Enzyme Stability Research Articles

Biocatalysts for biomethanol production: Advancements and future prospects

Biomethanol, a renewable and sustainable alternative to traditional fossil-fuel-derived methanol, has garnered considerable attention as a potential solution to mitigate greenhouse gas emissions and dependence on non-renewable resources. The utilization of biocatalysts in biomethanol production offers a promising avenue to achieve environmentally friendly and economically viable processes. Paper highlights the biocatalytic pathways involved in biomethanol synthesis. Particular emphasis is placed on microbial biocatalysts, such as methanogenic archaea and certain bacteria, which possess the unique capability of converting carbon dioxide and hydrogen into methanol through a series of enzymatic reactions. Additionally, enzyme-based systems derived from various microorganisms and genetically engineered organisms are also discussed as potential biocatalysts for biomethanol synthesis. Paper also delves into the current challenges and limitations faced in harnessing biocatalysts for biomethanol production. These challenges include substrate availability, low conversion rates, enzyme stability, and process scalability. Several strategies to address these issues are highlighted, including metabolic engineering, synthetic biology, and bioprocess optimization techniques. The advantages of utilizing biocatalysts for biomethanol production are outlined. Biocatalytic routes offer the advantage of operating under mild conditions, which reduces energy consumption and minimizes the production of unwanted by-products. Furthermore, the utilization of renewable feedstocks, such as carbon dioxide captured from industrial emissions or waste streams, enhances the sustainability of the process. The final section discusses future prospects and potential research directions in the field of biocatalytic biomethanol production. Advances in biotechnology, omics technologies, and computational modeling are poised to accelerate the discovery and optimization of novel biocatalysts, thereby unlocking the full potential of biomethanol as a sustainable fuel and chemical precursor. The use of biocatalysts for biomethanol production offers an attractive approach to establish a green and circular economy. With ongoing research and technological advancements, the field holds significant promise for reducing carbon emissions and transitioning towards a more sustainable energy landscape. However, to fully realize the potential of biocatalytic biomethanol production, interdisciplinary collaboration and concerted efforts are required to address existing challenges.

In-Bridge Stereochemistry: A Determinant of Stapled Peptide Conformation and Activity.

Peptide side chain stapling has been proven to be an effective strategy for fine-tuning peptide properties. This innovative approach leads to the creation of stapled peptides characterized by stabilized α-helical conformations, enhanced protein-binding affinity, improved cell permeability, superior enzymatic stability, and numerous other advantages. Extensive research has explored the impact of various stapling bridges on the properties of these peptides, with limited investigation into the influence of bridge chirality, until very recently. In this concise review, we provide a brief overview of the current state of knowledge regarding the stereochemistry within the bridges of stapled peptides, offering insights into the potential applications of chiral bridges in the design and development of stapled peptides.

Co-Immobilization of Laccase and Mediator into Fe-Doped ZIF-8 Significantly Enhances the Degradation of Organic Pollutants.

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.

Aflatoxin Decontamination in Maize Steep Liquor Obtained from Bioethanol Production Using Laccases from Species within the Basidiomycota Phylum.

Maize (Zea mays L.) is an important crop in Argentina. Aspergillus section Flavi can infect this crop at the pre-harvest stage, and the harvested grains can be contaminated with aflatoxins (AFs). During the production of bioethanol from maize, AF levels can increase up to three times in the final co-products, known as, dry and wet distiller's grain with solubles (DDGS and WDGS), intended for animal feed. Fungal enzymes like laccases can be a useful tool for reducing AF contamination in the co-products obtained from this process. The aim of the present study was to evaluate the ability of laccase enzymes included in enzymatic extracts (EE) produced by different species in the Basidiomycota phylum to reduce AF (AFB1 and AFB2) accumulation under the conditions of in vitro assays. Four laccase activities (5, 10, 15, and 20 U/mL) exerted by nine isolates were evaluated in the absence and presence of vanillic acid (VA), serving as a laccase redox mediator for the degradation of total AFs. The enzymatic stability in maize steep liquor (MSL) was confirmed after a 60 h incubation period. The most effective EE in terms of reducing AF content in the buffer was selected for an additional assay carried out under the same conditions using maize steep liquor obtained after the saccharification stage during the bioethanol production process. The highest degradation percentages were observed at 20 U/mL of laccase enzymatic activity and 1 mM of VA, corresponding to 26% for AFB1 and 26.6% for AFB2. The present study provides valuable data for the development of an efficient tool based on fungal laccases for preventing AF accumulation in the co-products of bioethanol produced from maize used for animal feed.

