Enzyme-substrate Interactions Research Articles

The dithiol mechanism of class I glutaredoxins promotes specificity for glutathione as a reducing agent

Class I glutaredoxins reversibly reduce glutathione- and nonglutathione disulfides with the help of reduced glutathione (GSH) using either a monothiol mechanism or a dithiol mechanism. The monothiol mechanism exclusively involves a single glutathionylated active-site cysteinyl residue, whereas the dithiol mechanism requires the additional formation of an intramolecular disulfide bond between the active-site cysteinyl residue and a resolving cysteinyl residue. While the oxidation of glutaredoxins by glutathione disulfide substrates has been extensively characterized, the enzyme-substrate interactions for the reduction of S-glutathionylated glutaredoxins or intramolecular glutaredoxin disulfides are still poorly characterized. Here we compared the thiol-specificity for the reduction of S-glutathionylated glutaredoxins and the intramolecular glutaredoxin disulfide. We show that S-glutathionylated glutaredoxins rapidly react with a plethora of thiols and that the 2nd glutathione-interaction site of class I glutaredoxins lacks specificity for GSH as a reducing agent. In contrast, the slower reduction of the partially strained intramolecular glutaredoxin disulfide involves specific interactions with both carboxylate groups of GSH at the 1st glutathione-interaction site. Thus, the dithiol mechanism of class I glutaredoxins promotes specificity for GSH as a reducing agent, which might explain the prevalence of dithiol glutaredoxins in pro- and eukaryotes.

Prediction of SHP2-E76K binding sites based on molecular dynamics simulation and Markov algorithm

SHP2-E76K, a mutant encoded by the PTPN11 gene, was associated with various solid tumors, such as lung cancer, glioblastoma, and intellectual disability. SHP2-E76K has become potential drug targets, while there was no effective inhibitor against the mutant currently. At present, the crystal complex structure of SHP099 with SHP2-E76K has been reported in the RCSB PDB protein data bank, however, the dynamic structure of SHP099 binding to the active center of SHP2-E76K protein was still lacking. Therefore, this study used molecular dynamics simulation and Markov model to characterize the kinetics of the inhibitor SHP099 with SHP2-E76K enzyme and to determine the active binding site, which would give a hint of a vital enzyme-substrate interaction in atomistic detail that proposed the potential to be applied for the discovery of more effective SHP2-E76K inhibitors and, in broader terms, dynamic protein-drug interactions.

Selective Adsorption of Lignin Peroxidase on Lignin for Biocatalytic Conversion of Poplar Wood Biomass to Value-Added Chemicals.

Lignin-first biorefineries aim to maximize the valorization of lignin by prioritizing its conversion over other biomass components. This study investigates the selective adsorption of lignin peroxidase (LiP) isozymes from white rot fungi Phanerochaete chrysosporium on lignin, aiming to enhance the biocatalytic conversion of poplar wood biomass into value-added chemicals. The research focuses on the adsorption characteristics of ten recombinant LiP isozymes, particularly PcLiP03, which exhibited the highest adsorption capacity on lignin and negligible adsorption on cellulose. Adsorption isotherms and structural analyses revealed that hydrophobic interactions significantly contribute to the selective adsorption of PcLiP03. Applying PcLiP03 in a continuous stirring cell system effectively converted lignin in unpretreated poplar wood to 2,6-dimethoxy-1,4-benzoquinone under ambient and mild conditions, highlighting its potential for selective lignin depolymerization in integrated biorefineries. This work underscores the importance of enzyme-substrate interactions and offers a promising approach for more efficient and sustainable biomass utilization.

