Pepsin immobilization on activated carbon and functionalized with glutaraldehyde and genipin for the synthesis of antioxidant peptides of goat casein.

In this article, the synthesis of antioxidant peptides in the enzymatic hydrolysis of caprine casein was analyzed at three different time points (60 min, 90 min, and 120 min) using immobilized pepsin on activated and modified carbon (AC, ACF, ACG 50, ACG 100). The immobilization assays revealed a reduction in the biocatalysts' activity compared to the free enzyme. Among the modified ones, ACG 50 exhibited greater activity and better efficiency for reuse cycles, with superior values after 60 min and 90 min. Peptide synthesis was observed under all studied conditions. Analyses (DPPH, ß-carotene/linoleic acid, FRAP) confirmed the antioxidant potential of the peptides generated by the immobilized enzyme. However, the immobilized enzyme in ACG 50 and ACG 100, combined with longer hydrolysis times, allowed the formation of peptides with an antioxidant capacity greater than or equivalent to those generated by the free enzyme, despite reduced enzymatic activity.

Immobilized Dipeptidase in Manganese Ion-Loaded Polyethylenimine-Induced Calcium Phosphate Nanocrystals for Carnosine Synthesis.

Carnosine is a natural bioactive dipeptide with important physiological functions widely used in food and medicine. Dipeptidase (PepD) from Serratia marcescens can catalyze the reverse hydrolytic reaction of ß-alanine with l-histidine to synthesize carnosine in the presence of Mn2+. However, it remains challenging to practice carnosine biosynthesis due to the low activity and high cost of the enzyme. Therefore, the development of biocatalysts with high activity and stability is of significance for carnosine synthesis. Here, we proposed to chelate Mn2+ to polyethylenimine (PEI) that induced rapid formation of calcium phosphate nanocrystals (CaP), and Mn-PEI@CaP was used for PepD immobilization via electrostatic interaction. Mn-PEI@CaP as the carrier enhanced the stability of the immobilized enzyme. Moreover, Mn2+ loaded in the carrier acted as an in situ activator of the immobilized PepD for facilitating the biocatalytic process of carnosine synthesis. The as-prepared immobilized enzyme (PepD-Mn-PEI@CaP) kept similar activity with free PepD plus Mn2+ (activity recovery, 102.5%), while exhibiting elevated thermal stability and pH tolerance. Moreover, it exhibited about two times faster carnosine synthesis than the free PepD system. PepD-Mn-PEI@CaP retained 86.8% of the original activity after eight cycles of batch catalysis without the addition of free Mn2+ ions during multiple cycles. This work provides a new strategy for the co-immobilization of PepD and Mn2+, which greatly improves the operability of the biocatalysis and demonstrates the potential of the immobilized PepD system for efficient carnosine synthesis.

Bioremediation of Hazardous Pollutants Using Enzyme-Immobilized Reactors.

Bioremediation uses the degradation abilities of microorganisms and other organisms to remove harmful pollutants that pollute the natural environment, helping return it to a natural state that is free of harmful substances. Organism-derived enzymes can degrade and eliminate a variety of pollutants and transform them into non-toxic forms; as such, they are expected to be used in bioremediation. However, since enzymes are proteins, the low operational stability and catalytic efficiency of free enzyme-based degradation systems need improvement. Enzyme immobilization methods are often used to overcome these challenges. Several enzyme immobilization methods have been applied to improve operational stability and reduce remediation costs. Herein, we review recent advancements in immobilized enzymes for bioremediation and summarize the methods for preparing immobilized enzymes for use as catalysts and in pollutant degradation systems. Additionally, the advantages, limitations, and future perspectives of immobilized enzymes in bioremediation are discussed.

Towards Greener and More Cost-efficient Biosynthesis of Pharmaceuticals and Fragrance Molecules.