Polyelectrolyte composite hydrogels based on a derivative of functional dietary fiber for long-term gastric retention and drug delivery

Gastric retention drug delivery is experiencing significant demand for hydrogels that exhibit in situ controllable forming in the stomach, resistance against severe gastric degradation, and exceptional biocompatibility. However, current studies lack comprehensive investigations on these requirements. In this study, resistant starch is chosen as a kind of novel dietary fiber with remarkable gastric retention effects compared to conventional natural polymers. To address its limited solubility, resistant starch was modified by carboxymethyl groups. Moreover, a semi-dissolution acidification sol–gel transition method was employed to enable in situ gelation under gastric acid conditions and fabricate physically crosslinked polyelectrolyte composite hydrogels (CMRS/CTS) based on carboxymethylated resistant starch and chitosan. This method obviates the need for toxic chemical crosslinkers and initiators, ensuring favorable biocompatibility. The influence of carboxymethyl substitution degree and the anion-to-cation ratio of the composite hydrogels on the mechanical, rheological, and swelling properties was thoroughly investigated. Compared to conventional carboxymethyl starch-based hydrogels, CMRS/CTS maintained a relatively intact structure even after immersion in simulated gastric fluid for 30 days owing to the enzymatic stability of the hydrogel. Upon drug loading, CMRS/CTS exhibited sustained and controlled drug release, facilitating prolonged therapeutic effects. Simultaneously achieving in situ gastric drug release, biocompatibility, resistance to degradation, and sustained release, CMRS/CTS represents a promising avenue for the development of long-term gastric retention hydrogels.

Opioid/Dopamine Receptor Binding Studies, NMR and Molecular Dynamics Simulation of LENART01 Chimera, an Opioid-Bombesin-like Peptide.

The design and development of hybrid compounds as a new class of drug candidates remains an excellent opportunity to improve the pharmacological properties of drugs (including enzymatic stability, efficacy and pharmacokinetic and pharmacodynamic profiles). In addition, considering various complex diseases and/or disorders, the conjugate chemistry approach is highly acceptable and justified. Opioids have long been recognized as the most potent analgesics and serve as the basic pharmacophore for potent hybrid compounds that may be useful in pain management. However, a risk of tolerance and physical dependence exists. Since dopamine receptors have been implicated in the aforementioned adverse effects of opioids, the construction of a hybrid with dual action at opioid and dopamine receptors is of interest. Herein, we present nuclear magnetic resonance (NMR) spectroscopy and molecular dynamics simulation results for LENART01, an opioid-ranatensin hybrid peptide. Apart from molecular docking, protein-ligand interactions were also assessed in vitro using a receptor binding assay, which proved LENART01 to be bound to mu-opioid and dopamine receptors, respectively.

Xylanase immobilization using activated carrier of gellan gum-agar beads: Improved stability and catalytic activity for the production of antioxidant and anti-proliferative xylooligosaccharides

Immobilization of xylanase produced by the fungus, Aspergillus flavus, was achieved utilizing the activated gellan gum-agar as a carrier. The immobilization conditions were optimized based on Box-Behnken design which comprised the addition of agar (1.23%) to gellan-gum (GL) and processing the obtained beads at pH 7.7 with 1.62% polyethyleneimine (PEI) as the optimum conditions. Alteration of the surface morphologies and elemental constituents of the carrier during the synthesis and immobilization process were inspected using energy-dispersive X-ray analysis (EDX) and scan electron microscope (SEM). The yield and efficiency of the immobilization process were 45.85% and 97.41%, respectively. The immobilized enzyme possessed the highest activity at pH 5–8 and temperature up to 70 °C with activation energy lower than half that of the free form. Higher half-lives and Gibbs free energy were achieved for the immobilized enzyme, sustaining the suitability of the used carrier for promoting enzyme stability. Moreover, the negative entropy values for both free enzyme and the immobilized forms estimated the high stability of the enzyme during the thermal deactivation process. The Km value of the immobilized form was lower than that of the free enzyme, reflecting its enhanced affinity toward xylan substrate. Furthermore, the immobilized enzyme retained 99.3 and 85.7% of its initial xylan hydrolyzing activity after 7, and 11 cycles, respectively. Finally, the applicability of the immobilized form in the hydrolysis of xylan and production of antioxidant and breast cancer anti-proliferative xylooligosaccharide mixture was also estimated.