Structural study and molecular docking insights into laccase-mediated dye degradation

Industrial wastewater often contains harmful dyes, posing significant environmental challenges. This study used computational tools to investigate the interactions between the low-degradability Azo dye, Reactive Blue 19, and laccase enzyme from two fungi species, Lentinus sp. WR2 and Trametes versicolor, as well as the protein Xyn5B (GH30) from Bacillus sp. BP7. Physiochemical characterization indicated acidic properties for Lentinus sp. WR2 and T. versicolor, while basic characteristics for Bacillus sp. BP7. Secondary structure (SOPMA analysis) revealed that most laccases had random coiling, enhancing protein flexibility. The negative GRAVY index revealed favorable water interaction for Lentinus sp. WR2 and Bacillus sp. BP7. The ERRAT plot validated the laccase 3D structure. The molecular docking results were compared using active site prediction and docking score. Lentinus sp. WR2 is linked with Reactive Blue 19 through a single residual amino acid THR 168; T. versicolor is linked through GLY 101, TRP 98, and TRP 512; and Bacillus sp. BP7 exhibited four linkages: VAL 262, TYR 263, TRP 117, and THR 260. The molecular docking results showed that Xyn5B demonstrated laccase activity and bound effectively with Reactive Blue 19, outperforming a commonly used fungal laccase in dye degradation. These findings enhance our understanding of enzyme-substrate interactions and highlight the potential of Xyn5B in eco-friendly dye degradation.

Enzyme catalytic efficiency prediction: employing convolutional neural networks and XGBoost.

In the intricate realm of enzymology, the precise quantification of enzyme efficiency, epitomized by the turnover number (k cat), is a paramount yet elusive objective. Existing methodologies, though sophisticated, often grapple with the inherent stochasticity and multifaceted nature of enzymatic reactions. Thus, there arises a necessity to explore avant-garde computational paradigms. In this context, we introduce "enzyme catalytic efficiency prediction (ECEP)," leveraging advanced deep learning techniques to enhance the previous implementation, TurNuP, for predicting the enzyme catalase k cat. Our approach significantly outperforms prior methodologies, incorporating new features derived from enzyme sequences and chemical reaction dynamics. Through ECEP, we unravel the intricate enzyme-substrate interactions, capturing the nuanced interplay of molecular determinants. Preliminary assessments, compared against established models like TurNuP and DLKcat, underscore the superior predictive capabilities of ECEP, marking a pivotal shift in silico enzymatic turnover number estimation. This study enriches the computational toolkit available to enzymologists and lays the groundwork for future explorations in the burgeoning field of bioinformatics. This paper suggested a multi-feature ensemble deep learning-based approach to predict enzyme kinetic parameters using an ensemble convolution neural network and XGBoost by calculating weighted-average of each feature-based model's output to outperform traditional machine learning methods. The proposed "ECEP" model significantly outperformed existing methodologies, achieving a mean squared error (MSE) reduction of 0.35 from 0.81 to 0.46 and R-squared score from 0.44 to 0.54, thereby demonstrating its superior accuracy and effectiveness in enzyme catalytic efficiency prediction. This improvement underscores the model's potential to enhance the field of bioinformatics, setting a new benchmark for performance.

Engineering Bacterial Chitinases for Industrial Application: From Protein Engineering to Bacterial Strains Mutation! A Comprehensive Review of Physical, Molecular, and Computational Approaches.

Bacterial chitinases are integral in breaking down chitin, the natural polymer in crustacean and insect exoskeletons. Their increasing utilization across various sectors such as agriculture, waste management, biotechnology, food processing, and pharmaceutical industries highlights their significance as biocatalysts. The current review investigates various scientific strategies to maximize the efficiency and production of bacterial chitinases for industrial use. Our goal is to optimize the heterologous production process using physical, molecular, and computational tools. Physical methods focus on isolating, purifying, and characterizing chitinases from various sources to ensure optimal conditions for maximum enzyme activity. Molecular techniques involve gene cloning, site-directed mutation, and CRISPR-Cas9 gene editing as an approach for creating chitinases with improved catalytic activity, substrate specificity, and stability. Computational approaches use molecular modeling, docking, and simulation techniques to accurately predict enzyme-substrate interactions and enhance chitinase variants' design. Integrating multidisciplinary strategies enables the development of highly efficient chitinases tailored for specific industrial applications. This review summarizes current knowledge and advances in chitinase engineering to serve as an indispensable guideline for researchers and industrialists seeking to optimize chitinase production for various uses.