Enzymes are natural catalysts which are gaining momentum in chemical synthesis due to their exquisiteselectivity and their biodegradability. However, the cost-efficiency and the sustainability of the overall biocatalytic process must be enhanced to unlock completely the potential of enzymes for industrial applications. To reach this goal, enzyme immobilization and the integration into continuous flow reactors have been the cornerstone of our research. We showed key examples of the advantages of those tools for the biosynthesis of antivirals, anticancer drugs, and valuable fragrance molecules. By combining new strategies to immobilize biocatalysts, innovative bioengineering approaches, and process development, the performance of the reactions could be boosted up to 100-fold.

An enzymatic continuous-flow reactor based on a pore-size matching nano- and isoporous block copolymer membrane.

Continuous-flow biocatalysis utilizing immobilized enzymes emerged as a sustainable route for chemical synthesis. However, inadequate biocatalytic efficiency from current flow reactors, caused by non-productive enzyme immobilization or enzyme-carrier mismatches in size, hampers its widespread application. Here, we demonstrate a general-applicable and robust approach for the fabrication of a high-performance enzymatic continuous-flow reactor via integrating well-designed scalable isoporous block copolymer (BCP) membranes as carriers with an oriented and productive immobilization employing material binding peptides (MBP). Densely packed uniform enzyme-matched nanochannels of well-designed BCP membranes endow the desired nanoconfined environments towards a productive immobilized phytase. Tuning nanochannel properties can further regulate the complex reaction process and fortify the catalytic performance. The synergistic design of enzyme-matched carriers and efficient enzyme immobilization empowers an excellent catalytic performance with >1 month operational stability, superior productivity, and a high space-time yield (1.05 × 105 g L-1 d-1) via a single-pass continuous-flow process. The obtained performance makes the designed nano- and isoporous block copolymer membrane reactor highly attractive for industrial applications.

Enzyme Immobilization by Inkjet Printing on Reagentless Biosensors for Electrochemical Phosphate Detection.

Enzyme-based biosensors commonly utilize the drop-casting method for their surface modification. However, the drawbacks of this technique, such as low reproducibility, coffee ring effects, and challenges in mass production, hinder its application. To overcome these limitations, we propose a novel surface functionalization strategy of enzyme crosslinking via inkjet printing for reagentless enzyme-based biosensors. This method includes printing three functional layers onto a screen-printed electrode: the enzyme layer, crosslinking layer, and protective layer. Nanomaterials and substrates are preloaded together during our inkjet printing. Inkjet-printed electrodes feature a uniform enzyme deposition, ensuring high reproducibility and superior electrochemical performance compared to traditional drop-casted ones. The resultant biosensors display high sensitivity, as well as a broad linear response in the physiological range of the serum phosphate. This enzyme crosslinking method has the potential to extend into various enzyme-based biosensors through altering functional layer components.

L-Lactate Electrochemical Biosensor Based on an Integrated Supramolecular Architecture of Multiwalled Carbon Nanotubes Functionalized with Avidin and a Recombinant Biotinylated Lactate Oxidase.

L-Lactate is an important bioanalyte in the food industry, biotechnology, and human healthcare. In this work, we report the development of a new L-lactate electrochemical biosensor based on the use of multiwalled carbon nanotubes non-covalently functionalized with avidin (MWCNT-Av) deposited at glassy carbon electrodes (GCEs) as anchoring sites for the bioaffinity-based immobilization of a new recombinant biotinylated lactate oxidase (bLOx) produced in Escherichia coli through in vivo biotinylation. The specific binding of MWCNT-Av to bLOx was characterized by amperometry, surface plasmon resonance (SPR), and electrochemical impedance spectroscopy (EIS). The amperometric detection of L-lactate was performed at -0.100 V, with a linear range between 100 and 700 µM, a detection limit of 33 µM, and a quantification limit of 100 µM. The proposed biosensor (GCE/MWCNT-Av/bLOx) showed a reproducibility of 6.0% and it was successfully used for determining L-lactate in food and enriched serum samples.

Development of an Innovative Biosensor Based on Graphene/PEDOT/Tyrosinase for the Detection of Phenolic Compounds in River Waters.

