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This article provides an overview of proteases and their immobilization on activated carbon-based supports. Proteases are enzymes that break down proteins and have various applications in industries such as food science. Immobilizing proteases on activated carbon offers advantages such as reusability and enhanced efficiency. The article discusses different immobilization methods and highlights the potential of activated carbon as a support. It also explores the modification of supports to improve their properties. The article concludes by describing specific proteases and their characteristics and applications. Another text focuses on bromelain and ficin, two proteases with broad pH ranges used in industries such as meat tenderization and brewing. The text also discusses enzyme immobilization and the use of activated carbon as a support. Various methods and supports for protease immobilization are described, with activated carbon being a versatile and cost-effective option. The surface of activated carbon can be modified to enhance enzyme immobilization. However, further research is needed in this area. Another document provides a list of methods for enzyme immobilization, along with their efficiencies and enzyme activities. The methods include physical adsorption, covalent bonding, and the use of various support materials. The enzymes used in the processes include pepsin, trypsin, papain, and protease from sunflower seeds. The efficiencies and activities vary depending on the method and enzyme. Lastly, a document presents tables summarizing studies on the use of activated carbon as a support for enzyme immobilization. The tables provide information on the [Extracted from the article]

Antibiotic-resistant bacteria are rapidly emerging, and the increasing prevalence of multidrug-resistant (MDR) Acinetobacter baumannii poses a severe threat to humans and healthcare organizations, due to the lack of innovative antibacterial drugs. Endolysins, which are peptidoglycan hydrolases encoded by a bacteriophage, are a promising new family of antimicrobials. Endolysins have been demonstrated as an effective therapeutic agent against bacterial infections of A. baumannii and many other Gram-positive and Gram-negative bacteria. Endolysin research has progressed from basic in vitro characterization to sophisticated protein engineering methodologies, including advanced preclinical and clinical testing. Endolysin are therapeutic agent that shows antimicrobial properties against bacterial infections caused by drug-resistant Gram-negative bacteria, there are still barriers to their implementation in clinical settings, such as safety concerns with outer membrane permeabilizers (OMP) use, low efficiency against stationary phase bacteria, and stability issues. The application of protein engineering and formulation techniques to improve enzyme stability, as well as combination therapy with other types of antibacterial drugs to optimize their medicinal value, have been reviewed as well. In this review, we summarize the clinical development of endolysin and its challenges and approaches for bringing endolysin therapies to the clinic. This review also discusses the different applications of endolysins. [ABSTRACT FROM AUTHOR]

De novo enzyme design has sought to introduce active sites and substrate-binding pockets that are predicted to catalyse a reaction of interest into geometrically compatible native scaffolds1,2, but has been limited by a lack of suitable protein structures and the complexity of native protein sequence-structure relationships. Here we describe a deep-learning-based 'family-wide hallucination' approach that generates large numbers of idealized protein structures containing diverse pocket shapes and designed sequences that encode them. We use these scaffolds to design artificial luciferases that selectively catalyse the oxidative chemiluminescence of the synthetic luciferin substrates diphenylterazine3 and 2-deoxycoelenterazine. The designed active sites position an arginine guanidinium group adjacent to an anion that develops during the reaction in a binding pocket with high shape complementarity. For both luciferin substrates, we obtain designed luciferases with high selectivity; the most active of these is a small (13.9 kDa) and thermostable (with a melting temperature higher than 95 °C) enzyme that has a catalytic efficiency on diphenylterazine (kcat/Km = 106 M-1 s-1) comparable to that of native luciferases, but a much higher substrate specificity. The creation of highly active and specific biocatalysts from scratch with broad applications in biomedicine is a key milestone for computational enzyme design, and our approach should enable generation of a wide range of luciferases and other enzymes.

Taenia solium can cause human taeniasis and/or cysticercosis. The latter can in some instances cause human neurocysticercosis which is considered a priority in disease‐control strategies and the prevention of mental health problems. Glutathione transferases are crucial for the establishment and long‐term survival of T. solium; therefore, we structurally analyzed the 24‐kDa glutathione transferase gene (Ts24gst) of T. solium and biochemically characterized its product. The gene promoter showed potential binding sites for transcription factors and xenobiotic regulatory elements. The gene consists of a transcription start site, four exons split by three introns, and a polyadenylation site. The gene architecture is conserved in cestodes. Recombinant Ts24GST (rTs24GST) was active and dimeric. Anti‐rTs24GST serum showed slight cross‐reactivity with human sigma‐class GST. A 3D model of Ts24GST enabled identification of putative residues involved in interactions of the G‐site with GSH and of the H‐site with CDNB and prostaglandin D2. Furthermore, rTs24GST showed optimal activity at 45 °C and pH 9, as well as high structural stability in a wide range of temperatures and pHs. These results contribute to the better understanding of this parasite and the efforts directed to fight taeniasis/cysticercosis.

Sulfoxides are remarkably useful in asymmetric synthesis and drug development. Herein, we propose a new electroenzymatic cascade route to selectively synthesize methyl phenyl sulfoxide rather than methyl phenyl sulfone by the oxidation of thioanisole. Chloroperoxidase (CPO) functionalized with 1-ethyl-3-methylimidazolium bromide (ILEMB) is encapsulated into dendritic N-doped mesoporous carbon nanospheres (NMCNs) modified with polyethyleneimine (NMCNs-PEI) to form a biohybrid (CPO-ILEMB@NMCNs-PEI). The in situ generation of hydrogen peroxide (H2O2) species by the two-electron oxygen reduction reaction (2e−ORR) on NMCNs-PEI initiates the subsequent selective oxidation of thioanisole by CPO-ILEMB. NMCNs-PEI shows not only high electroactivity for the 2e−ORR but also good biocompatibility for CPO-ILEMB immobilization. The mesopores in NMCNs-PEI can provide a protective space for the encapsulated enzyme, ensuring the operational stability of the enzyme and avoiding the escape of H2O2, which result in high catalytic activity. The experimental results suggest that the catalytic efficiency of the electroenzymatic cascade system with the CPO-ILEMB@NMCNs-PEI biohybrid is up to 4.5 times higher than that of the free CPO-ILEMB catalytic system with H2O2 generated by NMCNs-PEI for the oxidation of thioanisole to produce methyl phenyl sulfoxide. [ABSTRACT FROM AUTHOR]

Glutathione S-transferase omega 1 (GstO1) catalyzes deglutathionylation and plays an important role in the protein glutathionylation cycle in cells. GstO1 contains four conserved cysteine residues (C32, C90, C191, C236) found to be mutated in patients with associated diseases. In this study, we investigated the effects of cysteine mutations on the structure and function of GstO1 under different redox conditions. Wild-type GstO1 (WT) was highly sensitive to hydrogen peroxide (H2O2), which caused precipitation and denaturation at a physiological temperature. However, glutathione efficiently inhibited the H2O2-induced denaturation of GstO1. Cysteine mutants C32A and C236A exhibited redox-dependent stabilities and enzyme activities significantly different from those of WT. These results indicate that C32 and C236 play critical roles in GstO1 regulation by sensing redox environments and explain the pathological effect of cysteine mutations found in patients with associated diseases. [ABSTRACT FROM AUTHOR]

