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Multienzyme cascades (MECs) have gained much attention in synthetic chemistry but remain far from being a reliable synthetic tool. Here we report a four‐enzyme cascade comprising a cofactor‐independent and a cofactor self‐sustaining bienzymatic modules for the enantioselective benzylic C−H amination of arylalkanes, a challenging transformation from bulk chemicals to high value‐added chiral amines. The two modules were subsequently optimized by enzyme co‐immobilization with microenvironmental tuning, and finally integrated in a gas‐liquid segmented flow system, resulting in simultaneous improvements in enzyme performance, mass transfer, system compatibility, and productivity. The flow system enabled continuous C−H amination of arylalkanes (up to 100 mM) utilizing the sole cofactor NADH (0.5 mM) in >90 % conversion, achieving a high space‐time yield (STY) of 3.6 g ⋅ L−1 ⋅ h−1, which is a 90‐fold increase over the highest value previously reported. [ABSTRACT FROM AUTHOR]

The aqueous reprocessing of spent nuclear fuel generates a considerable amount of plutonium alongside contaminated stainless steel, necessitating meticulous handling for safe decommission and long‐term management. The present work investigated the co‐immobilization of CeO2 (as an inactive surrogate for PuO2) with metallic Fe and Cr (representing a simplified stainless steel) within a zirconolite ceramic wasteform, nominally targeting Ca1−xCexZrTi2−2xFexCrxO7. After sintering at 1400°C under an air atmosphere, the zirconolite phase constituted 90.8–95.1 wt.% of the product across the solid solution range of 0.05 ≤ x ≤ 0.25, alongside perovskite and baddeleyite secondary phases in varying proportion; no evidence of the unincorporated CeO2 or metallic and oxidized Fe or Cr were identified. Above x = 0.30 CeO2 was detected inferring, the solubility limit was reached. A polytype transformation from zirconolite 2M to 3T was confirmed by X‐ray diffraction and selected area electron diffraction results, with the relative fraction of the 3T phase gradually increasing to 49.5 wt.% at x = 0.30. Deconvolution of X‐ray photoelectron spectroscopy data revealed the partial reduction of Ce4+–Ce3+, whereas Fe and Cr species maintained trivalent, in agreement with the targeted substitution scheme. Benefitting from the excellent chemical flexibility of zirconolite structured compounds, the co‐immobilization approach may be an effective disposal pathway for Pu‐containing wastes and contaminated stainless steel residues. [ABSTRACT FROM AUTHOR]

Abstract Reductive amination by amine dehydrogenases is a green and sustainable process that produces only water as the by-product. In this study, a continuous flow process was designed utilizing a packed bed reactor filled with co-immobilized amine dehydrogenase wh84 and glucose dehydrogenase for the highly efficient biocatalytic synthesis of chiral amino alcohols. The immobilized amine dehydrogenase wh84 exhibited better thermo-, pH and solvent stability with high activity recovery. (S)-2-aminobutan-1-ol was produced in up to 99% conversion and 99% ee in the continuous flow processes, and the space-time yields were up to 124.5 g L-1 d-1. The continuous reactions were also extended to 48 h affording up to 91.8% average conversions. This study showcased the important potential to sustainable production of chiral amino alcohols in continuous flow processes.

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Reductive amination by amine dehydrogenases is a green and sustainable process that produces only water as the by-product. In this study, a continuous flow process was designed utilizing a packed bed reactor filled with co-immobilized amine dehydrogenase wh84 and glucose dehydrogenase for the highly efficient biocatalytic synthesis of chiral amino alcohols. The immobilized amine dehydrogenase wh84 exhibited better thermo-, pH and solvent stability with high activity recovery. (S)-2-aminobutan-1-ol was produced in up to 99% conversion and 99% ee in the continuous flow processes, and the space-time yields were up to 124.5 g L-1 d-1. The continuous reactions were also extended to 48 h affording up to 91.8% average conversions. This study showcased the important potential to sustainable production of chiral amino alcohols in continuous flow processes. [ABSTRACT FROM AUTHOR]

Rivastigmine is one of the several pharmaceuticals widely prescribed for the treatment of Alzheimer's disease. However, its practical synthesis still faces many issues, such as the involvement of toxic metals and harsh reaction conditions. Herein, we report a chemo‐enzymatic synthesis of Rivastigmine. The key chiral intermediate was synthesized by an engineered alcohol dehydrogenase from Lactobacillus brevis (LbADH). A semi‐rational approach was employed to improve its catalytic activity and thermal stability. Several LbADH variants were obtained with a remarkable increase in activity and melting temperature. Exploration of the substrate scope of these variants demonstrated improved activities toward various ketones, especially acetophenone analogs. To further recycle and reuse the biocatalyst, one LbADH variant and glucose dehydrogenase were co‐immobilized on nanoparticles. By integrating enzymatic and chemical steps, Rivastigmine was successfully synthesized with an overall yield of 66 %. This study offers an efficient chemo‐enzymatic route for Rivastigmine and provides several efficient LbADH variants with a broad range of potential applications. [ABSTRACT FROM AUTHOR]

The growing need in the current market for innovative solutions to obtain lactose-free (L-F) milk is caused by the annual increase in the prevalence of lactose intolerance inside as well as the newborn, children, and adults. Various configurations of enzymes can yield two distinct L-F products: sweet (β-galactosidase) and unsweet (β-galactosidase and glucose oxidase) L-F milk. In addition, the reduction of sweetness through glucose decomposition should be performed in a one-pot mode with catalase to eliminate product inhibition caused by H2O2. Both L-F products enjoy popularity among a rapidly expanding group of consumers. Although enzyme immobilization techniques are well known in industrial processes, new carriers and economic strategies are still being searched. Polymeric carriers, due to the variety of functional groups and non-toxicity, are attractive propositions for individual and co-immobilization of food enzymes. In the presented work, two strategies (with free and immobilized enzymes; β-galactosidase NOLA, glucose oxidase from Aspergillus niger, and catalase from Serratia sp.) for obtaining sweet and unsweet L-F milk under low-temperature conditions were proposed. For free enzymes, achieving the critical assumption, lactose hydrolysis and glucose decomposition occurred after 1 and 4.3 h, respectively. The tested catalytic membranes were created on regenerated cellulose and polyamide. In both cases, the time required for lactose and glucose bioconversion was extended compared to free enzymes. However, these preparations could be reused for up to five (β-galactosidase) and ten cycles (glucose oxidase with catalase). [ABSTRACT FROM AUTHOR]

Enzymatically derived selenium-enriched peptides from Cardamine violifolia (CV) can serve as valuable selenium supplements. However, the industrial application of free enzyme is impeded by its limited stability and reusability. Herein, this study explores the application of co-immobilized enzymes (Alcalase and Dispase) on amino resin for hydrolyzing CV proteins to produce selenium-enriched peptides. The successful enzyme immobilization was confirmed through scanning electron microscopy (SEM), energy dispersive X-ray (EDX), and Fourier-transform infrared spectroscopy (FTIR). Co-immobilized enzyme at a mass ratio of 5:1 (Alcalase/Dispase) exhibited the smallest pore size (7.065 nm) and highest activity (41 U/mg), resulting in a high degree of hydrolysis of CV protein (27.2%), which was obviously higher than the case of using free enzymes (20.7%) or immobilized Alcalase (25.8%). In addition, after a month of storage, the co-immobilized enzyme still retained a viability level of 41.93%, showing fairly good stability. Encouragingly, the selenium-enriched peptides from co-immobilized enzyme hydrolysis exhibited uniform distribution of selenium forms, complete amino acid fractions and homogeneous distribution of molecular weight, confirming the practicality of using co-immobilized enzymes for CV protein hydrolysis. [ABSTRACT FROM AUTHOR]

Core-shell structured magnetic wrinkled organosilica-based metal-enzyme integrated catalysts were synthesized, and their catalytic performances were studied in the chemoenzymatic dynamic kinetic resolution of chiral amines in an organic solvent, as well as in the chemoenzymatic synthesis of chiral alcohols in water. Structure-performance studies revealed the important influence of their tunable structure and composition on the optimization of activity, stability, and recyclability in chemoenzymatic catalysis.

