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Nanobiocatalysts (NBCs), which merge enzymes with nanomaterials, provide a potent method for improving enzyme durability, efficiency, and recyclability. This review highlights the use of eco-friendly synthesis methods to create sustainable nanomaterials for enzyme transport. We investigate different methods of immobilization, such as adsorption, ionic and covalent bonding, entrapment, and cross-linking, examining their pros and cons. The decreased environmental impact of green-synthesized nanomaterials from plants, bacteria, and fungi is emphasized. The review exhibits the various uses of NBCs in food industry, biofuel production, and bioremediation, showing how they can enhance effectiveness and eco-friendliness. Furthermore, we explore the potential impact of NBCs in biomedicine. In general, green nanobiocatalysts are a notable progression in enzyme technology, leading to environmentally-friendly and effective biocatalytic methods that have important impacts on industrial and biomedical fields.

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In this study, Bacillus tequilensis TB5 α-amylase from rice-milled by-products (rice bran and de-oiled rice bran) was successfully immobilized onto biologically synthesized magnetic nanoparticles fabricated with chitosan (MNP-Ch) and characterized via different biophysical techniques. Furthermore, the study emphasized incorporating this nanostructure framework (MNP@2mgchitosan_DORB-amy and MNP@3mgchitosan_RB-amy) to offer diverse applications, including enzymatic desizing, cleaning starchy stains, and treating synthetic starchy wastewater. An enzyme loading of > 90 % for both enzymes indicated increased binding sites due to the functional moieties of chitosan on the MNP. The Km was 0.28 and 0.31 mg/mL for the immobilized and free forms of DORB-amy, respectively, and 0.18 and 0.27 mg/mL for the immobilized and free forms of RB-amy, respectively. A low Km indicated an increased affinity of MNP-Ch-immobilized forms of enzymes toward the substrate. The performance of both immobilized enzymes improved at a wide range of pH and temperature, which may be attributed to the covalent binding of the enzyme on to the MNP-Ch. The nanobiocatalysts in the detergent act synergistically to fade the starchy stains. Furthermore, an 8-9 TEGEWA scale rating with > 11 % of starch removal was obtained through the biodesizing of starch-sized cotton fabric. The nanobiocatalyst efficiently decomposed starch and liberated 650-670 mg/L of reducing sugar from the synthetic wastewater, therefore offering promising opportunities for its exploration in a wastewater treatment plant. Thus, the study recommends the potential exploration of sturdy matrices like MNP to offer remarkable applications with maximum operational stability, easier recovery, and higher efficiency.

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The synergy between enzymes and nanotechnology (nano-biocatalysts) has created some of the most promising biomaterials fabricated by synergistically incorporating advanced nano-biotechnology. The incorporation of enzymes into nanotechnology is of great significance for making nanomaterials that are rarely harmful to the environment. However, the unique/specific physicochemical characteristics and supramolecular nature ascribed to functional nanostructures (nanomaterials), have made them novel, interesting, and exceptional matrices for the creation of nano-biocatalysts. These have a lot of potential for improving the enzyme stability, function, efficiency, kinetic characteristics, vulnerability to diffusional constraints, and engineering performance in bioprocessing. Hence, the nano-biocatalysts developed contain exceptional properties with many potential applications in diverse fields. This review covers a wide range of the nanotechnology and enzyme technology involved in producing nano-biocatalysts, including different mechanisms, strategies in nanomaterial enzyme immobilization, and various nanocarriers, as well as recent developments in controlling enzyme activity. The vast range of potential applications of nano-biocatalysts in various fields, including food, pharmaceuticals, biofuels, and bioremediation, has been discussed.