Laccases are lignolytic enzymes that catalyze and cleave various environmental pollutants. In this study, we isolated a novel laccase-producing bacterium, Bacillus aerius SP3, from compost soil using ABTS as the substrate. The production of extracellular laccase was improved through the one-factor-at-a-time (OFAT) method and response surface methodology using the Box–Behnken design (BBD) in submerged fermentation. Optimization of medium components, including 20 g/L starch, 10 g/L peptone, 3.36 mM CuSO4, and 72-h incubation time, resulted in a 3.46-fold increase in extracellular laccase production compared with the unoptimized medium. The extracellular laccase obtained from the culture supernatant was purified through 60% ammonium sulfate precipitation, dialysis, and ion exchange chromatography using a DEAE cellulose column. SDS-PAGE estimated the molecular weight of the isolated laccase to be approximately 60 kDa. The purified laccase also decolorized textile dyes, crystal violet and methylene blue, achieving 48% and 22% removal in the presence of ABTS as a mediator. The optimal temperature and pH for laccase activity were 35 °C and 7.0, respectively. The enzyme demonstrated stability up to 40 °C for 60 min and at pH 8.0 at 4 °C for 24 h. Cu2+ and Zn2+ enhanced laccase activity by 110%, while > 50% of activity was retained in the presence of heavy metals such as Hg2+ and Cd2+. Organic solvents, including 10% ethanol and methanol, increased laccase activity by 1.5 times. These results indicate that the laccase is active at neutral/alkaline pH, tolerant to heavy metals, and organic solvents. Enhancement of laccase activity by organic solvents is advantageous for chemical reactions in solvent environments and treating wastewater from dyeing industries. [ABSTRACT FROM AUTHOR]

The study of the relationship between the activity and stability of enzymes under crowding conditions in the presence of osmolytes is important for understanding the functioning of a living cell. The effect of osmolytes (trehalose and betaine) on the secondary and tertiary structure and activity of muscle glycogen phosphorylase b (Ph b) under crowding conditions created by PEG 2000 and PEG 20000 was investigated using dynamic light scattering, differential scanning calorimetry, circular dichroism spectroscopy, fluorimetry and enzymatic activity assay. At 25 °C PEGs increased Ph b activity, but PEG 20000 to a greater extent. Wherein, PEG 20000 significantly destabilized its tertiary and secondary structure, in contrast to PEG 2000. Trehalose removed the effects of PEGs on Ph b , while betaine significantly reduced the activating effect of PEG 20000 without affecting the action of PEG 2000. Under heat stress at 48 °C, the protective effect of osmolytes under crowding conditions was more pronounced than at room temperature, and the Ph b activity in the presence of osmolytes was higher in these conditions than in diluted solutions. These results provide important insights into the complex mechanism, by which osmolytes affect the structure and activity of Ph b under crowding conditions. • PEG 2000 and especially PEG 20000 destabilized structure of Ph b and increased its enzymatic activity. • Trehalose completely removed destabilizing effects of both PEGs but betaine only partialy • When heated in the presence of osmolytes, Ph b activity under crowding conditions was higher than in dilute solutions. • Protective effect of osmolytes under crowding conditions was most noticeable under heat stress. [ABSTRACT FROM AUTHOR]

Engineering human enzymes for therapeutic applications is attractive but introducing new amino acids may adversely affect enzyme stability and immunogenicity. Here we used a mammalian membrane-tethered screening system (ECSTASY) to evolve human lysosomal beta-glucuronidase (hBG) to hydrolyze a glucuronide metabolite (SN-38G) of the anticancer drug irinotecan (CPT-11). Three human beta-glucuronidase variants (hBG3, hBG10 and hBG19) with 3, 10 and 19 amino acid substitutions were identified that display up to 40-fold enhanced enzymatic activity, higher stability than E. coli beta-glucuronidase in human serum, and similar pharmacokinetics in mice as wild-type hBG. The hBG variants were two to three orders of magnitude less immunogenic than E. coli beta-glucuronidase in hBG transgenic mice. Intravenous administration of an immunoenzyme (hcc49-hBG10) targeting a sialyl-Tn tumor-associated antigen to mice bearing human colon xenografts significantly enhanced the anticancer activity of CPT-11 as measured by tumor suppression and mouse survival. Our results suggest that genetically-modified human enzymes represent a good alternative to microbially-derived enzymes for therapeutic applications. [Display omitted] [ABSTRACT FROM AUTHOR]

Taenia solium can cause human taeniasis and/or cysticercosis. The latter can in some instances cause human neurocysticercosis which is considered a priority in disease‐control strategies and the prevention of mental health problems. Glutathione transferases are crucial for the establishment and long‐term survival of T. solium; therefore, we structurally analyzed the 24‐kDa glutathione transferase gene (Ts24gst) of T. solium and biochemically characterized its product. The gene promoter showed potential binding sites for transcription factors and xenobiotic regulatory elements. The gene consists of a transcription start site, four exons split by three introns, and a polyadenylation site. The gene architecture is conserved in cestodes. Recombinant Ts24GST (rTs24GST) was active and dimeric. Anti‐rTs24GST serum showed slight cross‐reactivity with human sigma‐class GST. A 3D model of Ts24GST enabled identification of putative residues involved in interactions of the G‐site with GSH and of the H‐site with CDNB and prostaglandin D2. Furthermore, rTs24GST showed optimal activity at 45 °C and pH 9, as well as high structural stability in a wide range of temperatures and pHs. These results contribute to the better understanding of this parasite and the efforts directed to fight taeniasis/cysticercosis. [ABSTRACT FROM AUTHOR]

Laccase enzyme has gained popularity due to its wide range of substrates, use of only molecular oxygen as a co-factor and release of water as a by-product. In order to improve its reusability in harsh conditions, various supports have been explored for its immobilization. In this study, laccase was immobilized on zinc oxidechitosan/polyvinylpolypyrolidone (ZnONPs/CS/PVPP) and silver doped zinc oxidechitosan/polyvinylpolypyrolidone (Ag@ZnONPs/CS/PVPP) beads. The Ag@ZnONPs/CS/PVPP beads showed a higher immobilization yield and retained enzyme activity (76.0% and 50.0% respectively) compared to the ZnONPs/CS/PVPP beads (50.4% and 41.7% respectively). The biocatalysts demonstrated improved enzyme stability at higher temperatures and longer storage stability compared to the free enzyme hence show potential for further biotechnological applications. [ABSTRACT FROM AUTHOR]

Enzymatic reactions can be modulated by the incorporation of organic solvents, leading to alterations in enzyme stability, activity, and reaction rates. These solvents create a favorable microenvironment that enables hydrophobic reactions, facilities enzyme-substrate complex formation, and reduces undesirable water-dependent side reactions. However, it is crucial to understand the impact of organic solvents on enzymatic activity, as they can also induce enzyme inactivation. In this study, the enzymatic performance of Aspergillus oryzae α-amylase (Taka-amylase) in various organic solvents both experimentally and computationally was investigated. The results demonstrated that ethanol and ether sustain Taka-amylase activity up to 20% to 25% of the organic solvents, with ether providing twice the stability of ethanol. Molecular dynamics simulations further revealed that Taka-amylase has a more stable structure in ether and ethanol relative to other organic solvents. In addition, the analysis showed that the loop located near the active site in the AB-domain is a vulnerable site for enzyme destabilization when exposed to organic solvents. The ability of Taka-amylase to preserve the secondary loop structure in ether and ethanol contributed to the enzyme's activity. In addition, the solvent accessibility surface area of Taka-amylase is distributed throughout all enzyme structures, thereby contributing to the instability of Taka-amylase in the presence of most organic solvents. [ABSTRACT FROM AUTHOR]