Selective transformation of crocin-1 to crocetin-glucosyl esters by β-glucosidase (Lf18920) from Leifsonia sp. ZF2019: Insights from molecular docking and point mutations

Crocetin di/mono-glucosyl esters (crocin-4 and crocin-5) are rarely distributed in nature, limiting their potential applications in the food and pharmaceutical industries. In the present study, a novel GH3 family β-glucosidase Lf18920 was identified from Leifsonia sp. ZF2019, which selectively hydrolyzed crocin-1 (crocetin di-gentiobiosyl ester) to crocin-5 and crocin-4, but not to its aglycone, crocetin. Under the optimal condition of 40 °C and pH 6.0 for 120 min, Lf18920 almost completely hydrolyzed crocin-1, yielding 73.50±5.66 % crocin-4 and 16.19±1.38 % crocin-5. Molecular docking and point mutation studies revealed that Lf18920 formed a narrow binding channel that facilitated crocin-1 binding. Five single amino acid variants (D50A, D53A, W274A, G420A, and Q421A) were constructed, all of which showed reduced hydrolytic activity. Mutations at D50 and D53, located distal to the active site, increased binding energy and decreased hydrolytic activity, while mutations at W274, G420, and Q421, proximal to the active site, disrupted hydrolytic function. These findings suggest that the narrow binding channel and specific enzyme-substrate interactions are crucial for Lf18920’s selective hydrolytic activity. Overall, this study is the first to report a β-glucosidase capable of selectively transforming crocin-1 to crocetin di/mono-glucosyl esters, offering potential for synthesizing crocin-4 and crocin-5.

A method to detect enzymatic reactions with field effect transistor

Study of enzyme-substrate interactions is a task of great practical and scientific importance. This paper describes the application of ion-sensitive field effect transistors in quasi-equilibrium state for examination of enzymatic reactions. A reaction occurring in the liquid gate affects the chemical potential of electrons in this gate, and this phenomenon may be used to explore biochemical interactions. This strategy can be applied to detect interactions of enzymes with substrates, inhibitors and activators regardless of their optical and electrochemical properties. Using the developed method, the reactions catalyzed by the enzymes belonging to six different EC classes were analyzed, and Michaelis constants for their substrates were determibed. Km values obtained using the proposed method were in good agreement with those obtained with standard colorimetric and fluorimentric assays. Practical potential of the described method was demonstrated by studying the interactions of a diagnostically significant enzyme α-D-galactosidase with its natural and artificial substrates and its inhibitor. Km values for α-D-galactosidase using melibiose and raffinose as substrates and IC50 value for the enzyme inhibitor 1-deoxygalactonojirimycin were determined. The described method allows rapid and label-free investigation of enzyme interactions with substrates, inhibitors and activators for a wide range of biocatalysts.

Structural and biochemical insights into the molecular mechanism of N-acetylglucosamine/N-Acetylmuramic acid kinase MurK from Clostridium acetobutylicum

MurK is a MurNAc- and GlcNAc-specific amino sugar kinase, phosphorylates MurNAc and GlcNAc at the 6-hydroxyl group in an ATP-dependent manner, and contributes to the recovery of both amino sugars during the cell wall turnover in Clostridium acetobutylicum. Herein, we determined the crystal structures of MurK in complex with MurNAc, GlcNAc, and glucose, respectively. MurK represents the V-shaped fold, which is divided into a small N-terminal domain and a large C-terminal domain. The catalytic pocket is located within the deep cavity between the two domains of the MurK monomer. We mapped the significant enzyme-substrate interactions, identified key residues involved in the catalytic activity of MurK, and found that residues Asp77 and Arg78 from the β4-α2-loop confer structural flexibilities to specifically accommodate GlcNAc and MurNAc, respectively. Moreover, structural comparison revealed that MurK adopts closed-active conformation induced by the N-acetyl moiety from GlcNAc/MurNAc, rather than closed-inactive conformation induced by glucose, to carry out its catalytic reaction. Taken together, our study provides structural and functional insights into the molecular mechanism of MurK for the phosphorylation of both MurNAc and GlcNAc, sugar substrate specificity, and conformational changes upon sugar substrate binding.