Multi-enzymatic strategies have shown improvement in bioconversion during cofactor regeneration. In this study, purified l-arabinitol 4-dehydrogenase (LAD) and nicotinamide adenine dinucleotide oxidase (Nox) were immobilized via individual, mixed, and sequential co-immobilization approaches on magnetic nanoparticles, and were evaluated to enhance the conversion of l-arabinitol to l-xylulose. Initially, the immobilization of LAD or Nox on the nanoparticles resulted in a maximum immobilization yield and relative activity of 91.4% and 98.8%, respectively. The immobilized enzymes showed better pH and temperature profiles than the corresponding free enzymes. Furthermore, co-immobilization of these enzymes via mixed and sequential methods resulted in high loadings of 114 and 122 mg/g of support, respectively. Sequential co-immobilization of these enzymes proved more beneficial for higher conversion than mixed co-immobilization because of better retaining Nox residual activity. Sequentially co-immobilized enzymes showed a high relative conversion yield with broader pH, temperature, and storage stability profiles than the controls, along with high reusability. To the best of our knowledge, this is the first report on the mixed or sequential co-immobilization of LAD and Nox on magnetic nanoparticles for l-xylulose production. This finding suggests that selecting a sequential co-immobilization strategy is more beneficial than using individual or mixed co-immobilized enzymes on magnetic nanoparticles for enhancing conversion applications. [ABSTRACT FROM AUTHOR]

The co-immobilized laccase-mediator system (LMS) has broad development prospects in the fields of biocatalysis, biochemistry, and environmental remediation. In this study, laccase and mediator molecules were co-immobilized in functionally modified calcium alginate microspheres to improve their catalytic activity and stability and to systematically characterize the physicochemical properties and evaluate the catalytic performance of the co-immobilized laccase-mediator system. The co-immobilized LMS was used to catalyze the synthesis of theaflavins (TFs) from tea polyphenols, improve the catalytic efficiency, realize the recycling of the system, and explore the safety of the catalytic products. The results showed that the catalytic efficiency of the co-immobilized LMS was significantly improved and the relative activity remained above 50% after 10 cycles. The in vitro safety of the catalytic product was verified by Human hepatocellular carcinoma (HepG2) cell-related indexes. The results show that the co-immobilized laccase mediator system has great development potential in the industrial application of theaflavins.

In this study, bioethanol production was compared via a novel two-stage fermentation using Zymomonas mobilis and/or Pichia stipitis with low inoculum size (5%). A synthetic glucose (30 g/l)/xylose (20 g/l) medium simulated the real hydrolyzed lignocellulose, and the inhibitory effects of different compounds and high sugar and bioethanol concentrations were restricted to find strains synergistic interactions. Higher ethanol yield and sugar conversion alongside lower time and cost were also considered. Xylose was entirely consumed by single P. stipitis only, but glucose was completely fermented in all strategies. Maximum glucose and xylose conversion efficiencies were obtained by single Z. mobilis and P. stipitis cultures, respectively. Sequential culture provided the same glucose conversion and consumption rate (100% to 1.01 gg/l.h) and yield (0.50 ge/gg) as single Z. mobilis and the nearest xylose conversion efficiency (30% to 0.14 gx/l.h to 0.16 ge/gx) to single P. stipitis (47% to 0.34 gx/l.h to 0.27 ge/gx). However, this culture was inefficient since Z. mobilis was removed. By individually immobilizing the strains on stable calcium alginate beads, the sequential-co-immobilized strategy resulted in higher xylose conversion, yield, and overall productivity of 83%, 0.45 ge/gx, and 0.30 ge/l.h than the sequential-co culture (16%, 0.31 ge/gx, and 0.14 ge/l.h), respectively. This strategy provides synergistic interactions by allocating a part of glucose to rapid growth of P. stipitis and utilizing intermediate metabolites of xylose fermentation by Z. mobilis to enhance overall efficiency. The sequential-co-immobilized strategy was also examined in 2 L bioreactor where xylose uptake rate, xylose conversion, and ethanol productivity increased 0.04 gx/l.h, 11%, and 0.27 ge/l.h compared to the sequential-co, respectively. The sequential-co-immobilized culture was found as an efficient strategy in production of second-generation bioethanol from lignocellulose. [ABSTRACT FROM AUTHOR]

Co-immobilization of laccase and mediator 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) for wastewater treatment could simultaneously achieve the reusability of laccase and avoid secondary pollution caused by the toxic ABTS. Herein, Fe-induced mineralization was proposed to co-immobilize laccase and ABTS into a metal–organic framework (ZIF-8) within 30 min. Immobilized laccase (Lac@ZIF-8-Fe) prepared at a 1:1 mass ratio of Fe2+ to Zn2+ exhibited enhanced catalytic efficiency (2.6 times), thermal stability, acid tolerance, and reusability compared to free laccase. ABTS was then co-immobilized to form Lac+ABTS@ZIF-8-Fe (ABTS = 261.7 mg/g). Lac@ZIF-8-Fe exhibited significantly enhanced bisphenol A (BPA) removal performance over free laccase due to the local substrate enrichment effect and improved enzyme stability. Moreover, the Lac+ABTS@ZIF-8-Fe exhibited higher BPA removal efficiency than the free laccase+ABTS system, implying the presence of a proximity effect in Lac+ABTS@ZIF-8-Fe. In the successive malachite green (MG) removal, the MG degradation efficiency by Lac@ZIF-8-Fe was maintained at 96.6% at the fifth reuse with only an extra addition of 0.09 mM ABTS in each cycle. As for Lac+ABTS@ZIF-8-Fe, 58.5% of MG was degraded at the fifth cycle without an extra addition of ABTS. Taken together, this research has provided a novel strategy for the design of a co-immobilized laccase and ABTS system for the degradation of organic pollutants. [ABSTRACT FROM AUTHOR]

As an efficient and safe biocatalyst, enzymes have been widely used in many fields such as food, medicine and environmental governance, but it is difficult to realize further industrial application due to the poor environmental stability of free enzymes. Enzyme immobilization technology helps to improve the tolerance of free enzymes to sensitive environments and the stability during operation, and greatly reduces the application cost. This paper reviews the development and current situation of immobilization technology in the past five years, and summarizes the research progress of different immobilization methods (including traditional immobilization methods such as adsorption method and binding method and new immobilization methods such as coimmobilization enzyme method) and immobilization carriers (including natural material carriers, composite carriers and nanocarriers) in various fields. In general, compared with free enzymes, the immobilized enzyme system has been significantly improved in terms of stability and reusability. However, there are some shortcomings, such as lower recovery rate after immobilization, cumbersome and costly carrier synthesis pathway, and imperfect mechanism of immobilization enzyme.Finally, the development direction of the technology in the future was put forward based on these shortcomings. [ABSTRACT FROM AUTHOR]