Isomaltulose is a novel sweetener and is considered healthier than the common sugars, such as sucrose or glucose. It has been internationally recognized as a safe food product and holds vast potential in pharmaceutical and food industries. Sucrose isomerase is commonly used to produce isomaltulose from the substrate sucrose in vitro and in vivo. However, free cells/enzymes were often mixed with the product, making recycling difficult and leading to a significant increase in production costs. Immobilized cells/enzymes have the following advantages including easy separation from products, high stability, and reusability, which can significantly reduce production costs. They are more suitable than free ones for industrial production. Recently, immobilized cells/enzymes have been encapsulated using composite materials to enhance their mechanical strength and reusability and reduce leakage. This review summarizes the advancements made in immobilized cells/enzymes for isomaltulose production in terms of refining traditional approaches and innovating in materials and methods. Moreover, innovations in immobilized enzyme methods include cross-linked enzyme aggregates, nanoflowers, inclusion bodies, and directed affinity immobilization. Material innovations involve nanomaterials, graphene oxide, and so on. These innovations circumvent challenges like the utilization of toxic cross-linking agents and enzyme leakage encountered in traditional methods, thus contributing to enhanced enzyme stability. [ABSTRACT FROM AUTHOR]

Polycyclic aromatic hydrocarbons (PAHs) contaminated soil severely and are difficult to remediate. In this study, acid-modified chestnut inner shell biochar with abundant pore channels was used as the main raw materials for the immobilization of white-rot fungal crude enzyme. The maximum immobilization rate of crude enzymes (97.25%±6.20%) could be achieved under the optimal conditions of 24 h immobilization of 10 U/mL crude enzymes by 1 g biochar at 25℃ and pH = 5. Meanwhile, immobilization improved the stability of the crude enzyme. The relative activity of the immobilized crude enzyme increased by 59.32% and 49.73% (compared to the free crude enzymes) after 5 weeks of storage at 4°C and 25°C, respectively. It has been verified that chestnut-based immobilized crude enzyme can degrade 37% of benzo[a]pyrene in 10 days for PAHs-contaminated soils. An efficient, feasible, and low-cost remediation method for PAHs-contaminated soils was explored, which provides technical support for the application of crude enzymes in organic contaminated soils. [ABSTRACT FROM AUTHOR]

Emerging consumer demand for safer, more sustainable flavors and fragrances has created new challenges for the industry. Enzymatic syntheses represent a promising green production route, but the broad application requires engineering advancements for expanded diversity, improved selectivity, and enhanced stability to be cost-competitive with current methods. This review discusses recent advances and future outlooks for enzyme engineering in this field. We focus on carboxylic acid reductases (CARs) and unspecific peroxygenases (UPOs) that enable selective productions of complex flavor and fragrance molecules. Both enzyme types consist of natural variants with attractive characteristics for biocatalytic applications. Applying protein engineering methods, including rational design and directed evolution in concert with computational modeling, present excellent examples for property improvements to unleash the full potential of enzymes in the biosynthesis of value-added chemicals. • Carboxylic acid reductases (CARs) directly reduce carboxylic acids into aldehydes without a second enzyme for substrate activation. • Unspecific peroxygenases (UPOs) are self-sufficient mono peroxygenases that catalyze similar reactions as P450 enzymes. • Both groups of enzymes have broad substrate scopes and perform chemistries that are challenging to chemical methods. • We present enzyme engineering examples that improved and/or altered the catalytic property or stability of these enzymes. • The engineering achievements are steppingstones towards the ultimate industrial syntheses using CARs and UPOs. [ABSTRACT FROM AUTHOR]

This study aimed to establish a novel method for catalysing salidroside synthesis by immobilized β-D-glucosidase with deep eutectic solvents (DESs) as a new green non-aqueous medium. To this end, the catalytic properties of β-D-glucosidase (i.e. activity, stability, and enzymatic properties) were analysed within sixteen different DESs systems. Although most DESs significantly improved enzyme stability, they also reduced enzymatic activity. This was verified at the molecular level by employing fluorescence spectroscopy to evaluate conformational changes in β-D-glucosidase molecules. Subsequently, chitosan microspheres were used to immobilize β-D-glucosidase; the structures of the microspheres and immobilized enzyme were observed by scanning electron microscopy (SEM). Through single factor experiments and application of a response surface design, the optimal conditions for production of salidroside by the reaction of tyrosol with immobilized β-D-glucosidase were established. These conditions comprised 80 vol% ChCl/G (1:2), 50 °C reaction temperature, 5.8 pH, 100 h reaction time, 45 U/mL enzyme dosage, and a tyrosol/D-glucose molar ratio of 10. Under these conditions, the average conversion rate of the tyrosol substrate was 31.6%. Moreover, a salidroside crude product was obtained with a purity >70% (g/g) and a substrate conversion rate of immobilized β-D-glucosidase >50% of its initial value after five repeated uses. Collectively, this study establishes a novel green reaction system for the efficient preparation of salidroside and other glycoside compounds, while also providing a theoretical foundation for the analysis of β-D-glucosidase catalytic reactions in DESs systems. [ABSTRACT FROM AUTHOR]

High cost and low operational stability are the most important challenges limiting the possible use of laccase in the removal of textile dyes. To overcome these challenges, in this study, polyvinylpyrrolidone (PVP)-coated magnetic nanoparticles (MNPs) were produced and characterized. To our knowledge, this is the first study to explore the feasibility of immobilizing a Trametes trogii laccase enzyme on Fe3O4/PVP MNPs. The characterization of samples and the successful immobilization of laccase were verified by characterization methods. Besides, the biochemical properties and stability of the Fe3O4/PVP/Lac were evaluated in terms of optimum pH, optimum temperature, thermostability, thermodynamic and kinetic parameters, storage stability, operational stability, and decolorization efficiency of two different textile dyes. The optimum activities were recorded at pH 2.5 °C and 30 °C. The Fe3O4/PVP/Lac displayed remarkable thermal stability and activation energy for denaturation, enthalpy, Gibbs free energy, and entropy results confirmed the enhanced stability of Fe3O4/PVP/Lac against high temperatures. Meanwhile, the Fe3O4/PVP/Lac retained about 58% of its original activity after seven consecutive reuses, while it retained up to 25% of its original activity after 28 d of storage at room temperature. Km and Vmax for the Fe3O4/PVP/Lac were calculated to be 126 µM and 211 µmol/min, respectively. Finally, after 8 and 6 cycles of repeated use, the Fe3O4/PVP/Lac still decolorized 32.34% and 32.23% of Remazol Brilliant Blue R (RBBR) and Indigo Carmine (IC), respectively. As envisioned, this study suggests a promising way to solve the problems of high price and poor operational stability of the enzyme during biocatalytic decolorization of textile dyes in wastewaters. [ABSTRACT FROM AUTHOR]

Bating animal hides with trypsin is an essential step for making leathers. But the mechanism why the calcium in the hide influences the bating performance remains unclear. Here, the enzyme activity of trypsin was determined using N-alpha-benzoyl-L-arginine ethyl ester, bovine albumin (BSA) and collagen fibres as substrates in the presence and absence of calcium salts. The interactions of calcium with trypsin, BSA and collagen were analysed by fluorescence spectroscopy and molecular docking. Data reveal that calcium which binds to BSA and collagen improves the structural stability and enzyme resistance of hide proteins. As a result, a high calcium content of delimed hide is useful to obtain an intact surface of bated hide. However, a low calcium content of hide for pickling is necessary for producing leathers with high physical properties and uniform colour. According to these results, a novel bating system involving deliming, bating and redeliming operations was developed. This system provides a high calcium content of hide for bating and a low calcium content of hide for subsequent pickling and tanning, which effectively reduces the risk of hide damage and improves the physical properties and colour uniformity of leather. [Display omitted] • How calcium salts affect hydrolysis of hide proteins with trypsin in bating was revealed. • A high calcium content of delimed hide protects the grain surface of hide. • A low calcium content of hide is necessary for high physical properties of leather. • A novel system involving deliming, bating, and redeliming operations was designed. • This system effectively reduces the risk of hide damage and can ensure leather quality. [ABSTRACT FROM AUTHOR]