Transglutaminase mediates the hardening of fish egg envelope produced by duplication of factor XIIIA gene during the evolution of Teleostei.

During the fertilization of fish eggs, the hardening of the egg envelope is mediated by transglutaminase (hTGase). After fertilization, TGase undergoes processing. We isolated hTGase from extracts of unfertilized and water-activated rainbow trout eggs. Rainbow trout hTGase (Rt-hTGase) appeared as an 80 kDa protein, and its processed form was 55 kDa. Their N-terminal amino acid sequences were nearly identical, suggesting processing in the C-terminal region. The specific activities were not significantly different, indicating that C-terminal processing does not activate the enzyme itself. We cloned the cDNA by reverse transcription polymerase chain reaction (RT-PCR) using degenerate primers followed by RACE-PCR. The deduced amino acid sequence of the cDNA was similar to that of factor XIII subunit A (FXIIIA). Molecular phylogenetic and gene syntenic analyses clearly showed that hTGase was produced by duplication of FXIIIA during the evolution to Teleostei. The 55 kDa processed form of Rt-hTGase is predominantly composed of an enzyme domain predicted from the amino acid sequence of the cDNA. It is hypothesized that the C-terminal domain of Rt-hTGase binds to egg envelope proteins, and that processing allows the enzyme to move freely within the egg envelope, increasing substrate-enzyme interaction and thereby accelerating hardening.

Mechanistic Insight into the Enantioselective Degradation of Esterase QeH to (R)/(S)-Quizalofop-Ethyl with Molecular Dynamics Simulation Using a Residue-Specific Force Field.

The enantioselective mechanism of the esterase QeH against the two enantiomers of quizalofop-ethyl (QE) has been primitively studied using computational and experimental approaches. However, it is still unclear how the esterase QeH adjusts its conformation to adapt to substrate binding and promote enzyme-substrate interactions in the catalytic kinetics. The equilibrium processes of enzyme-substrate interactions and catalytic dynamics were reproduced by performing independent molecular dynamics (MD) runs on the QeH-(R)/(S)-QE complexes with a newly developed residue-specific force field (RSFF2C). Our results indicated that the benzene ring of the (R)-QE structure can simultaneously form anion-π and cation-π interactions with the side-chain group of Glu328 and Arg384 in the binding cavity of the QeH-(R)-QE complex, resulting in (R)-QE being closer to its catalytic triplet system (Ser78-Lys81-Tyr189) with the distances measured for the hydroxyl oxygen atom of the catalytic Ser78 of QeH and the carbonyl carbon atom of (R)-QE of 7.39 Å, compared to the 8.87 Å for (S)-QE, whereas the (S)-QE structure can only form an anion-π interaction with the side chain of Glu328 in the QeH-(S)-QE complex, being less close to its catalytic site. The computational alanine scanning mutation (CAS) calculations further demonstrated that the π-π stacking interaction between the indole ring of Trp351 and the benzene ring of (R)/(S)-QE contributed a lot to the binding stability of the enzyme-substrate (QeH-(R)/(S)-QE). These results facilitate the understanding of their catalytic processes and provide new theoretical guidance for the directional design of other key enzymes for the initial degradation of aryloxyphenoxypropionate (AOPP) herbicides with higher catalytic efficiencies.

Biocatalytic Synthesis of Ruxolitinib Intermediate via Engineered Imine Reductase.

