stability and reusability of CA, a silica-condensing peptide (R5) was fused with the fastest known CA from Sulfurihydrogenibium azorense (SazCA) to form R5-SazCA; the fusion protein successfully performed in vitro silicification. The entrapment efficiency reached 100% and the silicified form (R5-SazCA-SP) showed a high activity recovery of 91%. The residual activity of R5-SazCA-SP was two-fold higher than that of the free form when stored at 25 °C for 35 days; R5-SazCA-SP still retained 86% of its activity after 10 cycles of reuse. Comparing with an uncatalyzed reaction, the time required for the onset of CaCO3 formation was shortened by 43% and 33% with the addition of R5-SazCA and R5-SazCA-SP, respectively. R5-SazCA-SP shows great potential as a robust and efficient biocatalyst for CO2 sequestration because of its high activity, high stability, and reusability.

Partial Purification of Polyphenol Oxidase (PPO) from Mango Seed Kernel (MSK) Using Alcohol/Salt-Based Aqueous Two-Phase System (ATPS)

Background: Polyphenol oxidase (PPO) has applications in pharmaceutical, environmental, and food industries. However, conventional techniques for its purification are considered costly and laborious. Alternative methods of purification are needed. Methods: This study explored the use of an alcohol/salt-based Aqueous Two-Phase System (ATPS) to partially purify PPO from Mango Seed Kernel (MSK). Specifically, this study determined the effects of the 1-propanol/potassium phosphate buffer mass ratio on the partition parameters such as partition coefficients, activity recovery, and purification fold. Results: The enzyme partition coefficient (KE) was greater than 1, which signified that more PPO was partitioned in the top phase due to its hydrophobic nature. Specifically, the 3.22 1-propanol/ PPB mass ratio had the highest K E of 1.91 ± 0.16 and 1.46 ± 0.16 for runs 1 and 2, respectively. Moreover, the same mass ratio led to the highest activity recovery in the top phase reaching up to 73.99 ± 1.56% for run 1 and 64.27 ± 2.48% for run 2. In terms of purification fold, runs 1 and 2 had PF values of 0.55 ± 0.05 and 0.70 ± 0.01, respectively. Since the values obtained were low due to the large number of contaminants extracted together with the target enzyme in the top phase, the use of a back-extraction step recovered the PPO in the bottom phase and resulted in an activity recovery of 82.94 ± 1.05% and a purification fold of 7.01 ± 0.95. Conclusion: Results have shown that varying the mass ratio had significant effects on the partition parameters.

Improved adenylate cyclase activity via affinity immobilization onto co-modified GO with bio-inspired adhesive and PEI

Adenylate cyclase (AC) can efficiently catalyze the conversion of adenosine triphosphate (ATP) to cyclic adenosine-3′, 5′-monophosphate (cAMP). However, AC directly immobilized on substrate is not desirable due to enzyme inactivation. Herein, bio-inspired adhesive of polydopamine and polyethyleneimine (PDA/PEI) was used as flexible chains to graft on graphene oxide (GO), and the AC was directionally immobilized through affinity between metal ions and his-tags of AC. The properties of modified GO and the activity of immobilized AC were studied in detail. PDA/PEI layers have been proved to improve the amino density of GO surface for affinity groups decoration and adjust the interaction between AC and support. And modified GO by this novel method contributes to subsequent grafting and immobilization of AC by affinity. AC immobilized on modified GO exhibited high activity recovery with about 90 % of free AC, while enzyme immobilized on unmodified GO has been inactivated. This study offers a versatile approach for support modification and enzyme oriented immobilization. PDA/PEI functionalized GO can be used as a promising carrier to immobilize other his-tagged enzymes.

One-step selective affinity purification and immobilization of His-tagged enzyme by recyclable magnetic nanoparticles.