The development of technologies to ferment carbohydrates (mainly glucose and xylose) obtained from the hydrolysis of lignocellulosic biomass for the production of second-generation ethanol (2G ethanol) has many economic and environmental advantages. The pretreatment step of this biomass is industrially performed mainly by steam explosion with diluted sulfuric acid and generates hydrolysates that contain inhibitory compounds for the metabolism of microorganisms, harming the next step of ethanol production. The main inhibitors are: organic acids, furan, and phenolics. Several strategies can be applied to decrease the action of these compounds in microorganisms, such as cell immobilization. Based on data published in the literature, this overview will address the relevant aspects of cell immobilization for the production of 2G ethanol, aiming to evaluate this method as a strategy for protecting microorganisms against inhibitors in different modes of operation for fermentation. This is the first overview to date that shows the relation between inhibitors, cells immobilization, and fermentation operation modes for 2G ethanol. In this sense, the state of the art regarding the main inhibitors in 2G ethanol and the most applied techniques for cell immobilization, besides batch, repeated batch and continuous fermentation using immobilized cells, in addition to co-culture immobilization and co-immobilization of enzymes, are presented in this work. [ABSTRACT FROM AUTHOR]

Enzymatically derived selenium-enriched peptides from Cardamine violifolia (CV) can serve as valuable selenium supplements. However, the industrial application of free enzyme is impeded by its limited stability and reusability. Herein, this study explores the application of co-immobilized enzymes (Alcalase and Dispase) on amino resin for hydrolyzing CV proteins to produce selenium-enriched peptides. The successful enzyme immobilization was confirmed through scanning electron microscopy (SEM), energy dispersive X-ray (EDX), and Fourier-transform infrared spectroscopy (FTIR). Co-immobilized enzyme at a mass ratio of 5:1 (Alcalase/Dispase) exhibited the smallest pore size (7.065 nm) and highest activity (41 U/mg), resulting in a high degree of hydrolysis of CV protein (27.2%), which was obviously higher than the case of using free enzymes (20.7%) or immobilized Alcalase (25.8%). In addition, after a month of storage, the co-immobilized enzyme still retained a viability level of 41.93%, showing fairly good stability. Encouragingly, the selenium-enriched peptides from co-immobilized enzyme hydrolysis exhibited uniform distribution of selenium forms, complete amino acid fractions and homogeneous distribution of molecular weight, confirming the practicality of using co-immobilized enzymes for CV protein hydrolysis.

L -aspartate is an important chemical in the food and pharmaceutical industries. Presently, it is meaningful to realize a co-immobilized multi-enzyme biocatalyst that can synthesize L -aspartate in vitro through multi-step cascade reaction. Herein, the immobilization method was first employed to investigate the ability of immobilized double enzymes, isomerase Aa MaiA and aspartase AspB, to systhesize L -aspartate. The optimal temperature (50 ℃) of Aa MaiA-immobilized NH 2 -UiO66, a metal organic framework material, was significantly superior to that of its free counterpart. Compared to free enzymes, Aa MaiA@NH 2 -UiO66 retained 57.8% of enzyme activity and exhibited a half-life that was improved 2.59-fold at 50 ℃. The co-immobilized Aa MaiA/AspB@NH 2 -UiO66 (Aa MaiA: AspB = 6: 1, v/v) showed high protein loading (27.899 ± 1.945 mg/g) and 84.4% recovery of enzyme activity after 8 consecutive cycles. Compared to free Aa MaiA/AspB, the novel Aa MaiA/AspB@NH 2 -UiO66 exhibited far superior tolerance to organic solvents. For the first time, the prepared Aa MaiA/AspB@NH 2 -UiO66 was successfully applied as an efficient biocatalyst and converted 1 M maleate to L -aspartate with a yield of 86.8% within 1 h. Remarkably, Aa MaiA/AspB@NH 2 -UiO66 exhibited great potential for efficient biosynthesis of L -aspartate. [Display omitted] • Aa MaiA and AspB immobilized on NH 2 -UiO66 showed better thermal stability. • The conversion rate of Aa MaiA/AspB@NH 2 -UiO66 was 86.8% within 1 h. • Aa MaiA/AspB@NH 2 -UiO66 retained 84.4% initial activity after 8 consecutive cycles. • Aa MaiA/AspB@NH 2 -UiO66 exhibited good resistance against organic solvents. [ABSTRACT FROM AUTHOR]

Natural antioxidants have drawn growing interest for use in animal feed and the food industry. In the current study, essential oils (EOs) obtained from hydrodistillation of three mentha species, including Menthapiperita (peppermint), Mentha spicata (native spearmint) and Menthagracilis (Scotch spearmint), harvested in the Midwest region in the United States, were analyzed for their chemical composition using gas chromatography-mass spectrometry, and their antioxidant properties were assessed through chemical assays, in vitro cell culture modeling and in Caenorhabditis elegans (C. elegans). The activity of ferric iron reduction and free-radical scavenging capacity were assessed through chemical-based assays, including the reducing power assay, 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging assay, and Trolox equivalent antioxidant capacity assay (TEAC). Subsequently, the capacity of EOs to mitigate lipid peroxidation was analyzed at various doses using fresh liver homogenates from pigs. A porcine jejunum epithelial cell line (IPEC-J2) was employed as in vitro model to study the cellular antioxidant activity of the mint EOs. Finally, the effectiveness of mint EOs to alleviate acute systemic oxidative damage were evaluated in vivo using C. elegans. Data were analyzed by the MIXED procedure of SAS. Contrast statement was performed to assess linear or quadratic effects of mint EOs given at various doses. All three EOs are mostly composed of monoterpenes and their derivatives (76-90%), but differed in the major compounds, which are menthol and menthone (50%) in peppermint EO and carvone (70%) in spearmint EOs. Three mint EOs demonstrated prominent radical scavenging and Fe3+ reducing activity in chemical-based assays. In comparison with native and Scotch spearmint EOs, peppermint EO had the lowest (p < 0.05) half maximal effective concentration (EC50) in DPPH and TEAC assays and higher efficacy in the reducing power assay. All three EOs exhibited equivalent activity in mitigation of chemical-induced lipid peroxidation in liver tissues in a dose-dependent manner (linear, p < 0.001). The maximal cellular antioxidant activity (CAA) was observed at 5 µg/mL for peppermint, and 100 µg/mL for native and Scotch spearmint EOs. The addition of 25 µg/mL of both spearmint EOs increased (p < 0.05) cellular concentrations of glutathione in H2O2-treated IPEC-J2 cells, suggesting enhanced endogenous antioxidant defense. Supplementation of 100 µg/mL of peppermint or Scotch spearmint EO significantly increased (p < 0.05) the survival rate of C. elegans in response to H2O2-induced oxidative stress. The protective effect is comparable to that of supplementation of 10 µg/mL of ascorbic acid. However native spearmint EO failed to reduce the death rate within the same supplementation dose (10-200 μg/mL).

Lipase-catalyzed transesterification is a promising and sustainable approach to producing biodiesel. To achieve highly efficient conversion of heterogeneous oils, combining the specificities and advantages of different lipases is an attractive strategy. To this end, highly active Thermomyces lanuginosus lipase (1,3-specific) and stable Burkholderia cepacia lipase (non-specific) were covalently co-immobilized on 3-glycidyloxypropyltrimethoxysilane (3-GPTMS) modified Fe3O4 magnetic nanoparticles (co-BCL-TLL@Fe3O4). The co-immobilization process was optimized using response surface methodology (RSM). The obtained co-BCL-TLL@Fe3O4 exhibited a significant improvement in activity and reaction rate compared with mono and combined-use lipases, achieving 92.9% yield after 6 h under optimal conditions, while individually immobilized TLL, immobilized BCL and their combinations exhibited yields of 63.3%, 74.2% and 70.6%, respectively. Notably, co-BCL-TLL@Fe3O4 achieved 90–98% biodiesel yields after 12 h using six different feedstocks, demonstrating the perfect synergistic effect of BCL and TLL remarkably motivated in co-immobilization. Furthermore, co-BCL-TLL@Fe3O4 could maintain 77% of initial activity after nine cycles by removing methanol and glycerol from catalyst surface, accomplished by washing with t-butanol. The high catalytic efficiency, wide substrate adaptability and favorable reusability of co-BCL-TLL@Fe3O4 suggest that it will be an economical and effective biocatalyst for further applications. [ABSTRACT FROM AUTHOR]