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Global waste production is anticipated reach to 2.59 billion tons in 2030, thus accentuating issues of environmental pollution and health security. 37 % of waste is landfilled, 33 % is discharged or burned in open areas, and only 13.5 % is recycled, which makes waste management poorly efficient in the context of the circular economy. There is, therefore, a need for methods to recycle waste into valuable materials through the resource recovery process. Progress in the field of recycling is strongly dependent on the development of efficient, stable, and reusable yet inexpensive catalysts. In this case, growing attention has been paid to the development and application of nanobiocatalysts with promising features. The main purpose of this review paper is to: (i) introduce nanobiomaterials and describe their effective role in the preparation of functional nanobiocatalysts for the recourse recovery aims; (ii) provide production methods and the efficiency improvement of nanobaiocatalysts; (iii) give a comprehensive description of valued resource recovery for reducing toxic chemicals from the contaminated environment; (iv) describe various technologies for the valued resource recovery; (v) state the limitation of the valued resource recovery; (vi) and finally economic importance and current scenario of nanobiocatalysts strategies applicable for the resource recovery processes.

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Magnetic nanobiocatalysts (MNBCs) are a promising immobilization approach to ease enzyme recovery during bioprocessing. However, industrial adoption of MNBCs is unfeasible because MNBC-synthesis involves complex and potentially expensive processing steps including synthesis of silica-coated superparamagnetic iron oxide nanoparticles (Si-SPIONs). We developed a single-step process for Si-SPION synthesis using a tubular electrochemical system (TES) and investigated the effect of concentration of the Na2SiO3 coating agent on Si-SPION properties. The Si-SPIONs were used as a support for attachment of polymer-cellulase conjugate to make MNBCs. The spherical Si-SPIONs were 8-12 nm in diameter including a 2-nm silica coating. Na2SiO3 concentration in the reactor did not affect Si-SPION morphology, but increasing Na2SiO3 concentration reduced SPION productivity in the reactor. Protective properties of the SPION silica coatings were demonstrated by showing that they prevented dissolution of SPIONs in an acid solution for 48 h. Enzyme attachment was quantified as protein adsorption on Si-SPIONs which reached 55 µg/mg Si-SPION. The MNBCs were recovered and reused four times. The use of TES for Si-SPION synthesis is promising to reduce MNBC production complexity.

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Cascade reactions catalyzed by multi-enzymatic systems have attracted enormous scientific interest over the last decade. They are an emerging technology that significantly expands the applicability of biocatalysts in several biotechnological processes, such as the synthesis of high value-added products. Immobilization of enzymes on a solid carrier is a commonly used strategy to improve the stability and reuse of multiple enzyme systems. Magnetic nanoparticles have been applied as promising nanocarriers for either the immobilization of one enzyme or the co-immobilization of multiple enzymes. In this chapter, we describe the preparation of magnetic iron oxide nanoparticles γ-Fe2O3 modified with 3-(aminopropyl)-triethoxysilane (APTES), for the simultaneous covalent co-immobilization of oxidoreductases and hydrolytic enzymes, such as cellulase, ß-glucosidase (bgl), glucose oxidase (GOx), and horseradish peroxidase (HRP). Several spectroscopic techniques that are used to characterize the structure and the catalytic performance of such systems are also described.

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Multi-enzymatic assemblies offer the opportunity of bringing in proximity several enzymes that are enabled to work together for the catalysis of multi-step reactions. Especially, the development of robust nanobiocatalytic systems comprising of several enzymes has gained considerable attention over the last few years for the catalysis of complex reactions and the production of high added-value products. In the present chapter, we describe the methodology for the development of a bi-enzymatic nanobiocatalyst consisting of the enzymes ß-glucosidase from Thermotoga maritima and lipase A from Candida antarctica (CalA) co-immobilized on chitosan-coated magnetic nanoparticles. This nanobiocatalyst can be efficiently applied for the biotransformation of oleuropein to hydroxytyrosol, a reaction of increased biotechnological interest. Several techniques, as well as methodologies that are required for the characterization of the structure and the activity of such systems are also comprehensively described.