A promising strategy for combating bacterial infections through the use of light-triggered ROS in Ce6-immobilized hydrogels.

The reactive oxygen species (ROS) are composed of highly reactive molecules, including superoxide anions ( ), hydrogen peroxide (H2O2) and hydroxyl radicals. Researchers have explored the potential benefits of using hydrogel dressings that incorporate active substances to accelerate wound healing. The present investigation involved the development of a hyaluronic acid (HA) hydrogel capable of producing ROS using LED irradiation. The process of creating a composite hydrogel was created by chemically bonding Ce6 to an amide group. Our analysis revealed that the synthesized hydrogel had a well-structured amide bond, and the degree of cross-linking was assessed through swelling, enzyme stability and cytotoxicity tests. ROS production was found to be influenced by both the intensity and duration of light exposure. Furthermore, in situations where cell toxicity resulting from ROS generation in the hydrogel surpassed 70%, no detectable genotoxic consequences were evident, and antibacterial activity was confirmed to be directly caused by the destruction of bacterial membranes as a result of ROS damage. Furthermore, the utilization of the generated ROS influences the polarization of macrophages, resulting in the secretion of pro-inflammatory cytokines, which is a characteristic feature of M1 polarization. Subsequently, we validated the efficacy of a HA hydrogel that produces ROS to directly eradicate microorganisms. Furthermore, this hydrogel facilitated indirect antibacterial activity by stimulating macrophages to release pro-inflammatory cytokines. These cytokines are crucial for coordinating cell-mediated immune responses and for modulating the overall effectiveness of the immune system. Therefore, the Ce6-HA hydrogel has the potential to serve as an effective wound dressing solution for infected wounds because of its ability to produce substantial levels or a consistent supply.

A newly synthesized magnetic nanoparticle coated with glycidyl methacrylate monomer and 1,2,4-Triazole: Immobilization of α-Amylase from Bacillus licheniformis for more reuse, stability, and activity in the presence of H2O2

α-Amylase is a secretory enzyme commonly found in nature. The α-Amylase enzyme catalyzes the hydrolysis of α-D-(1,4)-glucosidic bonds in starch, glycogen, and polysaccharides. The chemical characterization of the composite carrier and the immobilized enzyme was performed, and the accuracy of the immobilization was confirmed by FTIR, SEM, and EDS analyses. The X-ray diffraction (XRD) analysis indicates that the magnetic nanoparticle retained its magnetic properties following the modification process. Based on the Thermogravimetric Analysis (TGA) outcomes, it was evident that the structural integrity of the FPT nanocomposite remained unchanged at 200°C. The optimal pH was determined to be 5.5, and no alteration was observed following the immobilization process. Purified α-amylases usually lose their activity rapidly above 50°C. In this study, Bacillus licheniformis α-Amylase enzyme was covalently immobilized on the newly synthesized magnetic composite carrier having more azole functional group. For novelty-designed immobilized enzymes, while there is no change in the pH and optimum operating temperature of the enzyme with immobilization, two essential advantages are provided to reduce enzyme costs: the storage stability and reusability are increased. Furthermore, our immobilization technique enhanced enzyme stability when comparing our immobilized enzyme with the reference enzyme in industrial applications. The activity of the immobilized enzyme was higher in presence of 1–3% H2O2.

An eminent approach towards next generation solvents for sustainable packaging and stability of enzymes: a comprehensive study of ionic liquid and deep eutectic solvent mixtures.