Morus alba leaves are a natural product with great antidiabetic potential. However, the therapeutic efficacy of natural products is usually achieved through the interaction of active compounds with specific targets. Among them, active compounds with multi‐target therapeutic functions are more effective than single‐target enzymes. In this study, a bienzyme system was constructed by co‐immobilizing α‐amylase and α‐glucosidase onto Fe3O4 for affinity screening of dual‐target active components in the complex extract from M. alba leaves. As a result, a potential active compound was selectively screened by ligand fishing, separated by high‐speed countercurrent chromatography using a solvent system of ethyl acetate–n‐butanol–water (3:2:5, v/v), and identified as rutin. In addition, the result of molecular docking showed that rutin could interact with the active center of α‐amylase and α‐glucosidase through multiple hydrogen bonds, van der Waals forces, etc. to play an inhibitory role. These results demonstrate the effectiveness of the polydopamine magnetically immobilized bienzyme system for dual‐target affinity screening of active substances. This study not only reveals the chemical basis of the antidiabetic activity of M. alba leaves from a dual‐target perspective, but also promotes the progress of multitarget affinity screening. [ABSTRACT FROM AUTHOR]

[Display omitted] • A bacterium-microalgae consortium co-immobilized filter had superior LIN efficiency. • The filter removed 98.54 % of LIN within 7 days. • The main LIN removed mechanism of the BMC system was bio-degradation. • LIN degraded products were less toxic than their substrate. • Eco-toxicity of ARGs was alleviated by the carriers' absortion. Lincomycin (LIN) in pharmaceutical wastewater would enter municipal wastewater treatment plants and decrease their performance, leading to residual LIN enter the natural environment and pose serious eco-risk. In this study, a bacterium-microalgae consortium co-immobilized filter (BMCCF) was established and used to remove LIN in artificial pharmaceutical wastewater treatment plants effluents (PWWTPE). LIN removal mechanisms and degradation products' eco-toxicity was studied, and the abundance change of class 1 integrase gene (intI1) and antibiotic resistance genes (ARGs) was monitored. As a result, 98.54% of 82 mg L-1 LIN was removed within 7 days, LIN removal was mainly attributed to bio-degradation by the Bacillus subtilis strain, and LIN degradation products were less toxic than their substrate. Therefore, the BMCCF established in this study provides a promising alternative for the bio-treatment of pharmaceutical wastewater containing high concentration of LIN. [ABSTRACT FROM AUTHOR]

In this study, we endeavored to catalyze the biosynthesis of D -phenyllactic (D-PLA) from L-Phenylalanine (L-Phe) through a one-pot method. However, the crucial enzymes for the biosynthesis of phenylpyruvate (PPA), amino acid oxidase (L-AAD), is a membrane-bound protein. Herein, we proposed a novel co-immobilization strategy of whole cells and enzymes, integrating them into ZIF-90 to achieve efficient biosynthesis of D-PLA. Consequently, we embarked on integrating both enzyme and E. coli into ZIF-90, ultimately obtaining the novel biocatalyst E. coli /LDH@ZIF-90. This achievement facilitated the cascade reaction between LDH and E. coli , enabling a streamlined one-pot bioconversion process. The morphology and structure of E. coli /LDH@ZIF-90 were thoroughly characterized using a range of methods, including XRD, SEM, FT-IR, CLSM, and XPS, which confirmed that the material had been successfully synthesized. Further activity experiments revealed that E. coli /LDH@ZIF-90 exhibited good stability even under harsh conditions. Additionally, the biocatalyst retained 76 % of its initial catalytic activity after completing six cycles. Moreover, when utilized for the biosynthesis of D-PLA, this system demonstrated an impressive conversion rate of 85.2 % after 12 h. The successful cascade catalysis from L-Phe to D-PLA underscored the potential of the enzyme-cell cascade catalytic system, offering valuable insights for its potential industrial applications. [ABSTRACT FROM AUTHOR]

To achieve an efficient and cost-effective conversion from waste cooking oil (WCO) to biodiesel, a facile one-pot co-immobilization of Aspergillus oryzae lipase (AOL) and Rhizomucor miehei lipase (RML) was developed by rapid encapsulation of magnetic chitosan microcapsules. The encapsulation process was accomplished by self-assembly of negatively charged magnetic nanoparticles (citrate modified Fe 3 O 4) on ionic cross-linked chitosan-citrate aggregates containing lipases. Integrating Tween 80 bio-imprinting of lipases, the co-immobilized AOL@RML (co-im Tween 80-AOL@RML) achieved a significant increase in activity and reached 98.5 % biodiesel yield under optimal conditions, which was 1.3 and 1.6 times higher than that of single immobilized AOL and RML, respectively. Notably, owing to the beneficial synergistic impact of methanol-tolerable AOL and thermal-stable RML, co-im Tween 80-AOL@RML displayed superior activity over single lipase counterparts under excess methanol and variable temperatures. The co-im Tween 80-AOL@RML could maintained 96 % activity after 3 cycles of biodiesel production; however, showed an obvious inactivation after continued reuse. Moreover, methanol-induced denaturation of lipases and glycerol-caused agglomeration of the magnetic nanoparticles during continued transesterification were ascertained by fourier transform infrared analysis and scanning electron microscopy. This study provides new pathways for constructing multi-lipase co-immobilzation as well as an economical and effective biocatalyst for biodiesel production from WCO. [Display omitted] • Lipase from Aspergillus oryzae and Rhizomucor miehei were co-immobilized. • The one-pot encaplsulation of magnetic microcapsules can be completed within 30 min. • The co-immobilized lipases catalyzed biodiesel production from waste cooking oil with a yield of 98.5 %. • Co-immobilization and bio-imprinting could increase tolerance to heat and methanol. • Structure alterations of lipase and magnetic microcapsules caused inactivation during repeated use. [ABSTRACT FROM AUTHOR]

Here, we present an immobilized enzyme cascade in a basket-type reactor allowing a one-pot, two-step enzymatic synthesis of L-erythrulose from D-serine and glycolaldehyde. Three enzymes, D-amino acid oxidase from Rhodotorula gracilis (DAAORg), catalase from bovine liver (CAT), and transketolase from Geobacillus stearothermophilus (TKgst) were covalently immobilized on silica monolithic pellets, characterized by an open structure of interconnected macropores and a specific surface area of up to 300 m2/g. Three strategies were considered: (i) separate immobilization of enzymes on silica supports ([DAAO][CAT][TK]), (ii) co-immobilization of two of the three enzymes followed by the third ([DAAO+CAT][TK]), and (iii) co-immobilization of all three enzymes ([DAAO+CAT+TK]). The highest L-erythrulose concentrations were observed for the co-immobilization protocols (ii) and (iii) (30.7 mM and 29.1 mM, respectively). The reusability study showed that the best combination was [DAAO + CAT][TK], which led to the same level of L-erythrulose formation after two reuse cycles. The described process paves the way for the effective synthesis of a wide range of α-hydroxyketones from D-serine and suitable aldehydes. [ABSTRACT FROM AUTHOR]