An enzyme catalyzed strategy for the synthesis of a chiral hydrazine from 3-cyclopentyl-3-oxopropanenitrile 5 and hydrazine hydrate 2 is presented. An imine reductase (IRED) from Streptosporangium roseum was identified to catalyze the reaction between 3-cyclopentyl-3-oxopropanenitrile 5 and hydrazine hydrate 2 to produce trace amounts of (R)-3-cyclopentyl-3-hydrazineylpropanenitrile 4. We employed a 2-fold approach to optimize the catalytic performance of this enzyme. First, a transition state analogue (TSA) model was constructed to illuminate the enzyme-substrate interactions. Subsequently, the Enzyme_design and Funclib methods were utilized to predict mutants for experimental evaluation. Through three rounds of site-directed mutagenesis, site saturation mutagenesis, and combinatorial mutagenesis, we obtained mutant M6 with a yield of 98% and an enantiomeric excess (ee) of 99%. This study presents an effective method for constructing a hydrazine derivative via IRED-catalyzed reductive amination of ketone and hydrazine. Furthermore, it provides a general approach for constructing suitable enzymes, starting from nonreactive enzymes and gradually enhancing their catalytic activity through active site modifications.

Quantitative principles of microbial metabolism shared across scales.

Metabolism is the complex network of chemical reactions occurring within every cell and organism, maintaining life, mediating ecosystem processes and affecting Earth's climate. Experiments and models of microbial metabolism often focus on one specific scale, overlooking the connectivity between molecules, cells and ecosystems. Here we highlight quantitative metabolic principles that exhibit commonalities across scales, which we argue could help to achieve an integrated perspective on microbial life. Mass, electron and energy balance provide quantitative constraints on their flow within metabolic networks, organisms and ecosystems, shaping how each responds to its environment. The mechanisms underlying these flows, such as enzyme-substrate interactions, often involve encounter and handling stages that are represented by equations similar to those for cells and resources, or predators and prey. We propose that these formal similarities reflect shared principles and discuss how their investigation through experiments and models may contribute to a common language for studying microbial metabolism across scales.

Structure-Assisted Design of Chitosanase Product Specificity for the Production of High-Degree Polymerization Chitooligosaccharides.

Chitosanases are valuable enzymatic tools in the food industry for converting chitosan into functional chitooligosaccharides (COSs). However, most of the chitosanases extensively characterized produced a low degree of polymerization (DP) COSs (DP = 1-3, LdpCOSs), indicating an imperative for enhancements in the product specificity for the high DP COS (DP >3, HdpCOSs) production. In this study, a chitosanase from Methanosarcina sp. 1.H.T.1A.1 (OUC-CsnA4) was cloned and expressed. Analysis of the enzyme-substrate interactions and the subsite architecture of the OUC-CsnA4 indicated that a Ser49 mutation could modify its interaction pattern with the substrate, potentially enhancing product specificity for producing HdpCOSs. Site-directed mutagenesis provided evidence that the S49I and S49P mutations in OUC-CsnA4 enabled the production of up to 24 and 26% of (GlcN)5 from chitosan, respectively─the wild-type enzyme was unable to produce detectable levels of (GlcN)5. These mutations also altered substrate binding preferences, favoring the binding of longer-chain COSs (DP >5) and enhancing (GlcN)5 production. Furthermore, molecular dynamics simulations and molecular docking studies underscored the significance of +2 subsite interactions in determining the (GlcN)4 and (GlcN)5 product specificity. These findings revealed that the positioning and interactions of the reducing end of the substrate within the catalytic cleft are crucial factors influencing the product specificity of chitosanase.

Correlation between Molecular Docking and the Stabilizing Interaction of HOMO-LUMO: Spirostans in CHK1 and CHK2, an In Silico Cancer Approach.