Phenolic compounds, originating from industrial, agricultural, and urban sources, can leach into flowing waters, adversely affecting aquatic life, biodiversity, and compromising the quality of drinking water, posing potential health hazards to humans. Thus, monitoring and mitigating the presence of phenolic compounds in flowing waters are essential for preserving ecosystem integrity and safeguarding public health. This study explores the development and performance of an innovative sensor based on screen-printed electrode (SPE) modified with graphene (GPH), poly(3,4-ethylenedioxythiophene) (PEDOT), and tyrosinase (Ty), designed for water analysis, focusing on the manufacturing process and the obtained electroanalytical results. The proposed biosensor (SPE/GPH/PEDOT/Ty) was designed to achieve a high level of precision and sensitivity, as well as to allow efficient analytical recoveries. Special attention was given to the manufacturing process and optimization of the modifying elements' composition. This study highlights the potential of the biosensor as an efficient and reliable solution for water analysis. Modification with graphene, the synthesis and electropolymerization deposition of the PEDOT polymer, and tyrosinase immobilization contributed to obtaining a high-performance and robust biosensor, presenting promising perspectives in monitoring the quality of the aquatic environment. Regarding the electroanalytical experimental results, the detection limits (LODs) obtained with this biosensor are extremely low for all phenolic compounds (8.63 × 10-10 M for catechol, 7.72 × 10-10 M for 3-methoxycatechol, and 9.56 × 10-10 M for 4-methylcatechol), emphasizing its ability to accurately measure even subtle variations in the trace compound parameters. The enhanced sensitivity of the biosensor facilitates detection and quantification in river water samples. Analytical recovery is also an essential aspect, and the biosensor presents consistent and reproducible results. This feature significantly improves the reliability and usefulness of the biosensor in practical applications, making it suitable for monitoring industrial or river water.

Molecular simulations guide immobilization of lipase on nest-like ZIFs with regulatable hydrophilic/hydrophobic surface.

The catalytic performance of immobilized lipase is greatly influenced by functional support, which attracts growing interest for designing supports to achieve their promotive catalytic activity. Many lipases bind strongly to hydrophobic surfaces where they undergo interfacial activation. Herein, the behavioral differences of lipases with distinct lid structures on interfaces of varying hydrophobicity levels were firstly investigated by molecular simulations. It was found that a reasonable hydrophilic/hydrophobic surface could facilitate the lipase to undergo interfacial activation. Building on these findings, a novel "nest"-like superhydrophobic ZIFs (ZIFN) composed of hydrophobic ligands was prepared for the first time and used to immobilize lipase from Aspergillus oryzae (AOL@ZIFN). The AOL@ZIFN exhibited 2.0-folds higher activity than free lipase in the hydrolysis of p-Nitrophenyl palmitate (p-NPP). Especially, the modification of superhydrophobic ZIFN with an appropriate amount of hydrophilic tannic acid can significantly improve the activity of the immobilized lipase (AOL@ZIFN-TA). The AOL@ZIFN-TA exhibited 30-folds higher activity than free lipase, and still maintained 82% of its initial activity after 5 consecutive cycles, indicating good reusability. These results demonstrated that nanomaterials with rational arrangement of the hydrophilic/hydrophobic surface could facilitate the lipase to undergo interfacial activation and improve its activity, displaying the potential of the extensive application.

Evaluation of free and immobilized cellulase on chitosan-modified magnetic nanoparticles for saccharification of sorghum residue.