Due to a surge in demand and cost, there is a requirement for alternative energy sources by devising strategies for the efficient production of biofuels. Immobilized microbial systems for the conversion of biomass to fuels have become progressively important. Cassava (Manihot esculentum) processing waste, a massive byproduct of starch processing is utilized in this work for bioethanol production. This study was an attempt to design and develop an experimental lab-scale Vertical Mass-Flow type Bioreactor (VMFB) demarcated into aerobic and anaerobic zones to produce bioethanol. The upper aerobic zone was meant for saccharification and the lower anaerobic zone for fermentation, the technique is called Simultaneous Saccharification and Fermentation (SSF). The feasibility of co-immobilizing saccharification strains (A. awamori and D. bruxellensis) and fermentation strains (Z. mobilis) for bioethanol production through SSF from cassava agro-waste were tested. Polyurethane foam was used in the aerobic reaction zone and calcium-alginate beads immobilized microorganisms in the anaerobic reaction zone were employed as carriers for the immobilization. The main objective of this study was to understand the usability of agricultural waste, especially cassava processing waste as raw material for bioethanol production, using SSF technology a concentration of 8 % w/w ethanol was obtained. [ABSTRACT FROM AUTHOR]

Biohydrogen is a promising clean energy. Multiple researchers have successfully increased hydrogen production by augmenting nanomaterials with anaerobic hydrogen-producing microorganisms. Herein, a novel approach is developed in which a pure strain of Clostridium pasteurianum CH5 is co-immobilized with carboxylic-acid-functionalized multiwalled carbon nanotubes (MWCNT-COOH). The direct co-immobilization of C. pasteurianum CH5 with 800-mg/L MWCNT-COOH improves the hydrogen yield (HY) to 2.43 mol H2/mol glucose. When C. pasteurianum CH5 and 800 mg/L MWCNT-COOH are preincubated for 24 h, the HY increases to 2.38 mol H2/mol glucose, a 46.9% increase relative to the control (without co-immobilization) and a 133.3% increase relative to the conventional suspended growth of C. pasteurianum CH5 mixed with the same concentration of MWCNT-COOH. These results indicate that bringing nanomaterials into closer contact with microorganisms can serve as a feasible and simple approach for biohydrogen production. [Display omitted] • Bacteria grew successfully within the tested polyvinyl alcohol–boric gel granules. • Immobilization of microorganisms with MWCNT-COOH remarkably improved H 2 production. • Preincubation of microorganisms with MWCNT-COOH is recommended. • Application of 800 mg/mL MWCNT-COOH improved the H 2 production rate by 46.9%. [ABSTRACT FROM AUTHOR]

The g-C3N4/CF composite carrier was prepared by ultrasound-assisted maceration and high-temperature calcination. The enzyme immobilization using the g-C3N4/CF as the novel carrier to immobilize lactase and glucose isomerase was enhanced for lactulose production. The carbon fiber (CF) was mixed with melamine powder in the mass ratio of 1:8. The g-C3N4/CF composite carrier was obtained by calcination at 550 °C for 3 h. After the analysis of characteristics, the g-C3N4/CF was successfully composited with the carbon nitride and CF, displaying the improvement of co-immobilization efficiency with the positive effects on the stability of the enzyme. The immobilization efficiency of the co-immobilized enzyme was 37% by the novel carrier of g-C3N4/CF, with the enzyme activity of 13.89 U g−1 at 60 °C. The relative activities of co-immobilized enzymes maintained much more steadily at the wider pH and higher temperature than those of the free dual enzymes, respectively. In the multi-batches of lactulose production, the relative conversion rates in enzymes co-immobilized by the composite carrier were higher than that of the free enzymes during the first four batches, as well as maintaining about a 90% relative conversation rate after the sixth batch. This study provides a novel method for the application of g-C3N4/CF in the field of immobilizing enzymes for the production of lactulose. [ABSTRACT FROM AUTHOR]

Enzymes have several excellent catalytic features, and the last few years have seen a revolution in biocatalysis, which has grown from using one enzyme to using multiple enzymes in cascade reactions, where the product of one enzyme reaction is the substrate for the subsequent one. However, enzyme stability remains an issue despite the many benefits of using enzymes in a catalytic system. When enzymes are exposed to harsh process conditions, deactivation occurs, which changes the activity of the enzyme, leading to an increase in reaction time to achieve a given conversion. Immobilization is a well-known strategy to improve many enzyme properties, if the immobilization is properly designed and controlled. Enzyme co-immobilization is a further step in the complexity of preparing a biocatalyst, whereby two or more enzymes are immobilized on the same particle or support. One crucial problem when designing and using co-immobilized enzymes is the possibility of using enzymes with very different stabilities. This paper discusses different scenarios using two co-immobilized enzymes of the same or differing stability. The effect on operational performance is shown via simple simulations using Michaelis–Menten equations to describe kinetics integrated with a deactivation term. Finally, some strategies for overcoming some of these problems are discussed. [ABSTRACT FROM AUTHOR]

Glucosylation cascade consisting of Leloir glycosyltransferase and sucrose synthase with in situ regeneration system of expensive and low available nucleotide sugars is a game-changing strategy for enzyme-based production of glycoconjugates of relevant natural products. We designed a stepwise approach including co-expression and one-step purification and co-immobilization on glass-based EziG resins of sucrose synthase from Glycine max (GmSuSy) with promiscuous glucosyltransferase YjiC from Bacillus licheniformis to produce efficient, robust, and versatile biocatalyst suited for preparative scale flavonoid glucosylation. The undertaken investigations identified optimal reaction conditions (30 °C, pH 7.5, and 10 mM Mg2+) and the best-suited carrier (EziG Opal). The prepared catalyst exhibited excellent reusability, retaining up to 96% of initial activity after 12 cycles of reactions. The semi-preparative glucosylation of poorly soluble isoflavone Biochanin A resulted in the production of 73 mg Sissotrin (Biochanin A 7-O-glucoside). Additionally, the evaluation of the designed double-controlled, monocistronic expression system with two independently induced promoters (rhaBAD and trc) brought beneficial information for dual-expression plasmid design. Key points: • Simultaneous and titratable expression from two independent promoters is possible, although full control over the expression is limited. • Designed catalyst managed to glucosylate poorly soluble isoflavone. • The STY of Sissotrin using the designed catalyst reached 0.26 g/L∙h∙g of the resin. [ABSTRACT FROM AUTHOR]

Immobilization of microorganisms in biodegradable polymeric matrices constitutes a promising technology for plant growth promoting to overcome the challenging conditions of the rhizosphere. Previously, we demonstrated that beads prepared from blends of chitosan/starch of analytical grades ionically cross-linked are useful carriers for Azospirillum brasilense and Pseudomonas fluorescens. The aims of this work were to study A. brasilense Az39 and P. fluorescens ZME4 immobilization in industrial quality beads produced with a blend of chitosan/starch, to assess bacterial survival during long-term storage and biofilm distribution in the beads. We also proposed to analyze the consortia root colonization and its performance as plant growth–promoting bioinoculants compared to liquid counterpart. Our results revealed that A. brasilense Az39 and P. fluorescens ZME4 can coexist in industrial grade chitosan/starch beads, and this mixed immobilization benefits the survival rates of both species, even for more than a year under shelf storage. Confocal laser scanning microscopy with fluorescent dyed strains showed that both species remain mainly in different locations inside and over the beads. Additionally, maize seed treatment with beads-loaded bacteria resulted in growth promotion of roots in a similar manner than traditional liquid-based inoculation. The evidence collected here demonstrate that low-cost chitosan/starch beads are a suitable carrier for bacteria consortia and could be a reliable alternative to liquid inoculation in agronomic practices with additional benefits for industrial management. Key Points: • Mixed immobilization increases bacterial survival in chitosan/starch industrial beads • Beads increase competence of bacteria in rhizosphere of maize • Inoculation mediated by beads promotes plant growth of maize [ABSTRACT FROM AUTHOR]