The NiFe2O4 magnetic nanoparticles (NF‐MNPs) were prepared for one‐step selective affinity purification and immobilization of His‐tagged recombinant glucose dehydrogenase (GluDH). The prepared nanoparticles were characterized by a Fourier‐transform infrared spectrophotometer and microscopy. The immobilization and purification of His‐tagged GluDH on NF‐MNPs were investigated. The optimal immobilization conditions were obtained that mixed cell lysis and carriers in a ratio of 0.13 in pH 8.0 Tris‐HCl buffer at 30℃ and incubated for 2 h. The highest activity recovery and protein bindings were 71.39% and 38.50 μg mg–1 support, respectively. The immobilized GluDH exhibited high thermostability, pH‐stability and it can retain more than 65% of the initial enzyme after 10 cycles for the conversion of glucose to gluconolactone. Comparing with a commercial Ni‐NTA resin, the NF‐MNPs displayed a higher specific affinity with His‐tagged recombinant GluDH.

Facile preparation of immobilized naringinase on polyethylenimine/dopamine‐coated hydrothermal carbon spheres with high performance

AbstractBACKGROUNDMussel‐inspired surface chemistry is recognized as a simple, efficient, and mild surface modification method, and has become a research hotspot in many fields. Hydrothermal carbon is a bio‐based material that is referred to as environmentally friendly renewable resource. In this study, the polyethylenimine (PEI)/dopamine (DA) was coated on the surface of hydrothermal carbon spheres using the co‐deposition method, making it possible to immobilize naringinase with high activity and operation stability. Subsequently, the optimal modification and immobilization conditions as well as enzyme properties were investigated.RESULTSThe naringinase activity reached up to 461.14 U g–1 carrier, which was much higher than those of the previous works. Besides, the residual naringinase activity still kept 76.44% of the initial enzyme activity after one month of storage, and maintained 62.01% of the initial enzyme activity after eight reuse cycles.CONCLUSIONTherefore, the strategy of the immobilized enzyme on PEI/DA coated hydrothermal carbon could be recognized as a promising and universal enzyme immobilization method with the advantages of high relative enzyme activity, high enzyme loading rate, high enzyme activity recovery rate, good operational stability, and facile and environmental friendly preparation route. © 2021 Society of Chemical Industry (SCI).

Fabrication of hollow covalent-organic framework microspheres via emulsion-interfacial strategy to enhance laccase immobilization for tetracycline degradation

• Hollow microspheres H-COF-OMe were prepared by emulsion interfacial polymerization. • Interfacial-defects in H-COF-OMe were formed by confined growth on emulsion droplets. • Lac@H-COF-OMe showed both high laccase loading and activity recovery. • Lac@H-COF-OMe displayed excellent structure stability and recyclability. • Lac@H-COF-OMe exhibited better adsorption uptake and faster degradation rate for tetracycline. Hollow covalent organic framework microsphere (H-COF-OMe) using TAPB and DMTP with enriched interfacial defects was fabricated via emulsion interfacial polymerization and in turn applied as a novel host for high laccase loading and tetracycline (TC) degradation. Benefited from the space-confined growth in the multiphase solvent interface, H-COF-OMe exhibited hollow spherical microstructure, high surface area and unique defects-rich interface. Attributed to these intriguing aspects, H-COF-OMe achieved maximum loading capacity of 567 mg/g and activity recovery of 85% for laccase. H-COF-OMe efficiently stabilized the active conformation of laccase from structural distortion via multiple binding sites, which endowed Lac@H-COF-OMe significantly higher pH, thermal, and storage stabilities, and reusability than free laccase and Lac@COF-OMe. Interestingly, the hollow morphology and defective interface of Lac@H-COF-OMe accelerated the diffusion of TC and shortened the reaction pathway, which endowed it with markedly enhanced TC degradation and recycling performance than many state-of-the-art catalysts. Significantly, Lac@H-COF-OMe (20 mg) could achieve 99% degradation of 50 mg/L tetracycline (50 mL) within 100 min. Monitoring of the intermediate products indicated that Lac@H-COF-OMe showed outstanding detoxification performance of the degradation products. This work suggested a novel COF synthesis strategy as laccase immobilization supporters for high TC degradation, which makes it as a promising candidate for degradation of organic pollutants.

Carrier-Free Immobilization of α-Galactosidase as Nano-Biocatalysts for Synthesizing Prebiotic α-Galacto-Oligosaccharides.