Enzymatic hydrolysis plays a pivotal role in transforming lignocellulosic biomass. Addressing alternate techniques to optimize the utilization of cellulolytic enzymes is one strategy to improve its efficiency and lower process costs. Cellulases are highly specific and environmentally benign biocatalysts that break down intricate polysaccharides into simple forms of sugars. In contrast to the most difficult and time-consuming enzyme immobilization processes, in this research, we studied simple, mild, and successful techniques for immobilization of pure cellulase on magnetic nanocomposites using glutaraldehyde as a linker and used in the application of sorghum residue biomass. Fe3O4 nanoparticles were coated with chitosan from the co-precipitation method, which served as an enzyme carrier. The nanoparticles were observed under XRD, Zeta Potential, FESEM, VSM, and FTIR. The size morphology results presented that the Cs@Fe3O4 have 42.2 nm, while bare nanoparticles (Fe3O4) have 31.2 nm in size. The pure cellulase reaches to 98.07% of loading efficiency and 71.67% of recovery activity at optimal conditions. Moreover, immobilized enzyme's pH stability, thermostability, and temperature tolerance were investigated at suitable conditions. The kinetic parameters of free and immobilized enzyme were estimated as Vmax; 29 ± 1.51 and 27.03 ± 2.02 µmol min-1 mg-1, Km; 4.7 ± 0.49 mM and 2.569 ± 0.522 mM and Kcat; 0.13 s-1, and 0.89 s-1. Sorghum residue was subjected to 2% NaOH pre-treatment at 50 â„ƒ. Pre-treated biomass contains cellulose of 64.8%, used as a raw material to evaluate the efficiency of reducing sugar during hydrolysis and saccharification of free and immobilized cellulase, which found maximum concentration of glucose 5.42 g/L and 5.12 g/L on 72 h. Thus, our study verifies the use of immobilized pure cellulase to successfully hydrolyze raw material, which is a significant advancement in lignocellulosic biorefineries and the reusability of enzymes.

Various Strategies for the Immobilization of a Phospholipase C from Bacillus cereus for the Modulation of Its Biochemical Properties.

In this study, the effect of various immobilization methods on the biochemical properties of phospholipase C (PLC) from Bacillus cereus obtained from the oily soil located in Sfax, Tunisia, was described. Different supports were checked: octyl sepharose, glyoxyl agarose in the presence of N-acetyl cysteine, and Q-sepharose. In the immobilization by hydrophobic adsorption, a hyperactivation of the PLCBc was obtained with a fold of around 2 times. The recovery activity after immobilization on Q-sepharose and glyoxyl agarose in the presence of N-acetyl cysteine was 80% and 58%, respectively. Furthermore, the biochemical characterization showed an important improvement in the three immobilized enzymes. The performance of the various immobilized PLCBc was compared with the soluble enzyme. The derivatives acquired using Q-sepharose, octyl sepharose, and glyoxyl agarose were stable at 50 °C, 60 °C, and 70 °C. Nevertheless, the three derivatives were more stable in a large range of pH than the soluble enzyme. The three derivatives and the free enzyme were stable in 50% (v/v) ethanol, hexane, methanol, and acetone. The glyoxyl agarose derivative showed high long-term storage at 4 °C, with an activity of 60% after 19 days. These results suggest the sustainable biotechnological application of the developed immobilized enzyme.

Integrated Process for Schizochytrium Oil Extraction, Enzymatic Modification of Lipids and Concentration of DHA Fatty Acid Esters Using Alternative Methodologies.

Marine microalgae Schizochytrium sp. have a high content of docosahexaenoic acid (DHA), an omega-3 fatty acid that is attracting interest since it prevents certain neurodegenerative diseases. The obtention of a bioactive and purified DHA fatty acid ester using a whole-integrated process in which renewable sources and alternative methodologies are employed is the aim of this study. For this reason, lyophilized Schizochytrium biomass was used as an alternative to fish oil, and advanced extraction techniques as well as enzymatic modification were studied. Microalgal oil extraction was optimized via a surface-response method using pressurized liquid extraction (PLE) obtaining high oil yields (29.06 ± 0.12%) with a high concentration of DHA (51.15 ± 0.72%). Then, the enzymatic modification of Schizochytrium oil was developed by ethanolysis using immobilized Candida antarctica B lipase (Novozym® 435) at two reaction temperatures and different enzymatic loads. The best condition (40 °C and 200 mg of lipase) produced the highest yield of fatty acid ethyl ester (FAEE) (100%) after 8 h of a reaction attaining a cost-effective and alternative process. Finally, an enriched and purified fraction containing DHA-FAEE was obtained using open-column chromatography with a remarkably high concentration of 93.2 ± 1.3% DHA. The purified and bioactive molecules obtained in this study can be used as nutraceutical and active pharmaceutical intermediates of marine origin.