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Biocatalysts are a biomolecule of interest for various biotechnological applications. Non-reusability and poor stability of especially enzymes has always limited their applications in large-scale processing units. Nanotechnology paves a way by conjugating the biocatalysts on different matrices. It predominantly enables nanomaterials to overcome the limited efficacy of conventional biocatalysts. Nanomaterial conjugated nanobiocatalyst have enhanced catalytic properties, selectivity, and stability. Nanotechnology extended the flexibility to engineer biocatalysts for various innovative and predictive catalyses. So developed nanobiocatalyst harbors remarkable properties and has potential applications in diverse biotechnological sectors. This article summaries various developments made in the area of nanobiocatalyst towards their applications in biotechnological industries. Novel nanobiocatalyst engineering is an area of critical importance for harnessing the biotechnological potential.

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Nanobiocatalysts are one of the most promising biomaterials produced by synergistically integrating advanced biotechnology and nanotechnology. These have a lot of potential to improve enzyme stability, function, efficiencyand engineering performance in bioprocessing. Functional nanostructures have been used to create nanobiocatalystsbecause of their specific physicochemical characteristics and supramolecular nature. This review covers a wide range of nanobiocatalysts including polymeric, metallic, silica and carbon nanocarriers as well as their recent developments in controlling enzyme activity. The enormous potential of nanobiocatalysts in bioprocessing in designing effective laboratory trials forapplications in various fields such as food, pharmaceuticals, biofuel, and bioremediation is also discussed extensively.

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The ever-growing demand for cost-effective and innocuous biocatalytic transformations has prompted the rational design and development of robust biocatalytic tools. Enzyme immobilization technology lies in the formation of cooperative interactions between the tailored surface of the support and the enzyme of choice, which result in the fabrication of tremendous biocatalytic tools with desirable properties, complying with the current demands even on an industrial level. Different nanoscale materials (organic, inorganic, and green) have attracted great attention as immobilization matrices for single or multi-enzymatic systems. Aiming to unveil the potentialities of nanobiocatalytic systems, we present distinct immobilization strategies and give a thorough insight into the effect of nanosupports specific properties on the biocatalysts' structure and catalytic performance. We also highlight the development of nanobiocatalysts for their incorporation in cascade enzymatic processes and various types of batch and continuous-flow reactor systems. Remarkable emphasis is given on the application of such nanobiocatalytic tools in several biocatalytic transformations including bioremediation processes, biofuel production, and synthesis of bioactive compounds and fine chemicals for the food and pharmaceutical industry.

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This study reports the isolation and partial purification of transaminase from the wild species of Bacillus licheniformis. Semi-purified transaminase was immobilized on copper nanoflowers (NFs) synthesized through sonochemical method and explored it as a nanobiocatalyst. The conditions for the synthesis of transaminase NFs [TA@Cu3(PO4)2NF] were optimized. Synthesized NFs revealed the protein loading and activity yield-60 ± 5% and 70 ± 5%, respectively. The surface morphology of the synthesized hybrid NFs was examined by scanning electron microscopy (SEM) and transmission electron microscopy (TEM), which revealed the average size to be around 1 ± 0.5 µm. Fourier-transform infrared (FTIR) was used to confirm the presence of the enzyme inside the immobilized matrix. In addition, circular dichroism and florescence spectroscopy were also used to confirm the integrity of the secondary and tertiary structures of the protein in the immobilized material. The transaminase hybrid NFs exhibited enhanced kinetic properties and stability over the free enzyme and revealed high reusability. Furthermore, the potential application of the immobilized transaminase hybrid NFs was demonstrated in the resolution of racemic α-methyl benzylamine.

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Abstract Owing to the excellent catalytic potential, β-galactosidase (EC: 3.2.1.23) has been exploited as an important industrial enzyme for obtaining galactooligosaccharides (GOS) and lactose-free products in dairy industries. Moreover, novel technologies have been implemented in the recent past for preparing and modifying nanoparticles (NPs) for immobilizing therapeutically and industrially important enzymes. Nanoparticles based enzyme immobilization (NBEI) offered more stability and robustness to the enzymes due to their fixed conformation and hence extend their applications in broader areas. A quick overview of the results exhibited greater activity for the enzymes immobilized on NPs as compared to enzyme immobilized on 2-D matrices. Based on these findings, this review was aimed to emphasize the recent development achieved for immobilizing β-galactosidase on NPs with their specific utilization in obtaining dairy products. These studies includes β-galactosidases from various sources that were immobilized on various NPs for hydrolyzing lactose in batch and continuous reactors, and for the production of GOS in biotechnology industries. NBEI of β-galactosidase offered profound stability for transporting substrate and product for enzymatic reactions, apart from cost effective advantage due to reusable nature of immobilized enzyme.