The capability of multi-component reactions in rapid immobilization of enzymes was considered for co-immobilization of Thermomyces lanuginous lipase (TLL) and Candida antarctica lipase B (CALB) [TLL: CALB]; Rhizomucor miehei lipase (RML) and CALB [RML: CALB] on amine-functionalized silica-coated magnetic nanoparticles (Fe3O4@SiO2-NH2). Immobilization of different ratios of lipases was performed within 3 h under mild conditions; producing specific activity ranging from 29 to 35 U/mg for TLL:CALB and 21–34 U/mg for RML:CALB. The co-immobilized derivatives showed improved co-solvent and thermal stability compared to the corresponding free enzymes. All the derivatives were also used to catalyze the transesterification of waste cooking oil with methanol to produce biodiesel (fatty acid methyl esters). Response surface method (RSM) and a central composite rotatable design (CCRD) were used to study the effects of different factors on the FAME yield. Fe3O4@SiO2-NH2-RML-CALB and Fe3O4@SiO2-NH2-TLL-CALB had maximum FAME yields of 99–80%, respectively. [ABSTRACT FROM AUTHOR]

Metal-Organic Frameworks (MOFs) are porous crystalline materials formed by self-assembly of metal centers and organic ligands through coordination. MOFs have the characteristics of strong stability, large specific surface area, high porosity, and reusability. As a new type of immobilized carrier material, it has a wide range of application prospects. In this article, the synthesis method of biological enzyme immobilized on MOFs carrier is mainly summarized and analyzed, the characteristics and application of MOFs-biological enzyme composite material, and the future research direction of the synthetic method of enzyme immobilized on MOFs carrier are prospected. A theoretical basis would be provided for the development research and applications in the biological and pharmaceutical industries.

The artificial cascade system with a coenzyme-independent decarboxylase and oxygenase was a very attractive approach for bioconversion of ferulic acid into vanillin. However, its potential was impeded by the low activity and thermostability of oxygenase. In this study, a combined strategy including the protein engineering of a carotenoid cleavage oxygenase from Thielavia terrestris (Tt CCO) and its linker-mediated co-immobilization with a decarboxylase from Bacillus atrophaeus (Ba PAD) was explored with aims to improve the catalytic efficiency and robustness of this artificial cascade system. We proved that engineering the enzyme by inserting an extra short peptide between E450 to E476 greatly affected Tt CCO substrate specificity, catalytic activity, and thermostability. Among three variants, Tt CCO2 displayed better thermostability and had 1.4-, 55- and 1.3-fold higher activity than Tt CCO on 4-vinylguaiacol, resveratrol, and isoeugenol, respectively. The excellent protein and enzyme activity yield of immobilization was achieved by linker-mediated immobilization of 4lp- Tt CCO2 @ mesoporous Y zeolite. A 100% conversion rate of ferulic acid and a 55% of the molar conversion yield of vanillin were obtained by co-immobilized 4lp- Tt CCO2/4lp- Ba PAD@mesoporous Y zeolite. This work demonstrated an effective engineering strategy for more efficient and robust oxygenases and the potential of this bi-enzyme cascade system for converting ferulic acid into vanillin. [Display omitted] • A new carotenoid cleavage oxygenase (Tt CCO) was identified from Thielavia terrestris. • A more efficient and robust oxygenase was engineered by inserting a short peptide. • High protein and activity yield were achieved by linker-mediated immobilization. • High molar conversion yield was obtained by co-immobilized 4lp- Tt CCO2/4lp- Ba PAD. [ABSTRACT FROM AUTHOR]

Cascading enzyme complexes can enhance cascade enzymatic activity through substrate channeling, which succeed in industrial process development, such as synthesis of pharmaceutical, cosmetic, and functional foods. The variety of reactions provided by enzyme has promoted biocatalysts been as a green economic sustainable production tool in modern synthetic chemistry. Nevertheless, co-immobilized cascading enzyme complexes are favorable to large-scale industrial manufacture. The advantages of using immobilization include not only improved cascade enzymatic activity but also enhanced enzyme stability and ease of recovery for reuse. Most noteworthily, characteristics of enzyme catalysis, immobilization methods, corresponding reaction kinetics are the key factors for activity and stability of enzyme complexes. As to sufficient coordination, the development in biology and materials science, such as the research and development of loading materials with nano characteristics and more stable properties, probably has provided for design of ideal multienzyme complexes devices. Based on different bonding methods and loading materials, recent advanced in bioreactions via co-immobilized multi-enzyme were described in this review. [ABSTRACT FROM AUTHOR]

As shown in the figure, with the increase of the amount of cross-linking agent, the relative enzyme activity of PMCSM@GOD@CAT firstly increased and then decreased, and the highest relative enzyme activity was 132.45 ± 3.35% when the amount of cross-linking agent was 200 L. When the concentration of cross-linking agent was low, the cross-linking between the enzyme and the carrier was incomplete. Keywords: catalase; co-immobilization; glucose oxidase; porous magnetic microsphere EN catalase co-immobilization glucose oxidase porous magnetic microsphere 989 1001 13 09/20/22 20220901 NES 220901 1 Introduction Sodium gluconate (SG) is rich in hydroxyl groups and is a deeply processed product of glucose. 3.4 Effect of the amount of GOD and CAT on the enzyme activity of GOD In the process of immobilization of the enzyme in the carrier, the relative amount of the enzyme and the carrier has a certain influence on the relative enzyme activity. When the pore size of the microspheres was 790.15 ± 250.91 nm, the immobilized enzyme activity reached 16.93 ± 0.14 U. At the same time, the enzyme load was the highest, which was 58.28 ± 2.64 mg/g. Different amount of GOD and CAT will affect the activity of immobilized enzyme, thus affecting the yield of SG. [Extracted from the article]

A reoccurring flaw of most yeast immobilization systems that limits the potential of the technique is leakage of the cells from the matrix. Leakage may be due to weakly adherent cells, deterioration of the matrix, or to new growth and loss of non-adherent daughter cells. Yeast biocapsules are a spontaneous, cost effective system of immobilization whereby Saccharomyces cerevisiae cells are attached to the hyphae of Penicillium chrysogenum, creating hollow spheres that allow recovery and reutilization. This attachment is based on naturally occurring adherent properties of the yeast cell surface. We hypothesized that proteins associated with flocculation might play a role in adherence to fungal hyphae. To test this hypothesis, yeast strains with overexpressed and deleted flocculation genes (FLO1, FLO5, and FLO11) were evaluated for biocapsule formation to observe the impact of gene expression on biocapsule diameter, number, volume, dry mass, and percent immobilized versus non-immobilized cells. Overexpression of all three genes enhanced immobilization and resulted in larger diameter biocapsules. In particular, overexpression of FLO11 resulted in a five fold increase of absorbed cells versus the wild type isogenic strain. In addition, deletion of FLO1 and FLO11 significantly decreased the number of immobilized yeast cells compared to the wild type BY4742. These results confirm the role of natural adherent properties of yeast cells in attachment to fungal hyphae and offer the potential to create strongly adherent cells that will produce adherent progeny thereby reducing the potential for cell leakage from the matrix.