A magnetic beads-based ligand fishing method for rapid discovery of monoterpene indoles as monoamine oxidase A inhibitors from Hunteria zeylanica.

In this study, a novel magnetic bead-based ligand fishing method was developed for rapid discovery of monoterpene indoles as monoamine oxidase A inhibitors from natural products. In order to improve the screening efficiency, two different magnetic beads, i.e. amine and carboxyl terminated magnetic beads, were comprehensively compared in terms of their ability to immobilize monoamine oxidase A (MAOA), biocatalytic activity and specific adsorption rates for affinity ligands. Carboxyl terminated magnetic beads performed better for MAOA immobilization and demonstrated superior performance in ligand fishing. The MAOA immobilized magnetic beads were applied to screen novel monoamine oxidase inhibitors in an alkaloid-rich plant, Hunteria zeylanica. Twelve MAOA affinity ligands were screened out, and ten of them were identified as monoterpene indole alkaloids by HPLC-Obitrap-MS/MS. Among them, six ligands, namely geissoschizol, vobasinol, yohimbol, dihydrocorynanthenol, eburnamine and (+)-isoeburnamine which exhibited inhibitory activity against MAOA with low IC50 values. To further explore their inhibitory mechanism, enzyme kinetic analysis and molecular docking studies were conducted.

Immobilized lipase enzyme on green synthesized magnetic nanoparticles using Psidium guava leaves for dye degradation and antimicrobial activities.

Zinc ferrite nanoparticles (ZnF NPs) were synthesized by a green method using Psidium guava Leaves extract and characterized via structural and optical properties. The surface of ZnF NPs was stabilized with citric acid (CA) by a direct addition method to obtain (ZnF-CA NPs), and then lipase (LP) enzyme was immobilized on ZnF-CA NPs to obtain a modified ZnF-CA-LP nanocomposite (NCs). The prepared sample's photocatalytic activity against Methylene blue dye (MB) was determined. The antioxidant activity of ZnF-CA-LP NCs was measured using 1,1-diphenyl-2-picryl hydrazyl (DPPH) as a source of free radicals. In addition, the antibacterial and antibiofilm capabilities of these substances were investigated by testing them against gram-positive Staphylococcus aureus (S. aureus ATCC 25923) and gram-negative Escherichia coli (E. coli ATCC 25922) bacterial strains. The synthesized ZnF NPs were discovered to be situated at the core of the material, as determined by XRD, HRTEM, and SEM investigations, while the CA and lipase enzymes were coated in this core. The ZnF-CA-LP NCs crystallite size was around 35.0 nm at the (311) plane. Results obtained suggested that 0.01 g of ZnF-CA-LP NCs achieved 96.0% removal of 5.0 ppm of MB at pH 9.0. In-vitro zone of inhibition (ZOI) and minimum inhibitory concentration (MIC) results verified that ZnF-CA-LP NCs exhibited its encouraged antimicrobial activity against S. aureus and E. coli (20.0 ± 0.512, and 27.0 ± 0.651 mm ZOI, respectively) & (1.25, and 0.625 µg/ml MIC, respectively). ZnF-CA-LP NPs showed antibiofilm percentage against S. aureus (88.4%) and E. coli (96.6%). Hence, ZnF-CA-LP NCs are promising for potential applications in environmental and biomedical uses.

Colorimetric and fluorometric determination of uric acid by a suspension-based assay using enzyme-immobilized micro-sized particles.

Daily monitoring of serum uric acid levels is very important to provide appropriate treatment according to the constitution and lifestyle of individual hyperuricemic patients. We have developed a suspension-based assay to measure uric acid by adding a sample solution to the suspension containing micro-sized particles immobilized on uricase and horseradish peroxidase (HRP). In the proposed method, the mediator reaction of uricase, HRP, and uric acid produces resorufin from Amplex red. This resorufin is adsorbed onto enzyme-immobilized micro-sized particles simultaneously with its production, resulting in the red color of the micro-sized particles. The concentration of resorufin on the small surface area of the microscopic particles achieves a colorimetric analysis of uric acid with superior visibility. In addition, ethanol-induced desorption of resorufin allowed quantitative measurement of uric acid using a 96-well fluorescent microplate reader. The limit of detection (3σ) and RSD (n = 3) were estimated to be 2.2 × 10-2 µg/mL and ≤ 12.1%, respectively. This approach could also be applied to a portable fluorometer.