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Nanobiocatalysts were produced via immobilization of CalB lipase on polyurethane (PU) based nanoparticles and their application on the synthesis of important industrial products was evaluated. Nanoparticles of polyurethane functionalized with poly(ethylene glycol) (PU-PEG) were synthetized through miniemulsion polymerization and the addition of crosslinking agents were evaluated. The nanoparticles were employed as support for CalB and the kinetic parameters were reported. The performance of new biocatalysts was evaluated on the hydrolysis reaction of p-NPB and on the enantioselective hydrolysis of (R,S)-mandelic acid. The esterification reaction was evaluated on the production of ethyl esters of Omega-3. The effect of poly(ethylene glycol) molar mass (400, 4000 or 6000 Da)on the biocatalyst activity was also analyzed. The PU-PEG6000-CalB showed the highest value of the kinetic parameters, highlighting the high reaction rate. The addition of trehalose as crosslinking agent improved the thermal stability of the biocatalysts. PU-PEG400-CalB was the most active nanobiocatalyst, exhibiting a ethyl esters production of 43.72 and 16.83 mM.U -1 using EPA and DHA, respectively. The nanobiocatalyst was also applied in enantiomeric resolution of mandelic acid, showing promising enantiomeric ratios. The results obtained in this work present alternative and sustainable routes for the synthesis of important compounds used on food and pharmaceutical industries.

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In view of the potential applications of immobilized enzymes, partially purified Lignin Peroxidase (LiP) from Pseudomonas fluorescens LiP-RL5 was immobilized on Graphene Oxide functionalized MnFe2O4 nanoparticles (10 nm, synthesized by sol-gel auto-combustion) to fabricate a new hyperactive and thermostable nanobiocatalyst and thereafter characterized by using standard techniques. Immobilized LiP was quite stable at 50 °C with the half-life of 14 h and showed higher tolerance towards various metal ions and solvents than free LiP. Immobilized LiP retained 50% of enzyme activity even after nine consecutive runs. When tested against various textile dyes, the immobilized LiP was found quite effective with higher dye decolourization efficiency (up to 88%) within 1 h of incubation at 30 °C. The results of this research effort confirmed that the immobilization of LiP and fabrication of nanobiocatalyst increase the efficacy, stability, and reusability of the enzyme which could be efficiently utilized under harsh industrial conditions.

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Lytic polysaccharide monooxygenases (LPMOs) with synergistic effect on polysaccharide hydrolase represent a revolution in biotechnology, which may accelerate the conversion of biomass to the second-generation biofuels. Discovering more hydrolases that have synergism with LPMOs will considerably expand the knowledge and application of biomass degradation. The LPMOs named CgAA9 were verified to exhibit 1.52-fold synergism when incubated with ß-1,3-xylanase at a molar ratio of 3:1. The ion chromatography results proved that CgAA9 did not alter the endogenous hydrolysis mode of ß-1,3-xylanase. Meanwhile, to decrease the operational cost of enzymes, a novel strategy for immobilizing LPMOs and ß-1,3-xylanases based on the biomimetic silica nanoparticles was developed. It enabled preparation of immobilized enzymes directly from the cell lysate. The immobilization efficiency and activity recovery reached 84.6 and 81.4%. They showed excellent reusability for 12 cycles by retaining 68% of initial activity. The optimum temperature for both free and immobilized biocatalyst were 40 and 37 °C, indicating they were ideal candidates for typical simultaneous saccharification and fermentation (SSF) in ethanol production from algea biomass. This was the first report on the synergy between LPMOs and ß-1,3-xylanase, and the strategy for enzyme self-immobilization was simple, timesaving, and efficient, which might have great potentials in algae biomass hydrolysis. KEY POINTS: • The lytic polysaccharide monooxygenases (LPMOs) from Chaetomium globosum were firstly verified to boost the hydrolysis of ß-1,3-xylanases for ß-1,3-xylan. • A novel strategy for simple preparation of SpyCather-modifed silica nanopartilcles and intelligent immobilization of target enzymes from the cell lysate was proposed. • The immobilized LPMOs and ß-1,3-xylanases could be reasonable alternatives for typical simultaneous saccharification and fermentation (SSF) in manipulation of algae biomass.