α-Galacto-oligosaccharides (α-GOSs) have great functions as prebiotics and therapeutics. This work established the method of batch synthesis of α-GOSs by immobilized α-galactosidase for the first time, laying a foundation for industrial applications in the future. The α-galactosidase from Aspergillus niger L63 was immobilized as cross-linked enzyme aggregates (CLEAs) nano-biocatalyst through enzyme precipitating and cross-linking steps without using carriers. Among the tested agents, the ammonium sulfate showed high precipitation efficacy and induced regular structures of α-galactosidase CLEAs (Aga-CLEAs) that had been analyzed by scanning electron microscopy and Fourier-transform infrared spectroscopy. Through optimization by response surface methodology, the ammonium sulfate-induced Aga-CLEAs achieved a high activity recovery of around 90% at 0.55 U/mL of enzymes and 36.43 mM glutaraldehyde with cross-linking for 1.71 h. Aga-CLEAs showed increased thermal stability and organic solvent tolerance. The storage ability was also improved since it maintained 74.5% activity after storing at 4 °C for three months, significantly higher than that of the free enzyme (21.6%). Moreover, Aga-CLEAs exhibited excellent reusability in the α-GOSs synthesis from galactose, retaining above 66% of enzyme activity after 10 batch reactions, with product yields all above 30%.

One-step purification and freeze stability of papain at acidic pH values

Papain is a proteolytic enzyme of great commercial value. It is a cysteine protease highly expressed in Carica papaya fruit latex, but also present in papaya leaves. Purification procedures mostly deal with the latex and include a combination of precipitation and/or chromatographic techniques. Due to its solubility, structure and activity characteristics, the pH and salt content play significant roles in handling papain extracts. Here we report a simple, rapid and easily scalable procedure for papain purification from papaya leaves, which contain different contaminants as compared to papaya latex. Sodium chloride precipitation of contaminants at pH 5 followed by ammonium sulphate precipitation resulted in the removal of other leaf proteins and protein fragments from papain solution and about a 3-fold purification. The procedure also benefits from the suppression of autoproteolysis and preservation of the native structure, as confirmed by FTIR analysis, and the high recovery of activity of over 80%.

Virus-templated magnetic composite hydrogels for surface immobilization of mimic-free-lipase.

Surface immobilization of enzymes on magnetic-recoverable carriers is of great interest and importance for the biocatalysis of relatively large molecules. In this work, the nanosized amino-rich filamentous M13 virus, a versatile biological scaffold, was applied as the unique soft backbone for lipase immobilization. Based on the structure and capsid proteins of M13 phages, the magnetic-recoverable mimic-free-lipases (MFLs) composed of the M13 hydrogels and magnetic particles were developed in two designs. In the first design, nanosized wild M13 phages were crosslinked into a phage hydrogel through the N-terminals of pVIII peptides while NH2-Fe3O4 magnetic nanoparticles (MNPs) were attached to the M13 virus through glutaraldehyde, forming the M13-(NH2-Fe3O4) magnetic phage hydrogel. In the second design, special M13 with Fe3O4 affinity pIII-peptide (FAP-M13) was biopanned for strongly binding towards bare Fe3O4 with the "hook"-like pIII-peptide (N-LPLSTQH-C). TEM observation confirmed the direct grasp of FAP-M13 on bare Fe3O4, forming the magnetic (FAP-M13)-Fe3O4 virus hydrogel. Lipases were uniformly anchored on the phage surface of nanoscale by crosslinking with the N-terminals of pVIII peptides, and then lipase@M13-(NH2-Fe3O4) and lipase@(FAP-M13)-Fe3O4 MFLs were constructed. For both MFLs, high activity recovery yield (>95%) and efficient magnetic separation were characterized. Significantly reduced MNP-usage-amount and enhanced lipase-loading-amount both by about 40 folds were obtained, compared with the conventional NH2-Fe3O4 carriers. The quantified Km and Vmax/Km values were almost equal to those of the free lipases, verifying free-enzyme-mimicking features of the MFLs. High pH-tolerance, wide temperature adaptability, enhanced thermal stability and stable magnetic separation capability of both MFLs were also observed. In particular, the (FAP-M13)-Fe3O4 magnetic virus hydrogel simply using bare Fe3O4 MNPs would be more convenient and economical in the scaled-up biocatalysis.