Encapsulation of an enzyme-immobilized smart polymer membrane in a metal-organic framework for enhancement of catalytic performance.

Encapsulation of enzymes within porous materials has shown great promise for protecting enzymes from denaturation, increasing their tolerance to harsh environments and promoting their industrialization. However, controlling the conformational freedom of the encapsulated enzymes to enhance their catalytic performance remains a great challenge. To address this issue, herein, following immobilization of GOx and HRP on a thermo-responsive porous poly(styrene-maleic-anhydride-N-isopropylacrylamide) (PSMN) membrane, a GOx-HRP@PSMN@HZIF-8 composite was fabricated by encapsulating GOx-HRP@PSMN in hollow ZIF-8 (HZIF-8) with liposome (L) as the sacrificial template. The improved conformational freedom for enzymes arising from the hollow cavity formed in ZIF-8 through the removal of L enhanced the mass transfer and dramatically promoted the catalytic activity of the composite. Interestingly, at high temperature, the coiled PN moiety in PSMN provided the confinement effect for GOx-HRP, which also significantly boosted the catalytic performance of the composites. Compared to the maximum catalytic reaction rates (Vmax) of GOx-HRP@PSMN@LZIF-8, the free enzyme and GOx-HRP@ZIF-8, the Vmax of the GOx-HRP@PSMN@HZIF-8 composite exhibited an impressive 17.8-fold, 10.8-fold and 6.0-fold enhancement at 37 °C, respectively. The proposed composites successfully demonstrated their potential as catalytic platforms for the colorimetric detection of glucose in a cascade reaction. This study paves a new way for overcoming the current limitations of immobilizing enzymes in porous materials and the use of smart polymers for the potential fabrication of enzyme@polymer@MOF composites with tunable conformational freedom and confinement effect.

DNA Framework-Programmed Nanoscale Enzyme Assemblies.

Multienzyme assemblies mediated by multivalent interaction play a crucial role in cellular processes. However, the three-dimensional (3D) programming of an enzyme complex with defined enzyme activity in vitro remains unexplored, primarily owing to limitations in precisely controlling the spatial topological configuration. Herein, we introduce a nanoscale 3D enzyme assembly using a tetrahedral DNA framework (TDF), enabling the replication of spatial topological configuration and maintenance of an identical edge-to-edge distance akin to natural enzymes. Our results demonstrate that 3D nanoscale enzyme assemblies in both two-enzyme systems (glucose oxidase (GOx)/horseradish peroxidase (HRP)) and three-enzyme systems (amylglucosidase (AGO)/GOx/HRP) lead to enhanced cascade catalytic activity compared to the low-dimensional structure, resulting in ∼5.9- and ∼7.7-fold enhancements over homogeneous diffusional mixtures of free enzymes, respectively. Furthermore, we demonstrate the enzyme assemblies for the detection of the metabolism biomarkers creatinine and creatine, achieving a low limit of detection, high sensitivity, and broad detection range.

Catalytic Synthesis of (S)-CHBE by Directional Coupling and Immobilization of Carbonyl Reductase and Glucose Dehydrogenase.