In this work, we assessed genipin activation of chitosan particles and pectinase immobilization parameters, and after applying these biocatalysts for fruit juice clarification. Support activation pH and enzyme load, along with immobilization pH, were evaluated. Pectinex Ultrapulp was used for enzyme immobilization. The immobilized enzyme maintained around 60% initial activity after 5 h at 60ºC, compared to free enzyme's rapid decline in <1 h. Orange juice clarification showed no statistical difference between free and immobilized enzyme, while grape juice clarification exhibited a significant increase with immobilized enzyme compared to free enzyme. In pectin (0.1%) hydrolysis batch test, the immobilized preparation retained 40% relative initial activity over 20 batches. For fruit juice clarification stability, an improved washing approach, to remove adsorbed juice particles in the biocatalysts, helped maintain 40% initial capabilities after 10 cycles. This study demonstrates the potential of genipin-activated chitosan particles for immobilizing pectinase and enhancing fruit juice clarification. [Display omitted] • Biocatalyst presented 21.9% of immobilization yield and activity of 851.9 U.g−1 using 100 mg.g−1 of protein. • Immobilized pectinase kept 60% of initial activity after 5 h at 60 °C, outperforming free enzyme. • Grape juice clarification was improved with immobilized pectinase compared to the free enzyme. • Juice particles tended to be adsorbed by the support particles after reutilization process. • Improved washing approach sustains 40% initial capabilities after 10 cycles. [ABSTRACT FROM AUTHOR]

The enzyme catalysis conversion of lignocellulosic biomass into valuable chemicals and fuels showed a bright outlook for replacing fossil resources. However, the high cost and easy deactivation of free enzymes restrict the conversion process. Immobilization of enzymes in metal–organic frameworks (MOFs) is one of the most promising strategies due to MOF materials' tunable building units, multiple pore structures, and excellent biocompatibility. Also, MOFs are ideal support materials and could enhance the stability and reusability of enzymes. In this paper, recent progress on the conversion of cellulose, hemicellulose, and lignin by MOF-immobilized enzymes is extensively reviewed. This paper focuses on the immobilized enzyme performances and enzymatic mechanism. Finally, the challenges of the conversion of lignocellulosic biomass by MOF-immobilized enzyme are discussed. [ABSTRACT FROM AUTHOR]

The use of biocompatible ionic liquids (bio-ILs) as a "green" solvent with uniquely tunable cations and anions, specific selectivity, and many other desirable physicochemical properties in biocatalysis has shown great promises to address many challenges. A plethora of studies have demonstrated ILs possess a range of exceptional capabilities in coordinating biocatalysis such as stabilizing enzyme structure, enhancing enzyme-substrate interaction, increasing substrate solubility and product separation/removal, and improving enzyme immobilization/recycling. Therefore, bio-ILs have been explored for biocatalysis in a wide range of homogenous as well as multiphasic systems including biphasic, multiphasic, microemulsion, and whole-cell systems. This review provides an overview of IL-enabled biocatalysis including enzymatic and whole-cell reactions in homogeneous and multiphasic systems. First, the properties of IL concentration, specific ion effect, pH, viscosity, alkyl chain length, hydrophobicity, and polarity on the structures and activities of enzymes are discussed together with characterization techniques to elucidate the mechanisms of IL-enzyme interactions. Second, while homogenous biocatalysis systems containing ILs are briefly introduced and summarized, particular attention is given to multiphasic systems. Furthermore, recent progress in using bio-ILs to modify emerging new enzyme carrier materials including carbonaceous and magnetic nanomaterials, gel microspheres, and membranes as well as their effect on increasing enzyme stability and recycling are highlighted. Finally, we provide our expert opinion for more innovative research in the field and future outlook for the industrial application of ILs in biocatalysis. [ABSTRACT FROM AUTHOR]

The use of edible oils and fats in dairy products is becoming increasingly important in the food industry because of their complementary functional properties. Most of these products are produced using food-grade enzymes as processing aids because processes involving enzymes are considered mild and environmentally friendly for regulatory purposes. The poor stability and recovery of enzymes in their native state limit their performance, and to enhance their activity, stability, and reusability, enzymes are often immobilised—a process that involves attaching them to a solid support. Additionally, immobilisation enables enzymes to selectively target specific substrates or products, making them highly efficient. These features have led to the increased use of immobilised enzymes in dairy and lipid processing and enzymes have been used to produce a broad range of products such as whey protein concentrates and isolates, peptide–lipid conjugates, lipid concentrates, structured lipids, and human milk fat substitutes. Therefore, this article reviews the current progress on different enzyme preparations and their use in lipid and dairy processing. It also summarises opportunities in enzyme-catalysed valorisation of dairy and lipid waste streams with the ultimate goals of sustainable food production and reductions in waste. [ABSTRACT FROM AUTHOR]

Biocatalytic oxidations are an emerging technology for selective C−H bond activation. While promising for a range of selective oxidations, practical use of enzymes catalyzing aerobic hydroxylation is presently limited by their substrate scope and stability under industrially relevant conditions. Here, we report the engineering and practical application of a non‐heme iron and α‐ketoglutarate‐dependent dioxygenase for the direct stereo‐ and regio‐selective hydroxylation of a non‐native fluoroindanone en route to the oncology treatment belzutifan, replacing a five‐step chemical synthesis with a direct enantioselective hydroxylation. Mechanistic studies indicated that formation of the desired product was limited by enzyme stability and product overoxidation, with these properties subsequently improved by directed evolution, yielding a biocatalyst capable of >15,000 total turnovers. Highlighting the industrial utility of this biocatalyst, the high‐yielding, green, and efficient oxidation was demonstrated at kilogram scale for the synthesis of belzutifan. [ABSTRACT FROM AUTHOR]

PETase exhibits a high degradation activity for polyethylene terephthalate (PET) plastic under moderate temperatures. However, the effect of non-active site residues in the second shell of PETase on the catalytic performance remains unclear. Herein, we proposed a crystal structure- and sequence-based strategy to identify the key non-active site residue. D186 in the second shell of PETase was found to be capable of modulating the enzyme activity and stability. The most active PETaseD186N improved both the activity and thermostability with an increase in Tm by 8.89 °C. The PET degradation product concentrations were 1.86 and 3.69 times higher than those obtained with PETaseWT at 30 and 40 °C, respectively. The most stable PETaseD186V showed an increase in Tm of 12.91 °C over PETaseWT. Molecular dynamics (MD) simulations revealed that the D186 mutations could elevate the substrate binding free energy and change substrate binding mode, and/or rigidify the flexible Loop 10, and lock Loop 10 and Helix 6 by hydrogen bonding, leading to the enhanced activity and/or thermostability of PETase variants. This work unraveled the contribution of the key second-shell residue in PETase in influencing the enzyme activity and stability, which would benefit in the rational design of efficient and thermostable PETase. [ABSTRACT FROM AUTHOR]