Laccase immobilization on core-shell magnetic metal-organic framework microspheres for alkylphenol ethoxylate compound removal

An amino-functionalized magnetic core-shell structure metal-organic framework (MOF), Fe3O4-NH2@MIL-100 (Fe), has been successfully constructed by a step-by-step method. The composite microspheres exhibited excellent magnetic characteristics, large surface area, and pore size, which were employed for the Trametes versicolor laccase immobilization. The immobilized laccase exhibited high activity recovery (72.05%) and significantly improved stability. The reactive activity remained 75% of its initial activity after storage for 4 weeks. Additionally, when the ambient reached 80 °C, the good stability enabled the immobilization laccase to keep 33% residual activity even after 6 h storage. Application of the immobilized laccase for environmental pollutant alkylphenol polyethoxylate (APEO) removal was also investigated. The results showed the immobilized laccase can remove 100% octylphenol polyethoxylate (OPEO) and 98.16% nonylphenol polyethoxylate (NPEO) in 200 min, and can still achieve 63.42% and 46.17% removal efficiency for OPEO and NPEO after reusing four cycles, respectively. The novel MOF based core-shell magnetic composite microspheres introduced by this study showed promising applications as laccase immobilization support, and both the immobilized laccase and its support had great potential in wastewater treatment.

Metal ions coordinated immobilization of phenylalanine dehydrogenase by GO‐PEI with high activity recovery and enhanced stability

AbstractBACKGROUNDDehydrogenases dependent on NAD(P)H are widely applied for biosynthesis of chiral chemicals. However, they are unstable and easy denaturated compared with hydrolases. A new strategy for dehydrogenases immobilization with the advantages of easy operation and high activity recovery should be developed. Biocompatible hybrid materials (GO‐PEI‐M) have been obtained by metal ions coordinated polyethylenimine (PEI) functionalized graphene oxide (GO).RESULTSThe immobilized enzyme recovery rates of GO‐PEI‐Mg (magnesium ion coordinated GO‐PEI) and GO‐PEI‐Mn (manganese ion coordinated GO‐PEI). In addition, the stability of GO‐PEI‐Mn‐PheDH (Phenylalanine Dehydrogenase) and GO‐PEI‐Mg‐PheDH was greatly improved, and it maintained 65% and 79% activity, respectively, after seven times reuse. In addition, the stability of GO‐PEI‐Mn‐PheDH (Phenylalanine Dehydrogenase) and GO‐PEI‐Mg‐PheDH was greatly improved. After seven times reuse, GO‐PEI‐Mn‐PheDH and GO‐PEI‐Mn‐PheDH and maintained 65% and 79% activity, respectively. An assembly mechanism was proposed from the perspectives of multi‐level interactions, including hydrogen bonding, electrostatic forces, and coordinated bonds.CONCLUSIONGO‐PEI‐Metal ion hybrid materials offer a promising carrier for enzyme immobilization with the advantages of biocompatibility and relatively strong interaction. © 2020 Society of Chemical Industry

Preparation of Chitosan/Magnetic Porous Biochar as Support for Cellulase Immobilization by Using Glutaraldehyde.

In this work, porous biochar was obtained from sugarcane bagasse by alkali activation and pyrolysis and then magnetized with γ-Fe2O3 by calcination. After functionalization with chitosan and activation with glutaraldehyde, the as-prepared chitosan/magnetic porous biochar served as a support to immobilize cellulase by covalent bonds. The immobilization amount of cellulase was 80.5 mg cellulase/g support at pH 5 and 25 °C for 12 h of immobilization. To determine the enzymatic properties, 1% carboxymethyl cellulose sodium (CMC) (dissolved in 0.1 M buffer) was considered as a substrate for hydrolysis at different pH values (3–7) and temperatures (30–70 °C) for 30 min. The results showed that the optimum pH and temperature of the free and immobilized cellulase did not change, which were pH 4 and 60 °C, respectively. The immobilized cellulase had a relatively high activity recovery of 73.0%. However, it also exhibited a higher Michaelis–Menten constant (Km) value and a slower maximum reaction velocity (Vmax) value compared to the free enzyme. In the reusability assay, the immobilized cellulase showed initial glucose productivity of 330.9 mg glucose/g CMC and remained at 86.0% after 10 uses. In conclusion, the chitosan/magnetic porous biochar has great potential applications as a support for enzyme immobilization.