Hybrid ionic fluids (HIFs) are newly emerging and fascinating sustainable solvent media, which are attracting a great deal of scientific interest in protecting the native structure of proteins. For a few decades, there has been a demand to consider ionic liquids (ILs) and deep eutectic solvents (DESs) as biocompatible solvent media for enzymes; however, in some cases, these solvent media also show limitations. Therefore, this work focuses on synthesising novel HIFs to intensify the properties of existing ILs and DESs by mixing them. Herein, HIFs have been synthesised by the amalgamation of a deep eutectic solvent (DES) and an ionic liquid (IL) with a common cation or anion. Later on, the stability and activity of hen's egg white lysozyme (Lyz) in the presence of biocompatible solvent media and HIFs were studied by various techniques such as UV-vis, steady-state fluorescence, circular dichroism (CD), Fourier transform infrared spectroscopy (FT-IR) and dynamic light scattering (DLS) measurements. This work emphasises the effect of a DES (synthesised using 1 : 2 choline chloride and malonic acid) [Maline], ILs (1-butyl-3-methylimidazolium chloride [BMIM]Cl or choline acetate [Chn][Ac]) and their corresponding HIFs on the structure and functionality of Lyz. Moreover, we also studied the secondary structure, thermal stability, enzymatic activity and thermodynamic profile of Lyz at pH = 7 in the presence of varying concentrations (0.1 to 0.5 M) of [BMIM]Cl and [Chn][Ac] ILs, Maline as a DES, and Maline [BMIM]Cl (HIF1) and Maline [Chn][Ac] (HIF2). Spectroscopic results elucidate that ILs affect the activity and structural stability of Lyz. In contrast, the stability and activity are inhibited by DES and are enhanced by HIFs at all the studied concentrations. Overall, the experimental results studied explicitly elucidate that the structure and stability of Lyz are maintained in the presence of HIF1 while these properties are intensified in HIF2. This study shows various applications in biocompatible green solvents, particularly in the stability and functionality of proteins, due to their unique combination where the properties counteract the negative effect of either DESs or ILs in HIFs.

Incorporation of CF3-pseudoprolines into polyproline type II foldamers confers promising biophysical features.

The development and the use of fluorinated polyproline-type II (PPII) foldamers are still underexplored. Herein, trifluoromethyl pseudoprolines have been incorporated into polyproline backbones without affecting their PPII helicity. The ability of the trifluoromethyl groups to increase hydrophobicity and to act as 19F NMR probes is demonstrated. Moreover, the enzymatic stability and the non-cytotoxicity of these fluorinated foldamers make them valuable templates for use in medicinal chemistry.

Optimization of active pectin coating formulation and its effect on surface morphology and shelf-life of white button mushroom (Agaricus bisporus)

The present work aimed to optimize an active pectin-based coating to preserve microbial and enzymatic stability and maintain the quality parameters of white button mushrooms (WBM), thus increasing their shelf stability. d-optimal mixture design was implemented to study the effect of pectin (X1), ascorbic acid (X2), and ethylene-diamine-tetra acetic acid (EDTA) (X3) on polyphenol oxidase (PPO) inhibition, browning index, and total phenolic content. The optimal coating was a mixture of 2.55% w/v pectin, 0.67% w/v ascorbic acid, and 0.78% w/v EDTA. Further, the optimally coated WBM was stored for 6 days at 20 ± 1 °C. The coating on WBM significantly prevented the growth of natural microflora in the sample. Similarly, a maximum of 84% PPO inhibition was achieved at the end of the storage period. A minimum browning index (BI) of 66 was observed after six days of storage, significantly lower than that of the uncoated sample with a BI of 72.72 ± 0.90. Maximum phenolic content of 10.23 mgGAE/g WBM (dry basis) was preserved till day 6, while the antioxidant capacity was 7.16 ± 0.20 mg GAEAOC/g WBM (dry basis). Based on microbial safety and overall quality parameters, the optimal pectin-based ascorbic acid and EDTA-enriched coating led to a shelf-life of 4 days against 2 days for uncoated mushrooms at 20 °C. The optimized coating of mushrooms resulted in competitive inhibition of PPO. Fourier transform infrared (FTIR) spectroscopy revealed that EDTA and optimized coating modulated the secondary structure conformation in the amide I region. Microstructural analysis of the mushroom cap surface through scanning electron microscopy (SEM) showed that the optimized coating formed a homogeneous protective barrier on the mushroom surface, leading to the closure of intercellular spaces on the surface. The optimized coating can help mitigate postharvest losses due to the high perishability of white button mushrooms and provides a valuable solution for the industry to enhance product quality and safety.

Structural insights into binding of polyglutamylated tetrahydrofolate by serine hydroxymethyltransferase 8 from soybean.