The purpose of this study is to employ the surface response approach to maximize the production of bacterial fibrinolytic enzyme. The Bacillus KDm99 bacterium stock was cultivated in nutrient broth, solid medium (nutrient agar-casein), and a special medium containing: peptone (0.5g), casein (0.1g), MgSO4 (0.02g), NaCl (0.02g), and KH2PO4 (0.05g). It was shown that the solid media had protease activity. The greatest protease activity was seen at a concentration of 50 microliters of enzyme with concentration (66.02 micro mole/min), 50 microliters of casein substrate with concentration (0.1%), 200 microliters of PBS-40 mm buffer with pH=8, and 300 microliters (Trichloroacetic acid)(5%) In order to optimize the bacterial growth environment for optimum enzyme synthesis, variables including supplies (carbon, nitrogen sources, metal ions, temperature, pH, agricultural residues, and animal fertilizers) were investigated. Following inoculation and 48 hours of incubation in a shaker incubator, the greatest efficiency of bacterial growth and enzyme production was assessed. The response method of variables at various levels has been modeled using the software (Design-Expert v13.0.0.0) in the presence of 0.5 mm NaCl concentration, Tween-20 organic solvent; body shampoo detergent (Zi-Musk) and ion's KH2PO4, FeSO4 enzyme Maximum activity and stability are displayed. Also, it was determined that Km is equivalent to 0.3 micromole and that Vm is equal to 0.8 micromole/min. The first hour saw an 80% maintenance of the temperature stability of the enzyme activity at 60°C, and the greatest enzyme activity was observed at pH=6 of the PBS buffer. [ABSTRACT FROM AUTHOR]

Wearable metabolite sensors are often limited by easily denaturable enzymes that only allow short‐duration monitoring. Although encapsulating enzymes in metal–organic frameworks (MOFs) shows promise of long‐term enzyme protection, it is typically accompanied by significantly decreased activity due to increased diffusion barrier, steric hindrance for enzyme‐substrate binding, and poor enzyme‐electronic interface. Herein, the co‐encapsulation of enzymes and ultrasmall arginine‐derived carbon dots (Argdot) into a mesoporous Zeolitic Imidazolate Framework‐8 (mZIF‐8) matrix and the enhancement effect of Argdot on enzyme stability and activity, which consequently improves the electrochemical sensor's long‐term sensitivity are investigated. Specifically, the glucose oxidase (GOx)‐Argdot@mZIF‐8 nanocomposite consistently exhibits 40% higher electrochemical sensitivity compared to control GOx@mZIF‐8, an improvement similarly demonstrated with another model enzyme lactate oxidase (LOx). Furthermore, GOx‐Argdot@mZIF‐8 displays excellent stability, retaining 100% of initial sensitivity over 30 days of repeated testing at 37 °C. A touch‐based glucose sensor prototype is demonstrated as an excellent reusable sensor to monitor finger‐tip sweat glucose levels over one month at room temperature. This enzyme encapsulation strategy is not only useful for developing reusable sweat sensors with long‐term monitoring capability, but also promising to expand the industry use of enzymes under harsh conditions. [ABSTRACT FROM AUTHOR]

Cytochrome P450 oxidoreductase (POR) is a crucial membrane-bound enzyme that facilitates the transfer of electrons to all cytochrome P450 (CYP450) enzymes. Several mutations in the POR gene have been reported to cause cytochrome P450 oxidoreductase deficiency (PORD), an autosomal recessive genetic disorder. This study explored the consequences of seven POR missense mutations (Y181D, A287P, R457H, R498P, C569Y, Y607C, and H628P), which have been documented in PORD patients, on the structural integrity and stability of the POR enzyme in vitro. The comparison between these mutants and the wild-type POR focused on in vitro protein expression, purification, and crystallization characteristics. The mutation-induced alterations in the POR architecture significantly influenced the protein's expression and crystallization capabilities. The magnitude of these effects on the enzyme's behavior varied from moderate to severe, contingent on the mutation's nature and position. This research illuminates the influence of specific mutations on POR stability, underlining the necessity of understanding mutation-driven effects on enzyme stability to devise personalized therapeutic approaches for PORD patients. Future studies will involve the functional characterization of these mutant enzymes to further understand their impact on the POR enzyme's activity and stability. [ABSTRACT FROM AUTHOR]

• Thermal stability of polysorbate-degradative enzymes was assessed with chemical proteomics and biophysical studies. • Brief heat treatment inactivated polysorbate-degradative enzymes and had minimal impact on the therapeutic antibody. • Heat inactivation of protein-A pool decelerated polysorbate degradation. Polysorbate degradation in biotherapeutics formulations is an industry-wide problem and mainly caused by residual host cell-derived enzymes. We present a proof-of-concept study of a control strategy which takes advantage of lower thermal stability of such enzymes relative to therapeutic proteins. We profiled heat sensitivity of host cell–derived enzyme activity with chemical proteomics and observed that PLA2G7 became inactive after brief heating. Further biophysical studies indicated that these enzymes were less thermally stable than a monoclonal antibody. Importantly, brief heat treatment had minimal impact on the stability of the antibody. Consequently, heat inactivation of polysorbate-spiked protein-A pool decelerated polysorbate degradation. This study suggests that heat inactivation of host cell–derived enzymes could be a control stragy for polysorbate degradation. [ABSTRACT FROM AUTHOR]

Antimicrobial peptides (AMPs), acknowledged as host defense peptides, constitute a category of predominant cationic peptides prevalent in diverse life forms. This study explored the antibacterial activity of α-conotoxin RgIA, and to enhance its stability and efficacy, D-amino acid substitution was employed, resulting in the synthesis of nine RgIA mutant analogs. Results revealed that several modified RgIA mutants displayed inhibitory efficacy against various pathogenic bacteria and fungi, including Candida tropicalis and Escherichia coli. Mechanistic investigations elucidated that these polypeptides achieved antibacterial effects through the disruption of bacterial cell membranes. The study further assessed the designed peptides' hemolytic activity, cytotoxicity, and safety. Mutants with antibacterial activity exhibited lower hemolytic activity and cytotoxicity, with Pep 8 demonstrating favorable safety in mice. RgIA mutants incorporating D-amino acids exhibited notable stability and adaptability, sustaining antibacterial properties across diverse environmental conditions. This research underscores the potential of the peptide to advance innovative oral antibiotics, offering a novel approach to address bacterial infections. [ABSTRACT FROM AUTHOR]

Xylanase is one of the critical enzymes for the degradation of xylan. The poor thermostability of xylanase hinders its industrial application. Disulfide bonds are important in stabilizing protein structure. In this study, the disulfide bonds of xylanase XynA were predicted by Disulfide by Design 2, Discovery Studio 2020 and sequence alignment. Mutants Mut1, Mut2 and Mut3 were generated and successfully expressed via assembling the disulfide bonds A1CT30C, T26CT44C, S28CS40C and T39CA49C. The half-life of Mut1, Mut2 and Mut3 was significantly increased at both 60 °C and 80 °C compared to XynA, respectively, and the T m of mutants were increased by 7–9 °C. Additionally, the specific activity of Mut1 was 1.24-fold that of XynA. Computer simulation showed that increased interactions and decreased net surface charge may be responsible for the increased thermostability, while the improved specific activity may be attributed to the flexibility of the "thumb" and more open cleft. To our knowledge, this is the first report of the simultaneous insertion of three disulfide bonds into the N-terminus of xylanase to improve enzyme stability while maintaining good activity, suggesting that inserting disulfide bonds at the suitable site of xylanase can achieve a balance between its thermal stability and specific activity. [Display omitted] • This is the first report of successfully inserting three disulfide bonds at N-terminus. • The first revealed a negative correlation between the N-terminus and the thumb. • Mutant Mut1 achieves double improvement in thermostability and catalytic activity. • The thermostability of enzyme improves with an increased number of disulfide bonds. • Flexible thumb and wider cleft of enzyme improved catalytic efficiency and activity. [ABSTRACT FROM AUTHOR]