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BACKGROUND: Microalgae, due to its well-recognized advantages have gained renewed interest as potentially good feedstock for biodiesel. Production of fatty acid methyl esters (FAMEs) as a type of biodiesel was carried out from Chlorella vulgaris bio-oil. Biodiesel was produced in the presence of nano-biocatalysts composed of immobilized lipase on functionalized superparamagnetic few-layer graphene oxide via a transesterification reaction. A hybrid of few-layer graphene oxide and Fe3O4 (MGO) was prepared and characterized. The MGO was functionalized with 3-aminopropyl triethoxysilane (MGO-AP) as well as with a couple of AP and glutaraldehyde (MGO-AP-GA). The Rhizopus oryzae lipase (ROL) was immobilized on MGO and MGO-AP using electrostatic interactions as well as on MGO-AP-GA using covalent bonding. The supports, MGO, MGO-AP, and MGO-AP-GA, as well as nano-biocatalyst, ROL/MGO, ROL/MGO-AP, and ROL/MGO-AP-GA, were characterized using FESEM, VSM, FTIR, and XRD. The few-layer graphene oxide was characterized using AFM and the surface charge of supports was evaluated with the zeta potential technique. The nano-biocatalysts assay was performed with an evaluation of kinetic parameters, loading capacity, relative activity, time-course thermal stability, and storage stability. Biodiesel production was carried out in the presence of nano-biocatalysts and their reusability was evaluated in 5 cycles of transesterification reaction. RESULTS: The AFM analysis confirmed the few-layer structure of graphene oxide and VSM also confirmed that all supports were superparamagnetic. The maximum loading of ROL (70.2%) was related to MGO-AP-GA. The highest biodiesel conversion of 71.19% achieved in the presence of ROL/MGO-AP-GA. Furthermore, this nano-biocatalyst could maintain 58.77% of its catalytic performance after 5 cycles of the transesterification reaction and was the best catalyst in the case of reusability. CONCLUSIONS: In this study, the synthesized nano-biocatalyst based on bare and functionalized magnetic graphene oxide was applied and optimized in the process of converting microalgae bio-oil to biodiesel for the first time and compared with bare lipase immobilized on magnetic nanoparticles. Results showed that the loading capacity, kinetic parameters, thermal stability, and storage stability improved by the functionalization of MGO. The biocatalysts, which were prepared via covalent bonding immobilization of enzyme generally, showed better characteristics.

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Surface modification of multiwalled carbon nanotubes (MWCNTs) could enhance the features of the nanomaterial as carrier for enzyme immobilization. In this strategy, magnetic MWCNTs were fabricated by incorporating them with cobalt and functionalization was carried out by aminated polydopamine. The surface modified MWCNTs were then used as a carrier for the immobilization of Candida rugosa lipase (CRL) via covalent binding using glutaraldehyde. The immobilized CRL maintained high activity, which was 3-folds of free CRL. The immobilized CRL exhibited excellent thermal resistance as validated by TGA and DTA technique and was found to be active in a broad range of pH and temperatures in comparison to free CRL. Systematic characterization via FT-IR spectroscopy, CD spectroscopy, SEM, TEM and confocal laser scanning microscopy confirmed the presence of CRL on the modified MWCNTs. Immobilized CRL presented an exquisite recycling performance as after ten consecutive reuses it retained around 84% of its initial hydrolytic activity and further showed high yield enzymatic synthesis of ethyl butyrate and isoamyl acetate having characteristic pineapple and banana flavour demonstrating 78% and 75% ester yield, respectively. The present work provides a novel perspective for lipase catalyzed biotechnological applications by adding a magnetic gain to intrinsic features of MWCNTs.