Tetrahydrofolate and its derivatives participate in one-carbon transfer reactions in all organisms. The cellular form of tetrahydrofolate (THF) is modified by multiple glutamate residues and polyglutamylation plays a key role in organellar and cellular folate homeostasis. In addition, polyglutamylation of THF is known to increase the binding affinity to enzymes in the folate cycle, many of which can utilize polyglutamylated THF as a substrate. Here, we use X-ray crystallography to provide a high-resolution view of interactions between the enzyme serine hydroxymethyltransferase (SHMT), which provides one carbon precursors for the folate cycle, and a polyglutamylated form of THF. Our 1.7 Å crystal structure of soybean SHMT8 in complex with diglutamylated 5-formyl-THF reveals, for the first time, a structural rearrangement of a loop at the entrance to the folate binding site accompanied by the formation of novel specific interactions between the enzyme and the diglutamyl tail of the ligand. Biochemical assays show that additional glutamate moieties on the folate ligand increase both enzyme stability and binding affinity. Together these studies provide new information on SHMT structure and function and inform the design of anti-folate agents.

Rational design of deep eutectic solvents for the stabilization of dehydrogenases: an artificial neural network prediction approach.

Stabilized enzymes are crucial for the industrial application of biocatalysis due to their enhanced operational stability, which leads to prolonged enzyme activity, cost-efficiency and consequently scalability of biocatalytic processes. Over the past decade, numerous studies have demonstrated that deep eutectic solvents (DES) are excellent enzyme stabilizers. However, the search for an optimal DES has primarily relied on trial-and-error methods, lacking systematic exploration of DES structure-activity relationships. Therefore, this study aims to rationally design DES to stabilize various dehydrogenases through extensive experimental screening, followed by the development of a straightforward and reliable mathematical model to predict the efficacy of DES in enzyme stabilization. A total of 28 DES were tested for their ability to stabilize three dehydrogenases at 30°C: (S)-alcohol dehydrogenase from Rhodococcus ruber (ADH-A), (R)-alcohol dehydrogenase from Lactobacillus kefir (Lk-ADH) and glucose dehydrogenase from Bacillus megaterium (GDH). The residual activity of these enzymes in the presence of DES was quantified using first-order kinetic models. The screening revealed that DES based on polyols serve as promising stabilizing environments for the three tested dehydrogenases, particularly for the enzymes Lk-ADH and GDH, which are intrinsically unstable in aqueous environments. In glycerol-based DES, increases in enzyme half-life of up to 175-fold for Lk-ADH and 60-fold for GDH were observed compared to reference buffers. Furthermore, to establish the relationship between the enzyme inactivation rate constants and DES descriptors generated by the Conductor-like Screening Model for Real Solvents, artificial neural network models were developed. The models for ADH-A and GDH showed high efficiency and reliability (R2 > 0.75) for in silico screening of the enzyme inactivation rate constants based on DES descriptors. In conclusion, these results highlight the significant potential of the integrated experimental and in silico approach for the rational design of DES tailored to stabilize enzymes.

Enzymatic Performance of Aspergillus oryzae α-Amylase in the Presence of Organic Solvents: Activity, Stability, and Bioinformatic Studies.

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.

Enzyme stability in polymer hydrogel-enzyme hybrid nanocarrier containing phosphorylcholine group.

Enzymes are biological catalysts with good biocompatibility and high efficiency and have been widely used in many fields, such as wastewater treatment, biosensors, and the medical industry. However, their inherently low stability under conditions of practical use limits further applications. Zwitterionic polymers possessing a pair of oppositely charged groups in their repeating units can increase protein stability because of their good biocompatibility and high water content. In this study, zwitterionic copolymer nanogels comprising poly(2-methacryloyloxyethyl phosphorylcholine (MPC)-co-methacrylic acid-N-hydroxy succinimide ester (MNHS)) (PMS) were synthesized via reversible addition-fragmentation chain-transfer polymerization (RAFT). β-Galactosidase (β-gal) was post-modified within zwitterionic polymer nanogels with a covalently-bound spacer and the activity was compared with that of directly immobilized β-gal and free β-gal. Compared with direct immobilization, covalent immobilization with a spacer could reduce the structural change of β-gal, as confirmed by the circular dichroism spectra. Although the activity of β-gal decreased after immobilization, the hybrids of the β-gal immobilized nanogels, termed hybrid nanogel-enzymes, demonstrated superior stability compared to the free enzymes. The hybrid nanogel-enzymes maintained their function against inactivation by organic solvents and proteinases owing to their high water content, anti-biofouling properties, and limited mass transfer. They can also withstand protein aggregation at high temperatures and maintain their activity. Compared to direct immobilization, immobilization with a spacer resulted in a dramatic increase in the enzyme activity and a slight decrease in the stability. These results indicate that polymer nanogels containing phosphorylcholine units are promising materials for enzyme immobilization, expanding the scope of enzyme applications.