In this work, the diamine oxidase (DAO) produced from pea seedlings was partially purified using ammonium sulphate precipitation, dialysis, and gel filtration chromatography. Partially purified DAO was immobilized covalently with the help of a cross-linker glutaraldehyde onto the chitosan- modified gold electrode. The properties of immobilized enzyme were evaluated by optimum pH, optimum temperature, activity recovery, and recyclability using putrescine dihydrochloride as substrate. After immobilization of DAO, optimum pH ranges from 6.0 to 7.0 did not change and optimum temperature of enzyme changed from 34 to 38°C in comparison to free DAO. The immobilized DAO preserved 81% activity of free DAO and permitted increased stability and reusability of the enzyme than its native form. After 15 cycles of usage, the immobilized DAO maintained around 51% of its original activity, and this activity prolonged for 30 days at 4°C. [ABSTRACT FROM AUTHOR]

A cochleate formulation was developed to enhance the oral bioavailability of revaprazan (RVP). Dimyristoyl phosphatidylcholine (DMPC) liposome containing dicetyl phosphate (DCP) successfully formed a cochleate after treatment with CaCl2, whereas that containing sodium deoxycholate did not. Cochleate was optimised using a D-optimal mixture design with three independent variables—DMPC (X1, 70.58 mol%), cholesterol (X2, 22.54 mol%), and DCP (X3, 6.88 mol%)—and three response variables: encapsulation efficiency (Y1, 76.92%), released amount of free fatty acid at 2 h (Y2, 39.82%), and released amount of RVP at 6 h (Y3, 73.72%). The desirability function was 0.616, showing an excellent agreement between the predicted and experimental values. The cylindrical morphology of the optimised cochleate was visualised, and laurdan spectroscopy confirmed the dehydrated membrane interface, showing an increased generalised polarisation value (approximately 0.5) over small unilamellar vesicle of RVP (RVP-SUV; approximately 0.1). The optimised cochleate showed greater resistance to pancreatic enzyme than RVP-SUV. RVP was released in a controlled manner, achieving approximately 94% release in 12 h. Following oral administration in rats, the optimised cochleate improved the relative bioavailability of RVP by approximately 274%, 255%, and 172% compared to RVP suspension, a physical mixture of RVP and the cochleate, and RVP-SUV, respectively. Thus, the optimised cochleate formulation might be a good candidate for the practical development of RVP. [ABSTRACT FROM AUTHOR]

Despite decades of laboratory and clinical trials, breast cancer remains the main cause of cancer-related disease burden in women. Considering the metabolism destruction effect of metformin (Met) and cancer cell starvation induced by glucose oxidase (GOx), after their efficient delivery to tumor sites, GOx and Met may consume a large amount of glucose and produce sufficient hydrogen peroxide in situ. Herein, a pH-responsive epigallocatechin gallate (EGCG)-conjugated low-molecular-weight chitosan (LC-EGCG, LE) nanoparticle (Met–GOx/Fe@LE NPs) was constructed. The coordination between iron ions (Fe3+) and EGCG in this nanoplatform can enhance the efficacy of chemodynamic therapy via the Fenton reaction. Met–GOx/Fe@LE NPs allow GOx to retain its enzymatic activity while simultaneously improving its stability. Moreover, this pH-responsive nanoplatform presents controllable drug release behavior. An in vivo biodistribution study showed that the intracranial accumulation of GOx delivered by this nanoplatform was 3.6-fold higher than that of the free drug. The in vivo anticancer results indicated that this metabolism destruction/starvation/chemodynamic triple-combination therapy could induce increased apoptosis/death of tumor cells and reduce their proliferation. This triple-combination therapy approach is promising for efficient and targeted cancer treatment. [Display omitted] • Met–GOx/Fe@LE NPs can retain GOx enzyme activity and improve its enzyme stability simultaneously. • Met–GOx/Fe@LE NPs presents the controllable release behavior in an acidic responsive manner. • Met–GOx/Fe@LE NPs can block the glycolysis process and induce starvation and chemodynamic therapy. • Met–GOx/Fe@LE NPs can increase death of tumor cells and decrease liver metastatic nodules. [ABSTRACT FROM AUTHOR]

Endolysins are produced by (bacterio)phages and play a crucial role in degrading the bacterial cell wall and the subsequent release of new phage progeny. These lytic enzymes exhibit a remarkable diversity, often occurring in a multimodular form that combines different catalytic and cell wall-binding domains, even in phages infecting the same species. Yet, our current understanding lacks insight into how environmental factors and ecological niches may have influenced the evolution of these enzymes. In this study, we focused on phages infecting Streptococcus thermophilus , as this bacterial species has a well-defined and narrow ecological niche, namely, dairy fermentation. Among the endolysins found in phages targeting this species, we observed limited diversity, with a singular structural type dominating in most of identified S. thermophilus phages. Within this prevailing endolysin type, we discovered a novel and highly conserved calcium-binding motif. This motif proved to be crucial for the stability and activity of the enzyme at elevated temperatures. Ultimately, we demonstrated its positive selection within the host's environmental conditions, particularly under the temperature profiles encountered in the production of yogurt, mozzarella, and hard cheeses that rely on S. thermophilus. [ABSTRACT FROM AUTHOR]

Background: Chitin, the main form of aminated polysaccharide in nature, is a biocompatible, polycationic, and antimicrobial biopolymer used extensively in industrial processes. Despite the abundance of chitin, applications thereof are hampered by difficulties in feedstock harvesting and limited structural versatility. To address these problems, we proposed a two-step cascade employing carbohydrate oxidoreductases and amine transaminases for plant polysaccharide aminations via one-pot reactions. Using a galactose oxidase from Fusarium graminearum for oxidation, this study compared the performance of CvATA (from Chromobacterium violaceum) and SpATA (from Silicibacter pomeroyi) on a range of oxidized carbohydrates with various structures and sizes. Using a rational enzyme engineering approach, four point mutations were introduced on the SpATA surface, and their effects on enzyme activity were evaluated. Results: Herein, a quantitative colorimetric assay was developed to enable simple and accurate time-course measurement of the yield of transamination reactions. With higher operational stability, SpATA produced higher product yields in 36 h reactions despite its lower initial activity. Successful amination of oxidized galactomannan by SpATA was confirmed using a deuterium labeling method; higher aminated carbohydrate yields achieved with SpATA compared to CvATA were verified using HPLC and XPS. By balancing the oxidase and transaminase loadings, improved operating conditions were identified where the side product formation was largely suppressed without negatively impacting the product yield. SpATA mutants with multiple alanine substitutions besides E407A showed improved product yield. The E407A mutation reduced SpATA activity substantially, supporting its predicted role in maintaining the dimeric enzyme structure. Conclusions: Using oxidase–amine transaminase cascades, the study demonstrated a fully enzymatic route to polysaccharide amination. Although the activity of SpATA may be further improved via enzyme engineering, the low operational stability of characterized amine transaminases, as a result of low retention of PMP cofactors, was identified as a key factor limiting the yield of the designed cascade. To increase the process feasibility, future efforts to engineer improved SpATA variants should focus on improving the cofactor affinity, and thus the operational stability of the enzyme. [ABSTRACT FROM AUTHOR]