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In the present study, we developed novel ß-glucosidase-based nano-biocatalysts for the bioconversion of oleuropein to hydroxytyrosol. Using non-covalent or covalent immobilization approaches, ß-glucosidases from almonds and Thermotoga maritima were attached for the first time on oxidized and non-oxidized porous carbon cuboids (PCC). Various methods were used for the characterization of the bio-nanoconjugates, such as Fourier transform infrared spectroscopy (FTIR), X-ray photoelectron spectroscopy (XPS), atomic force microscopy (AFM), and fluorescence spectroscopy. The oxidation state of the nanο-support and the immobilization procedure play a key role for the immobilization efficiency or the catalytic activity of the immobilized ß-glucosidases. The nano-biocatalysts were successfully used for the hydrolysis of oleuropein, which leads to the formation of its bioactive derivative, hydroxytyrosol (up to 2.4 g L-1), which is a phenolic compound with numerous health benefits. The bio-nanoconjugates exhibited high thermal and operational stability (up to 240 hours of repeated use), which indicated that they are efficient tools for various bio-transformations.

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Multiwalled carbon nanotubes molybdenum disulfide 3D nanocomposite (MWCNT-MoS2 NC) was successfully synthesized via eco-friendly hydrothermal method. The microstructural characterization of synthesized nanocomposite was carried out using different spectroscopic and microscopic techniques. Nanocomposite was activated using glutaraldehyde chemistry and used as a platform to immobilize Lens culinaris ß-galactosidase (Lsbgal) which resulted in 93% of immobilization efficiency. Attachment of Lsbgal onto nanocomposite was confirmed by AFM, FE-SEM, FTIR, and CLSM. The nanobiocatalyst showed broadening in operational pH and temperature working range. Remarkable increase in thermal stability was observed as compared to soluble enzyme. Nanobiocatalyst showed outstanding increase in storage stability, retained 92% of residual activity over a period of 8 months. This offers good reusability as it retained ∼50% residual activity up to 21 reuses and exhibited higher rate of lactose hydrolysis in whey. MWCNT-MoS2 NC conjugated to biomolecules can serve as a potential platform for fabrication of lactose biosensor.

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L-DOPA (3,4-dihydroxyphenyl-L-alanine) is a preferred drug for Parkinson's disease, and is currently in great demand every year worldwide. Biocatalytic conversion of L-tyrosine by tyrosinases is the most promising method for the low-cost production of L-DOPA in both research and industry. Yet, it has been hampered by low productivity, low conversion rate, and low stability of the biocatalyst, tyrosinase. An alternative tyrosinase TyrVs from Verrucomicrobium spinosum with more efficient expression in heterologous host and better stability than the commercially available Agaricus bisporus tyrosinase was identified in this study. Additionally, it was prepared as a novel nano-biocatalyst based on the distinct one-step in situ immobilization on the surface of polyhydroxyalkanoate (PHA) nano-granules. The resulting PHA-TyrVs nano-granules demonstrated improved L-DOPA-forming monophenolase activity of 9155.88 U/g (Tyr protein), which was 3.19-fold higher than that of free TyrVs. The nano-granules also exhibited remarkable thermo-stability, with an optimal temperature of 50 °C, and maintained more than 70% of the initial activity after incubation at 55 °C for 24 h. And an enhanced affinity of copper ion was observed in the PHA-TyrVs nano-granules, making them even better biocatalysts for L-DOPA production. Therefore, a considerable productivity of L-DOPA, amounting to 148.70 mg/L h, with a conversion rate of L-tyrosine of 90.62% can be achieved by the PHA-TyrVs nano-granules after 3 h of biocatalysis under optimized conditions, without significant loss of enzyme activity or L-DOPA yield after 8 cycles of repeated use. Our study provides an excellent and robust nano-biocatalyst for the cost-effective production of L-DOPA.

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