We propose a co-immobilized chemo-enzyme cascade system to mitigate random intermediate diffusion from the mixture of individual immobilized catalysts and achieve a one-pot reaction of multi-enzyme and reductant. Catalyzed by lipase and lipoxygenase, unsaturated lipid hydroperoxides (HPOs) were synthesized. 13(S)-hydroperoxy-9Z, 11E-octadecadienoic acid (13-HPODE), one compound of HPOs, was subsequently reduced to 13(S)-hydroxy-9Z, 11E-octadecadienoic acid (13-HODE) by cysteine. Upon the optimized conditions, 75.28 mg of 13-HPODE and 4.01 mg of 13-HODE were produced from per milliliter of oil. The co-immobilized catalysts exhibited improved yield compared to the mixture of individually immobilized catalysts. Moreover, it demonstrated satisfactory durability and recyclability, maintaining a relative HPOs yield of 78.5% after 5 cycles. This work has achieved the co-immobilization of lipase, lipoxygenase and the reductant cysteine for the first time, successfully applying it to the conversion of soybean oil into 13-HODE. It offers a technological platform for transforming various oils into high-value products. [Display omitted] • The formation of metal-amino acid frameworks was facilitated by the addition of PVP and L-cysteine. • In a one-pot cascade reaction, soybean oil was successfully converted into 13-HPODE and 13-HODE. • The in-situ co-immobilization of lipase, lipoxygenase and the reductant L-cysteine has been accomplished within MOF. [ABSTRACT FROM AUTHOR]

Agarose from biomass can be used to synthesize the rare sugar 3,6-anhydro- L -galactose (L -AHG), and the new synthesis route and functional properties of L -AHG have always been the focus of research. Here we developed a novel method to co-immobilize Aga50D and Bp GH117 onto streptavidin-coated magnetic nanoparticles and achieved the conversion of agarose to bioactive L -AHG in one pot. Results showed that enzymes were successfully immobilized on the carrier. The activity of co-immobilized enzymes was 2.5-fold higher than that of single immobilized enzymes. Compared with free enzymes, co-immobilized enzymes exhibited enhanced thermal stability. The co-immobilized enzymes retained 79.45 % relative activity at 40 °C for 3 h, while the free enzymes only possessed 21.40 % residual activity. After eight cycles, the co-immobilized enzymes still retained 73.47 % of the initial activity. After silica gel chromatography, the purity of L -AHG obtained by co-immobilized enzymes hydrolysis reached 83.02 %. Furthermore, bioactivity experiments demonstrated that L -AHG displayed better antioxidant and antibacterial effects than neoagarobiose. L -AHG had broad-spectrum antibacterial activity, while neoagarobiose and D -galactose did not show an obvious antibacterial effect. This study provides a feasible method for the production of L -AHG by a co-immobilized multi-enzyme system and confirms that L -AHG plays a key role in the bioactivity of neoagarobiose. [Display omitted] • Co-mmobilization of agarases based on biotin/streptavidin affinity system • Co-immobilized enzymes have higher stability and catalytic efficiency. • One-pot conversion of agarose into bioactive 3,6-anhydro- L -galactose • 3,6-Anhydro- L -galactose displayed broad-spectrum antibacterial activity. [ABSTRACT FROM AUTHOR]

The biocatalytic system comprised of RizA and acetate kinase (AckA) combines the specific synthesis of bioactive arginyl dipeptides with efficient ATP regeneration. Immobilization of this coupled enzyme system was performed and characterized in terms of activity, specificity and reusability of the immobilisates. Co-immobilization of RizA and AckA into a single immobilisate conferred no disadvantage in comparison to immobilization of only RizA, and a small addition of AckA (20:1) was sufficient for ATP regeneration. New variants of RizA were constructed by combining mutations to yield variants with increased biocatalytic activity and specificity. A selection of RizA variants were co-immobilized with AckA and used for the production of the salt-taste enhancers Arg-Ser and Arg-Ala and the antihypertensive Arg-Phe. The best variants yielded final dipeptide concentrations of 11.3 mM Arg-Ser (T81F_A158S) and 11.8 mM Arg-Phe (K83F_S156A), the latter of which represents a five-fold increase in comparison to the wild-type enzyme. T81F_A158S retained more than 50% activity for over 96 h and K83F_S156A for over 72 h. This study provides the first example of the successful co-immobilization of an l-amino acid ligase with an ATP-regenerating enzyme and paves the way towards a bioprocess for the production of bioactive dipeptides. [ABSTRACT FROM AUTHOR]

Lipases are considered the most widely used industrial biocatalysts, and play a significant role in diverse industrial and biotechnological applications such as in biodiesel. Biodiesel is a renewable and ecologically friendly biofuel, and has received significantly increased attention in recent years. The reaction media of biodiesel synthesis via the transesterification process of fats and oils could be considered cascade reactions. The use of a mixture of immobilized lipases as biocatalysts in biodiesel reactions has received much consideration since it is a 'greener technique' and has several advantages over free lipases. The selection of appropriate carrier materials and immobilization techniques fundamentally affects the immobilized lipases' catalytic performance for biodiesel production. Hence, this review discusses the recent developments for carrier materials and techniques for lipase immobilization as well as the current status of immobilized lipases mixtures and their application for biodiesel synthesis. The initial part of this review presents a brief introduction of lipases and their application for biodiesel preparation. New carrier materials and different basic and recent lipase immobilization techniques are also evaluated. Most significantly, studies carried out on mixtures of immobilized lipase‐catalyzed biodiesel for the last 15 years (2006–2021) are summarized. The economic and environmental evaluation of enzymatic biodiesel production is also presented. In the future, the application of mixed and co‐immobilized lipases may be enlarged to other enzymes and processes. © 2022 Society of Chemical Industry and John Wiley & Sons, Ltd [ABSTRACT FROM AUTHOR]

Inulin can be hydrolyzed by inulinases to yield inulo-oligosaccharides (IOSs), which have great application potential in the food and nutraceutical industries. However, conventional enzymatic production of IOSs is limited by long hydrolysis times and poor thermo-stability of inulinases. Here, the self-assembling protein scaffold EutM was engineered to co-immobilize exo-inulinase (EXINU) and endo-inulinase (ENINU) for synergistic hydrolysis of inulin to produce IOSs with 3 to 5 monosaccharide units (DP3–5 IOSs). The immobilization of EXINU/ENINU onto the EutM scaffold resulted in an increase of catalytic efficiency, a 65% increase of the V max of ENINU, as well as an increase of thermo-stability, with 4.26-fold higher residual activity of EXINU after 22 h-incubation at 50 °C. After optimization, two efficient production protocols were obtained, in which the yield and productivity of DP3–5 IOSs reached 80.38% and 70.86 g·(L·h)−1, respectively, which were at a high level in similar studies. Overall, this study provides an attractive self-assembling protein platform for the co-immobilization of inulinases, as well as optimized bioprocesses with great promise for the industrial production of DP3–5 IOSs. [Display omitted] • Inulin was efficiently hydrolyzed by synergistic action of EXINU and ENINU to produce DP3-5 IOSs. • Self-assembling protein scaffold EutM was used for the co-immobilization of EXINU/ENINU. • Improved catalytic efficiency and thermo-stability of ENINU/EXINU were obtained by using EutM. • A high level of DP3-5 IOSs productivity of 70.86 g·(L·h)-1 was obtained from 10% long chain inulin. [ABSTRACT FROM AUTHOR]

Core–shell immobilization of peroxide nanoparticles and horseradish peroxidase can be advantageous for removal of organic contaminants. In the present study, this novel technology was exploited for phenol removal from polluted water in order to prevent phenolic shock for the downstream microbial population. Since hydrogen peroxide production by calcium peroxide nanoparticles could be six times higher than magnesium peroxide (250 and 42 µmol H2O2 /g nanoparticles during a 10-min assay, respectively), calcium peroxide was selected as hydrogen peroxide supplier for further experiments. Regarding the immobilization matrix, diffusion properties, and mechanical strength of chitosan and alginate were compared. The mechanical strength was 3.0 ± 0.9 g for 6% of chitosan and 19.0 ± 3.3 g for 3% of alginate. According to the obtained results, the microcapsule core, embedding 1% w/v nanoparticles, was formed by cross-linking chitosan (6% w/v) with sodium tripolyphosphate. Subsequently, it was uniformly coated with an enzyme-containing alginate layer (3% w/v) cross-linked with calcium chloride. As the enzyme source, commercial horseradish peroxidase and partially purified peroxidase from horseradish roots were compared to evaluate the application of prepared microcapsules for industrial uses. The initial phenol removal rate of the optimized core–shell microcapsules was 0.0687 µmol phenol/h in a volume of reaction and gradually reduced to 50% of initial rate after 15 days in batch and column experiments. These results showed the high capacity of the proposed system for phenol removal from contaminated water streams which could be promising for various applications such as permeable reactive barriers for wastewater treatment or groundwater remediation. [ABSTRACT FROM AUTHOR]