Introduction: Continuous cropping affected the stability of soil enzyme activity and the structural characteristics of microbial community. Owing to challenges in the study of complex rhizosphere microbial communities, the composition and function of these microbial communities in farmland ecosystems remain elusive. Here, we studied the microbial communities of the rhizosphere of wine grapes with different years of continuous cropping and investigated their relationships with soil enzyme activity. Methods: Metagenomic sequencing was conducted on the rhizosphere soils from one uncultivated wasteland and four vineyards with varying durations of continuous cropping. Results: The predominant microbial were bacteria (98.39%), followed by archaea (1.15%) and eukaryotes (0.45%). Continuous cropping caused a significant increase in the relative abundance of Rhizobiales and Micrococcales but a marked decrease in Solirubrobacterales. At the genus level, 75, 88, 65, 132, and 128 microbial genera were unique to uncultivated wasteland, 5, 10, 15, and 20 years of continuous cropping, respectively. The relative abundance of genes with signal transduction function was the highest. The activity of all enzymes measured in this study peaked at 5 years of continuous cropping, and then decreased with 10 to 15 year of continuous cropping, but increased at 20 years again. In addition, soil enzyme activity, especially of alkaline phosphatase was significantly correlated with the diversity of the dominant microorganisms at the genus level. Moreover, the coupled enzyme activities had a greater impact on the diversity of the microbial community than that of individual enzymes. Conclusion: Our findings reveal the composition and function of the soil microbial communities and enzymes activity in response to changes in cropping years, which has important implications for overcoming continuous cropping obstacles and optimizing land use. [ABSTRACT FROM AUTHOR]

The bacterial enzyme chondroitinase ABCI (chABCI), which has been isolated from Proteus Vulgaris, is crucial in the treatment of spinal cord injuries. However, due to its short lifespan, the maintenance and clinical application of this enzyme are very constrained. In this study, the immobilization of this enzyme on hydroxyapatite has been carried out and assessed with the aim of enhancing the characteristics and efficiency of chABCI. Hydroxyapatite particles (HAPs) are a potential candidate for drug-delivery carriers because of their excellent biocompatibility, shape controllability, and high adsorption. The use of the nanometer scale allows efficient access to the enzyme's substrate. It demonstrates important biological application capabilities in this way. Field emission gun-scanning electron microscopy (FEG-SEM), X-ray diffraction (XRD), infrared spectroscopy (FT-IR), in vitro release study, and cytotoxicity test were used to characterize the drug nanosystem's properties. According to the findings, electrostatic bindings was formed between charged groups of the enzyme and hydroxyapatite nanoparticles. The results also demonstrated that immobilized chABCI on hydroxyapatite has beneficial properties, such as more manageable drug release, minimal toxicity and side effects, and a high potential to enhance the efficacy of drug delivery and decrease the need for repeated injections. Key points: It has been proven that hydroxyapatite increases the shelf life of chondroitinase when it is used as a carrier matrix. Stability of the enzyme loaded onto hydroxyapatite was enhanced significantly after immobilization. The proposed process can make the hydroxyapatite nanoparticles a useful stabilizer drug delivery vehicle. The release of the chondroitinase is more controllable than previous studies; therefore the stability of the nanosystem has been increased. [ABSTRACT FROM AUTHOR]

The employment of 2-deoxyribose-5-phosphate aldolase (DERA) stands as a prevalent biocatalytic route for synthesizing statin side chains. The main problem with this pathway is the low stability of the enzyme. In this study, mesocellular silica foam (MCF) with different pore sizes was used as a carrier for the covalent immobilization of DERA. Different functionalizing and activating agents were tested and kinetic modeling was subsequently performed. The use of succinic anhydride as an activating agent resulted in an enzyme hyperactivation of approx. 140%, and the stability almost doubled compared to that of the free enzyme. It was also shown that the pore size of MCF has a decisive influence on the stability of the DERA enzyme. [ABSTRACT FROM AUTHOR]

Bacterial isolates collected from the environment were screened for pectinolytic activity, and a strain with the highest activity was selected and identified as Bacillus subtilis Mz‐12. The presence of pectin hydrolase, lyase, and esterase activities was confirmed. Pectinase was purified and characterized. Enzyme production was optimized with respect to temperature, pH, and growth medium. Enzyme stability and activity were characterized under different temperatures and pH values. The results showed that this strain was capable of producing high yields of pectinase in commercial medium (Pharmamedia) 24.6 U/mL compared to other media. The purified pectinase of 22.3 kDa produced was constitutive in nature. The isolated enzyme from this strain displayed a wide range of temperature and pH stability, with the optimal activity observed at pH 9.0 and 50°C. These results indicate that the B. subtilis Mz‐12 strain is a favorable candidate for industrial enzyme production. The use of Pharmamedia is reported for first time for pectinase production. [ABSTRACT FROM AUTHOR]

The utilization of lipase in synthesis structured lipids (SLs) from natural oils confers additional health properties and effectively enhances their value-added potential. This paper provided a comprehensive review of recent studies on the enzymatic preparation of SLs, summarizing the advancements in structural design and immobilized enzyme engineering technology to improve the efficiency of their synthesis by enzymatic approaches. The sn-1,3 regiospecific lipase enabled the precise synthesis of SLs, while employing stepwise variable temperature reactions, supercritical fluid reactions, vacuum reactions, and ultrasonic-assisted methods could inhibit the acyl migration effects and further enhance product yield. Furthermore, structural design could enhance the catalytic efficiency, stability, and substrate specificity of lipases to elevate the efficiency and yield in enzymatic synthesis of SLs. Immobilized enzyme technology could enhance enzyme reactivity and stability while reducing the costs of enzymatic synthesis of SLs. In the future, the high-performance enzymes should be developed by combining structural modification and novel immobilization technology on the basis of the clearer understanding of their structure-function characteristics. These insights would contribute to innovation and advancement in efficient and precise enzymatic synthesis processes for SLs. [ABSTRACT FROM AUTHOR]

The lipase from Prunus dulcis almonds has been immobilized for the first time. For this purpose, two different supports, an octadecyl methacrylate particulate support, and aminated agarose (monoaminoethyl-N-aminoethyl) have been utilized. Both immobilized biocatalysts show improved enzyme stability, but great changes in enzyme specificity were detected. The enzyme immobilized via ion exchange maintained its activity intact versus p-nitrophenyl butyrate, while the enzyme immobilized on the hydrophobic support fully lost its activity versus this substrate, which was confirmed to be due to substrate adsorption on the support. However, this biocatalyst was much more active versus triacetin (more than 10-fold), R- or S- methyl mandelate at pH 7. At pH 9, a strong effect of using phosphate or bicarbonate as reaction buffers was detected. Using bicarbonate, the interfacially immobilized enzyme presented no activity versus R-isomer, but it was very active versus the S-isomer and triacetin. Using a phosphate buffer during the reaction, all compounds were recognized as substrates. The enzyme immobilized via ion exchange was significantly more active using phosphate; in fact, using bicarbonate, the enzyme was inactive versus both methyl mandelate isomers. This paper shows for the first time a great interaction between the effects of the immobilization protocol and buffer used during reaction on the enantiospecificity of lipases. [ABSTRACT FROM AUTHOR]