Checkpoint kinases 1 and 2 (CHK1 and CHK2) are enzymes that are involved in the control of DNA damage. At the present time, these enzymes are some of the most important targets in the fight against cancer since their inhibition produces cytotoxic effects in carcinogenic cells. This paper proposes the use of spirostans (Sp), natural compounds, as possible inhibitors of the enzymes CHK1 and CHK2 from an in silico analysis of a database of 155 molecules (S5). Bioinformatics studies of molecular docking were able to discriminate between 13 possible CHK1 inhibitors, 13 CHK2 inhibitors and 1 dual inhibitor for both enzymes. The administration, distribution, metabolism, excretion and toxicity (ADMETx) studies allowed a prediction of the distribution and metabolism of the potential inhibitors in the body, as well as determining the excretion routes and the appropriate administration route. The best inhibition candidates were discriminated by comparing the enzyme-substrate interactions from 2D diagrams and molecular docking. Specific inhibition candidates were obtained, in addition to studying the dual inhibitor candidate and observing their stability in dynamic molecular studies. In addition, Highest Occupied Molecular Orbital-Lowest Unoccupied Molecular Orbital (HOMO-LUMO) interactions were analyzed to study the stability of interactions between the selected enzymes and spirostans resulting in the predominant gaps from HOMOCHKs to LUMOSp (Highest Occupied Molecular Orbital of CHKs-Lowest Unoccupied Molecular Orbital of spirostan). In brief, this study presents the selection inhibitors of CHK1 and CHK2 as a potential treatment for cancer using a combination of molecular docking and dynamics, ADMETx predictons, and HOMO-LUMO calculation for selection.

Reshaping the binding pocket of D-tagaturonate epimerase UxaE to improve the epimerization activity of C4-OH for enabling green synthesis of d-tagatose

d-tagatose, a rare sugar with broad applications in the food and pharmaceutical industries, can be synthesized from d-fructose by epimerases, an approach with significant biosynthesis potential. However, so far there has been only one attempt to engineer tagatose 4‑epimerase through random mutagenesis. The lack of exploration has severely restricted the application of epimerases in the industrial production of d-tagatose. The previous research primarily focused on enhancing the catalytic activity of epimerases through random mutagenesis, with limited understanding of how substrate-adjacent residues affect enzyme efficiency, which was hence the subject of this study. First, using a semi-rational design approach, after three rounds of mutagenesis and screening, the mutant (S125D/S268A/Q124E) of D-tagaturonate epimerase (UxaE) from Thermotoga neapolitana was obtained, which increased d-tagatose production efficiency by 10.8-fold compared to the wild type, with a specific activity of 1505.6 U/g, representing the highest specific activity reported for a UxaE mutant to date. Molecular dynamics simulations showed that the substitution of Ser to Asp-125 enhanced substrate-enzyme interactions, while the substitution of Glu to Gln-124 reinforced the hydrogen bond network around the catalytic residue Glu-128, further reshaping the substrate-binding pocket and enhancing catalytic activity. This study for the first time explored the enhancement of the C4 epimerization function of epimerases by reshaping their substrate-binding pockets, thereby providing a feasible route for the industrial biosynthesis of d-tagatose.

Zwitterionic Polyelectrolyte Complex Vesicles Assembled from Homopoly(2-Oxazoline)s as Enzyme Catalytic Nanoreactors for Potent Anti-Tumor Efficiency.

Enzymes are known for their remarkable catalytic efficiency across a wide range of applications. Here, we present a novel and convenient nanoreactor platform based on zwitterionic polyelectrolyte complex vesicles (PCVs), assembled from oppositely charged homopoly(2-oxazoline)s, facilitating enzyme immobilization. We show remarkable enhancements in catalytic activity and stability by encapsulation of lipase as a model enzyme. Even as the temperature rises, the performance of the lipase remains robust. Further, the structural characteristics of PCVs, including hollow architecture and semipermeable membranes, endow them with unique advantages for enzyme cascade reactions involving glucose oxidase (GOx) and horseradish peroxidase (HRP). A decline in catalytic efficiency is shown when the enzymes are individually loaded and subsequently mixed, in contrast to the coloaded GOx-HRP-PCV group. We demonstrate that the vesicle structures establish confined environments where precise enzyme-substrate interactions facilitate enhanced catalytic efficiency. In addition, the nanoreactors exhibit excellent biocompatibility and efficient anti-tumor activity, which hold significant promise for biomedical applications within enzyme-based technologies.