Ethyl (S)-4-chloro-3-hydroxybutyrate ((S)-CHBE) is an important chiral intermediate in the synthesis of the cholesterol-lowering drug atorvastatin. Studying the use of SpyTag/SpyCatcher and SnoopTag/SnoopCatcher systems for the asymmetric reduction reaction and directed coupling coenzyme regeneration is practical for efficiently synthesizing (S)-CHBE. In this study, Spy and Snoop systems were used to construct a double-enzyme directed fixation system of carbonyl reductase (BsCR) and glucose dehydrogenase (BsGDH) for converting 4-chloroacetoacetate (COBE) to (S)-CHBE and achieving coenzyme regeneration. We discussed the enzymatic properties of the immobilized enzyme and the optimal catalytic conditions and reusability of the double-enzyme immobilization system. Compared to the free enzyme, the immobilized enzyme showed an improved optimal pH and temperature, maintaining higher relative activity across a wider range. The double-enzyme immobilization system was applied to catalyze the asymmetric reduction reaction of COBE, and the yield of (S)-CHBE reached 60.1% at 30 °C and pH 8.0. In addition, the double-enzyme immobilization system possessed better operational stability than the free enzyme, and maintained about 50% of the initial yield after six cycles. In summary, we show a simple and effective strategy for self-assembling SpyCatcher/SnoopCatcher and SpyTag/SnoopTag fusion proteins, which inspires building more cascade systems at the interface. It provides a new method for facilitating the rapid construction of in vitro immobilized multi-enzyme complexes from crude cell lysate.

A Study on the Efficient Preparation of α-Ketoglutarate with L-Glutamate Oxidase.

Alpha-ketoglutaric acid (α-KG), as an intermediate product of the tricarboxylic acid cycle, plays a crucial role in peptide and amino acid synthesis. In order to reduce costs and improve efficiency in the oxidative production of α-ketoglutaric acid, this study successfully synthesized and expressed L-glutamate oxidase (LGOXStr) from Streptomyces viridosporus R111 and catalase (KatGEsc) from Escherichia coli H736. Two immobilization methods and the conditions for one-step whole-cell catalysis of α-ketoglutaric acid were investigated. α-Ketoglutaric acid has broad applications in the pharmaceutical, food, and chemical industries. The specific research results are as follows: (1) By fusing the sfGFP tag, L-glutamate oxidase (LGOXStr r) and catalase (KatGEsc) were successfully anchored to the outer membrane of Escherichia coli cells, achieving one-step whole-cell catalysis of α-ketoglutaric acid with a conversion efficiency of up to 75%. (2) Through the co-immobilization of LGOXStr and KatGEsc, optimization of the preparation parameters of immobilized cells, and exploration of the immobilization method using E.coli@ZIF-8, immobilized cells with conversion rates of over 60% were obtained even after 10 cycles of reuse. Under the optimal conditions, the production rate of α-ketoglutaric acid reached 96.7% in a 12 h reaction, which is 1.1 times that of E. coli@SA and 1.29 times that of free cells.

Alloyed Trimetallic Nanocomposite as an Efficient and Recyclable Solid Matrix for Ideonella sakaiensis Lipase Immobilization.

In this work, a trimetallic (Ni/Co/Zn) organic framework (tMOF), synthesized by a solvothermal method, was calcinated at 400 and 600 °C and the final products were used as a support for lipase immobilization. The material annealed at 400 °C (Ni-Co-Zn@400) had an improved surface area (66.01 m2/g) and pore volume (0.194 cm3/g), which showed the highest enzyme loading capacity (301 mg/g) with a specific activity of 0.196 U/mg, and could protect the enzyme against thermal denaturation at 65 °C. The optimal pH and temperature for the lipase were 8.0 and 45 °C but could tolerate pH levels 7.0-8.0 and temperatures 40-60 °C. Moreover, the immobilized enzyme (Ni-Co-Zn@Lipase, Ni-Co-Zn@400@Lipase, or Ni-Co-Zn@600@Lipase) could be recovered and reused for over seven cycles maintaining 80, 90, and 11% of its original activity and maintained a residual activity >90% after 40 storage days. The remarkable thermostability and storage stability of the immobilized lipase suggest that the rigid structure of the support acted as a protective shield against denaturation, while the improved pH tolerance toward the alkaline range indicates a shift in the ionization state attributed to unequal partitioning of hydroxyl and hydrogen ions within the microenvironment of the active site, suggesting that acidic residues may have been involved in forming an enzyme-support bond. The high enzyme loading capacity, specific activity, encouraging stability, and high recoverability of the tMOF@Lipase indicate that a multimetallic MOF could be a better platform for efficient enzyme immobilization.