The lipases from Thermomyces lanuginosus (TLL) and Candida antarctica (B) (CALB) were immobilized on octyl-agarose beads at 1 mg/g (a loading under the capacity of the support) and by overloading the support with the enzymes. These biocatalysts were compared in their stabilities in 10 mM of sodium phosphate, HEPES, and Tris-HCl at pH 7. Lowly loaded CALB was more stable than highly loaded CALB preparation, while with TLL this effect was smaller. Phosphate was very negative for the stability of the CALB biocatalyst and moderately negative using TLL at both loadings. The stability of the enzymes in HEPES and Tris-HCl presented a different response as a function of the enzyme loading (e.g., using lowly loaded CALB, the stabilities were similar in both buffers, but it was clearly smaller in HEPES using the highly loaded biocatalysts). Moreover, the specific activity of the immobilized enzymes versus p-nitrophenol butyrate, triacetin and R- or S-methyl mandelate depended on the buffer, enzyme loading, and interaction between them. In some cases, almost twice the expected activity could be obtained using highly loaded octyl-CALB, depending on the buffer. A co-interaction between the effects on enzyme activity and the specificity of support enzyme loading and buffer nature was detected. [ABSTRACT FROM AUTHOR]

In the presented study, a variety of hybrid and single nanomaterials of various origins were tested as novel platforms for horseradish peroxidase immobilization. A thorough characterization was performed to establish the suitability of the support materials for immobilization, as well as the activity and stability retention of the biocatalysts, which were analyzed and discussed. The physicochemical characterization of the obtained systems proved successful enzyme deposition on all the presented materials. The immobilization of horseradish peroxidase on all the tested supports occurred with an efficiency above 70%. However, for multi-walled carbon nanotubes and hybrids made of chitosan, magnetic nanoparticles, and selenium ions, it reached up to 90%. For these materials, the immobilization yield exceeded 80%, resulting in high amounts of immobilized enzymes. The produced system showed the same optimal pH and temperature conditions as free enzymes; however, over a wider range of conditions, the immobilized enzymes showed activity of over 50%. Finally, a reusability study and storage stability tests showed that horseradish peroxidase immobilized on a hybrid made of chitosan, magnetic nanoparticles, and selenium ions retained around 80% of its initial activity after 10 repeated catalytic cycles and after 20 days of storage. Of all the tested materials, the most favorable for immobilization was the above-mentioned chitosan-based hybrid material. The selenium additive present in the discussed material gives it supplementary properties that increase the immobilization yield of the enzyme and improve enzyme stability. The obtained results confirm the applicability of these nanomaterials as useful platforms for enzyme immobilization in the contemplation of the structural stability of an enzyme and the high catalytic activity of fabricated biocatalysts. [ABSTRACT FROM AUTHOR]

Natural deep eutectic solvents (NADESs) can serve as solvents for enzymes, are biodegradable, and have low toxicities. Eight NADESs with different hydrogen bond acceptors and donors were tested to improve the stability and activity of a lipoxygenase from Basidiomycete Pleurotus sapidus (LOXPSA). Betaine:sorbitol:water (1:1:3, BSorbW) and betaine:ethylene glycol (1:3, BEtGly) had the best impact on the peroxidation of linoleic acid and the side reaction of piperine to the vanilla-like scented compound piperonal. The yield of piperonal in NADESs increased by 43% in BSorbW and 40% in BEtGly compared to the control. The addition of BSorbW also enhanced the enzyme's stability at various temperatures and increased its activity during incubation at 60 °C. The demonstrated improvement in lipoxygenase activity and stability indicates versatile applications in industry, expanding the potential uses of the enzyme. [ABSTRACT FROM AUTHOR]

ATP-dependent energy-consuming enzymatic reactions are widely used in cell-free biocatalysis. However, the direct addition of large amounts of expensive ATP can greatly increase cost, and enzymatic production is often difficult to achieve as a result. Although a polyphosphate kinase (PPK)-polyphosphate-based ATP regeneration system has the potential to solve this challenge, the generally poor thermal stability of PPKs limits the widespread use of this method. In this paper, we evaluated the thermal stability of a PPK from Sulfurovum lithotrophicum (SlPPK2). After directed evolution and computation-supported design, we found that SlPPK2 is very recalcitrant and cannot acquire beneficial mutations. Inspired by the usually outstanding stability of ancestral enzymes, we reconstructed the ancestral sequence of the PPK family and used it as a guide to construct three heat-stable variants of SlPPK2, of which the L35F/T144S variant has a half-life of more than 14 h at 60°C. Molecular dynamics simulations were performed on all enzymes to analyze the reasons for the increased thermal stability. The results showed that mutations at these two positions act synergistically from the interior and surface of the protein, leading to a more compact structure. Finally, the robustness of the L35F/T144S variant was verified in the synthesis of nucleotides at high temperature. In practice, the use of this high-temperature ATP regeneration system can effective ly avoid byproduct accumulation. Our work extends the temperature boundary of ATP regeneration and has great potential for industrial applications. [ABSTRACT FROM AUTHOR]

Lignocellulosic biomass holds promise as a renewable feedstock for various applications, but its efficient conversion requires cost-effective degradation strategies. The main objective of this study was to investigate the effect of the growth conditions of Cellulomonas phragmiteti in the production of (hemi)cellulosic supernatants. To meet this aim, different lignocellulosic residues were used as carbon sources for growth using defined mineral or nutritive culture media. Cell-free culture supernatants with xylanolytic activity were produced in all the conditions evaluated, but the highest xylanase activity (15.3 U/mL) was achieved in Luria–Bertani (LB) medium containing 1% waste paper. Under these conditions, almost negligible β-glucosidase, cellobiohydrolase, β-xylosidase, and α-arabinofuranosidase activity was detected. The xylanolytic supernatant showed tolerance to salt and displayed maximal catalytic efficiency at pH 6 and 45 °C, along with good activity in the ranges of 45–55 °C and pH 5–8. As it showed good stability at 45 °C, the supernatant was employed for the hydrolysis of birchwood xylan (50 g/L) under optimal conditions, releasing 10.7 g/L xylose in 72 h. Thus, C. phragmiteti was found to produce a xylanolytic enzymatic supernatant efficiently by utilizing the cheap and abundant lignocellulosic residue of waste paper, and the produced supernatant has promising attributes for industrial applications. [ABSTRACT FROM AUTHOR]

Hydrogen is consider as clean fuel with potential source of unlimited clean power. The global market for the hydrogen generation is expected to be $317.39 billion by 2030. The key factors responsible for hydrogen generation market includes high demand of conventional fuel in steel, cement and power generation industries and government initiatives for green and sustainable environment. It is of the utmost note that the major production of hydrogen is based on conversion from fossil fuels such as coal, petroleum etc. Among green hydrogen production method transformation of biomass to biohydrogen via anaerobic fermentation is considered as carbon neutral source of energy. However, the yield of hydrogen production is low and depends upon various factors such waste composition, bioreactor type, and microorganisms used its production, but production is limited due to shortage of raw materials. Since, the biomass such as agriculture waste residues are largely available as corn stover, sugar beet juice, beverage waste; water, wheat straw, rice straw, wood chips, sawdust, shrubs, and branches etc. The major hurdle in dark fermentation is lower biohydrogen yield due to inefficient conversion of carbohydrates into biohydrogen. Nanomaterials can help in valorization of agriculture residue for efficient biohydrogen production. Nano-technological application like increasing surface area, enzyme stability and saccharification efficiency of enzyme can contribute in biohydrogen production. Nanoparticles may result in a shift in the microbial community and the metabolic pathway for hydrogen production. This review explore the impending application of nanomaterial for valorization of agriculture biomass, biochemical pathways, and possible application of nanomaterial as individual or in composite to improve biohydrogen production. • Agriculture biomass is an inexpensive substrate that may be used to produce biohydrogen economically. • Comparing to traditional acid-based pretreatment, nanomaterial-mediated pretreatment is more environmentally friendly. • Availability of cellulose and hemicellulose for the microorganisms is increased by pretreatment of agricultural leftovers. • Nanomaterials can be used as nano-catalysts that increase the synthesis of biohydrogen. [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]