Advancements in enzyme immobilization on magnetic nanomaterials: toward sustainable industrial applications.

Enzymes are widely used in biofuels, food, and pharmaceuticals. The immobilization of enzymes on solid supports, particularly magnetic nanomaterials, enhances their stability and catalytic activity. Magnetic nanomaterials are chosen for their versatility, large surface area, and superparamagnetic properties, which allow for easy separation and reuse in industrial processes. Researchers focus on the synthesis of appropriate nanomaterials tailored for specific purposes. Immobilization protocols are predefined and adapted to both enzymes and support requirements for optimal efficiency. This review provides a detailed exploration of the application of magnetic nanomaterials in enzyme immobilization protocols. It covers methods, challenges, advantages, and future perspectives, starting with general aspects of magnetic nanomaterials, their synthesis, and applications as matrices for solid enzyme stabilization. The discussion then delves into existing enzymatic immobilization methods on magnetic nanomaterials, highlighting advantages, challenges, and potential applications. Further sections explore the industrial use of various enzymes immobilized on these materials, the development of enzyme-based bioreactors, and prospects for these biocatalysts. In summary, this review provides a concise comparison of the use of magnetic nanomaterials for enzyme stabilization, highlighting potential industrial applications and contributing to manufacturing optimization.

IN VITRO ASSESSMENT OF PROBIOTIC SURVIVAL IN SIMULATED GASTROINTESTINAL CONDITIONS AND ADHESIVE PROPERTIES

The study focused on evaluating the functional properties of four lactic acid bacteria (LAB) strains – Lactobacillus fermentum 30, Lactobacillus cellobiosus 36, Lacticaseibacillus paracasei 36/1, and Lac-ticaseibacillus paracasei 30/1 – and their associations, aiming to assess acid resistance, bile tolerance, enzyme stability, and adhesion capabilities using in vitro methods. The survival rates of these LAB strains and their associations were investigated under simulated gastric juice (SGJ) and simulated intestinal juice (SIJ). In SGJ, the association L. fermentum 30 + L. cellobiosus 36 showed higher survival rates compared to individual strains, with approximately 75% viability after 2 hours incubation. Similarly, the associa-tion L. paracasei 30/1 + L. paracasei 36/1 exhibited enhanced survival in SGJ, maintaining about 80% viability. In SIJ, both associations demonstrated improved survival compared to individual strains, with L. fermentum 30 + L. cellobiosus 36 and L. paracasei 30/1 + L. paracasei 36/1 maintaining approxi-mately 70% and 75% viability, respectively. The adhesion capabilities of these LAB strains and associa-tions were evaluated using human erythrocytes. All strains displayed high adhesive activity, particularly notable in L. paracasei 36/1 + L. paracasei 30/1, which demonstrated a significantly higher adhesion index compared to other strains. These findings highlight the robust survival and adhesive properties of the LAB strains and their associations under simulated gastrointestinal conditions. Further research is warranted to explore their potential applications in promoting gut health and combating gastrointestinal disorders.

Improving the thermostability and modulating the inulin profile of inulosucrase through rational glycine-to-proline substitution.

The flexibility of protein structure plays a crucial role in enzyme stability and catalysis. Among the amino acids, glycine is particularly important in conferring flexibility to proteins. In this study, the effects of flexible glycine residues in Lactobacillus reuteri 121 inulosucrase (LrInu) on stability and inulin profile were investigated through glycine-to-proline substitutions. Molecular dynamics (MD) simulations were employed to discover the flexible glycine residues, and eight glycine residues, including Gly217, Gly298, Gly330, Gly416, Gly450, Gly624, Gly627, Gly629, were selected for site-directed mutagenesis. The results demonstrated significant changes in both thermostability and inulin profiles of the variants. Particularly, the G624P and G627P variants showed reduced production of long-chain oligosaccharides compared to the WT. This can be ascribed to the increased rigidity of the active site, which is crucial for the induction-fit mechanism. Overall, this study provides valuable insights into the role of flexible glycine residues in the activity, stability, and inulin synthesis of LrInu.

Improvement of DERA activity and stability in the synthesis of statin precursors by immobilization on magnetic nanoparticles

Higher stability and hyperactivation of the DERA enzyme were achieved by covalent bonding to magnetic nanoparticles with succinic anhydride as an activating agent.