The number of active groups on the molecular chain of chitosan is limited. Tn order to further increase the amount of chitosan enzyme and the biocatalysis stability of immobilized enzyme, in this study, 3-aminopropyl triethoxysilane (A PTES) and gl utaric anhydride (GA) are used as surface modifiers to prepare carboxyl functionalized porous magnetic chitosan microspheres (PMCSM-COOH), and Glucose oxidase (GOT)) and Catalase (CAT) are successfully immobilized. The results show that GOT) relative enzyme activity reaches the highest value of (1 31.91 ±0.58) % when the mass ratio of PMCSM-C00H/GOT)/CAT is 100/9.34/1 0.7& Compared with the free enzyme, the temperature stability, acid-base stability and storage stability of the co-immobilized enzyme are improved, and the affinity with the substrate is also enhanced. After repeated use for 1 0 times, the co-immobilized enzyme still retains a high relative enzyme activity of (79.1 6 ± 1.98) %• The secondary structure of free enzyme and immobilized enzyme show that the relative a -helix content of immobilized enzyme is higher than that of free GOD, so that immobilized enzyme has better structural rigidity and stability than free enzyme. [ABSTRACT FROM AUTHOR]

[Display omitted] • Taguchi design-assisted Co-immobilization of Lipase. • Co-immobilization of Lipase A and B from Candida antarctica onto Chitosan (CALA-CALB-CHI). • CALA-CALB-CHI derivative evaluated in the kinetic resolution of halohydrins acetates. • (S)-chlorohydrin 3b produced with 98% ee, conversion of 46% and E > 200. • Molecular docking was performed to elucidate the hydrolysis interaction reaction. In the present communication, the simultaneous co-immobilization by covalent binding of lipase A from Candida antarctica (CALA) and lipase B from Candida antarctica (CALB) in glutaraldehyde (GLU) activated chitosan (CHI) was optimized using the Taguchi method. Under optimized conditions (pH 9, 5 mM, 6:1 (protein load/g of support and 1 h), it was possible to reach 80.00 ± 0.01% for the immobilization yield (IY) and 46.01 ± 0.35 U/g for the activity of the derivative (AtD); in this case, load protein and ionic strength were the only statistically significant parameters and, therefore, those that most influenced the immobilization process. Furthermore, at pH 7, CALA-CALB-CHI had a half-life 2–6 times longer than the mixture of CALA and CALB for a temperature range of 50−80 °C. CALA-CALB showed the highest activity at pH 7, whereas CALA-CALB-CHI, except at pH 7, was more active than the soluble lipase mixture in the pH range (5–9), especially at pH 9. CHI, CHI-GLU, and CALA-CALB-CHI were characterized by X-ray powder diffraction (XRPD), Fourier Transform Infrared Spectroscopy (FTIR), Scanning Electron Microscope (SEM), Thermogravimetry (TGA), and Energy Dispersive Spectroscopy (EDS), proving the immobilization of CALA and CALB in chitosan. CALA-CALB-CHI derivative evaluated in the kinetic resolution of halohydrins acetates rac -2-bromo-1-(2-chlorophenyl) ethyl acetate (2a) and rac -2-chloro-1-(2,4-dichlorophenyl) ethyl acetate (2b), to produce the corresponding halohydrins 3a-b , which are intermediates in the synthesis of the drugs chlorprelanine (antiarrhythmic) and luliconazol (antifungal), respectively. (S)-bromohydrin 3a was obtained with 79% enantiomeric excess (ee), whereas (S)-chlorohydrin 3b produced with 98% ee , conversion of 46% and E > 200. Additionally, molecular docking was performed to elucidate the hydrolysis interaction reaction between β-halohydrin acetates and lipases CALA-CALB. [ABSTRACT FROM AUTHOR]

BACKGROUND: Non‐aqueous biocatalytic system has exhibited advantages including outstanding substrate solubility, facilitation for product purification, suppression of unwanted water‐dependent side reactions and rare wastewater discharge. However, limited by enzyme stability, selectivity and cofactor recycling, there has been no successful attempt to use carbonyl reductases in non‐aqueous phase. RESULTS: In this study, a non‐aqueous catalytic system of carbonyl reductase was developed for the first time with a carbonyl reductase–cofactor co‐immobilized biocatalyst. Using n‐hexane as the reaction medium, the key chiral diol intermediate of the top‐selling hypolipidemic drug rosuvastatin, tert‐butyl (3R,5S)‐6‐chloro‐3,5‐dihydroxyhexanoate ((3R,5S)‐CDHH) was prepared in a continuous packed‐bed bioreactor, with a total yield of 95.51%, enantiomeric excess (e.e.) > 99.5% and diastereomeric excess (d.e.) > 99.5% in a 44‐batch reaction. The yield of per unit biocatalyst was 34.42 mg U−1, and the space–time yield was 91.01 g h−1 L−1. CONCLUSION: Our work corroborated the valuable potential of carbonyl reductases in non‐aqueous biosynthesis of chiral pharmaceuticals and fine chemicals. © 2021 Society of Chemical Industry (SCI). [ABSTRACT FROM AUTHOR]

Ni2+-functionalized porous ceramic/agarose composite beads (Ni-NTA Cerose) can be used as carrier materials to immobilize enzymes harboring a metal affinity tag. Here, a 6×His-tag fusion alcohol dehydrogenase Mu-S5 and glucose dehydrogenase from Bacillus megaterium (BmGDH) were co-immobilized on Ni-NTA Cerose to construct a packed bed reactor (PBR) for the continuous synthesis of the chiral intermediate (S)-(4-chlorophenyl)-(pyridin-2-yl) methanol ((S)-CPMA) NADPH recycling, and in situ product adsorption was achieved simultaneously by assembling a D101 macroporous resin column after the PBR. Using an optimum enzyme activity ratio of 2:1 (Mu-S5: BmGDH) and hydroxypropyl-β-cyclodextrin as co-solvent, a space-time yield of 1560 g/(L·d) could be achieved in the first three days at a flow rate of 5 mL/min and substrate concentration of 10 mM. With simplified selective adsorption and extraction procedures, (S)-CPMA was obtained in 84% isolated yield. [ABSTRACT FROM AUTHOR]

[Display omitted] • Recent works addressing the topic of co-immobilization of organic acids and bases are summarized. • The rational design and engineering of these catalysts for cooperative and tandem catalysis are reviewed. • The structure–activity relationships of these catalysts are discussed. • Interdisciplinary efforts are required for the development of ideal supports and procedures. Acid–base bifunctional catalysts have been extensively used for a series of C C bond formation reactions. Among these catalysts, organic acid–base catalysts have attracted wide attention due to their structural variety, conformational dynamics, and enantioselectivity. To prevent mutual deactivation, a simple, effective, and versatile strategy is the attachment of both acid and base onto the surfaces of supports. In particular, the co-immobilization of organic acids and bases not only enhances synergistic effects in cooperative catalysis but also improves yields to desired products by controlling the diffusion of intermediates in tandem catalysis, leading to significant improvements in energy and atom efficiency. In this review, we highlight recent works addressing the broad topic of the co-immobilization of organic acids and bases for cooperative and tandem catalysis. We mainly focus on the synthetic strategies for silica-supported organic acid–base catalysts and polymer-supported organic acid–base catalysts. Furthermore, we summarize and discuss the structure–activity relationships of these catalysts. Last, the remaining issues and prospects will be discussed to advance the rational design and engineering of co-immobilized acid–base bifunctional catalysts. [ABSTRACT FROM AUTHOR]