Efficient synthesis of an antiviral drug intermediate using overexpressed short‐chain dehydrogenase and cross‐linked enzyme aggregates stabilization

AbstractBACKGROUND(2R,3S)‐N‐tert‐Butoxycarbonyl‐3‐amino‐1‐chloro‐2‐hydroxy‐4‐phenylbutane (1b) is key for the synthesis of the antiviral drug atazanavir. It can be obtained via the stereoselective bioreduction of (3S)‐3‐(N‐Boc‐amino)‐1‐chloro‐4‐phenyl‐butanone (1a) with short‐chain dehydrogenase/reductase (SDR) (EC 1.1.1.1). To develop a practical and cost‐effective biocatalytic approach with high stability and reusability, an SDR mutant (muSDR) from Novosphingobium aromaticivorans was overexpressed in Escherichia coli cell and immobilized by cross‐linked enzyme aggregates with enhanced stability and high efficiency.RESULTSmuSDR gene was successfully cloned and overexpressed in Escherichia coli and reduced 1a to 1b with high stereoselectivity. Four resin‐bound muSDR and cross‐linked muSDR aggregates (muSDR‐CLEAs) were prepared and compared. With addition of Tween‐80, the muSDR‐CLEAs demonstrated much higher activity recovery than resin‐bound muSDR. Compared with free enzyme, muSDR‐CLEAs demonstrated enhanced thermal, pH, and storage stabilities and could obtain 1b with high substrate concentration, high de, and complete conversion. The reusability of CLEAs was also determined as no significant activity loss was observed after seven batches of reactions. The space time yield of the bioreduction using muSDR‐CLEAs was 226.6 g∙L−1∙day−1.CONCLUSIONOverexpressed muSDR was efficiently immobilized, and it demonstrated superior stability and reusability. The recombinant muSDR and muSDR‐CLEAs are practical potential biocatalysts for industrial production of the atazanavir precursor. © 2020 Society of Chemical Industry (SCI)

Chitosan-based CLEAs from Aspergillus niger type A feruloyl esterase: high-productivity biocatalyst for alkyl ferulate synthesis.

The enzymatic synthesis of alkyl ferulates is an important reaction in cosmetic and pharmaceutical chemistries, since it may allow to expand the biorefinery concept valorizing biomass wastes enriched in ferulic acid. However, robust biocatalysts for that purpose are scarce. Herein, we have immobilized the type A feruloyl esterase from Aspergillus niger (AnFaeA) as cross-linked enzyme aggregates, employing chitosan as co-feeder (ChCLEAs). High immobilization yields and relative activity recovery were attained in all assessed conditions (> 93%). Furthermore, we enhanced the thermal stability of the soluble enzyme 32-fold. AnFaeA-ChCLEAs were capable to quantitatively perform the solvent-free direct esterification of short- to medium-chain alkyl ferulates (C4-C12) in less than 24 h. By raising the operational temperature to 50 °C, AnFaeA-ChCLEAs transformed 350 mM ferulic acid into isopentyl ferulate with a space-time yield of 46.1 g of product × L-1 × day-1, 73-fold higher than previously reported. The overall sustainability of this alkyl ferulate production bioprocess is supported by the high total turnover number (TTN 7 × 105) and the calculated green metrics (E factor = 30). Therefore, we herein present a robust, efficient, and versatile heterogeneous biocatalyst useful for the synthesis of a wide diversity of alkyl ferulates. KEY POINTS: • CLEAs of feruloyl esterase A from A. niger using chitosan as co-feeder were obtained. • Microenvironment of the biocatalysts allowed to obtain C1 to C18 alkyl ferulates. • Biocatalyst at boundary conditions showed a high productivity of 46 g/L day. Graphical Abstract.