Substrate Characterization for Hydrolysis of Multiple Types of Aromatic Esters by Promiscuous Aminopeptidases.

Synthetic aromatic esters, widely employed in agriculture, food, and chemical industries, have become emerging environmental pollutants due to their strong hydrophobicity and poor bioavailability. This study attempted to address this issue by extracellularly expressing the promiscuous aminopeptidase (Aps) from Pseudomonas aeruginosa GF31 in B. subtilis, achieving an impressive enzyme activity of 13.7 U/mg. Notably, we have demonstrated, for the first time, the Aps-mediated degradation of diverse aromatic esters, including but not limited to pyrethroids, phthalates, and parabens. A biochemical characterization of Aps reveals its esterase properties and a broader spectrum of substrate profiles. The degradation rates of p-nitrobenzene esters (p-NB) with different side chain structures vary under the action of Aps, showing a preference for substrates with relatively longer alkyl side chains. The structure-dependent degradability aligns well with the binding energies between Aps and p-NB. Molecular docking and enzyme-substrate interaction elucidate that hydrogen bonding, hydrophobic interactions, and π-π stacking collectively stabilize the enzyme-substrate conformation, promoting substrate hydrolysis. These findings provide new insights into the enzymatic degradation of aromatic ester pollutants, laying a foundation for the further development and modification of promiscuous enzymes.

Efficient and rapid digestion of proteins with a dual-enzyme microreactor featuring 3-D pores formed by dopamine/polyethyleneimine/acrylamide-coated KIT-6 molecular sieve

The microreactor with two types of immobilized enzymes, exhibiting excellent orthogonal performance, represents an effective approach to counteract the reduced digestion efficiency resulting from the absence of a single enzyme cleavage site, thereby impacting protein identification. In this study, we developed a hydrophilic dual-enzyme microreactor characterized by rapid mass transfer and superior enzymatic activity. Initially, we selected KIT-6 molecular sieve as the carrier for the dual-IMER due to its three-dimensional network pore structure. Modification involved co-deposition of polyethyleneimine (PEI) and acrylamide (AM) as amine donors, along with dopamine to enhance material hydrophilicity. Remaining amino and double bond functional groups facilitated stepwise immobilization of trypsin and Glu-C. Digestion times for bovine serum albumin (BSA) and bovine hemoglobin (BHb) on the dual-IMER were significantly reduced compared to solution-based digestion (1 min vs. 36 h), resulting in improved sequence coverage (91.30% vs. 82.7% for BSA; 90.24% vs. 89.20% for BHb). Additionally, the dual-IMER demonstrated excellent durability, retaining 96.08% relative activity after 29 reuse cycles. Enhanced protein digestion efficiency can be attributed to several factors: (1) KIT-6’s large specific surface area, enabling higher enzyme loading capacity; (2) Its three-dimensional network pore structure, facilitating faster mass transfer and substance diffusion; (3) Orthogonality of trypsin and Glu-C enzyme cleavage sites; (4) The spatial effect introduced by the chain structure of PEI and glutaraldehyde’s spacing arm, reducing spatial hindrance and enhancing enzyme–substrate interactions; (5) Mild and stable enzyme immobilization. The KIT-6-based dual-IMER offers a promising technical tool for protein digestion, while the PDA/PEI/AM-KIT-6 platform holds potential for immobilizing other proteins or active substances.

Hydrolytic enzyme activity in high-rate anaerobic reactors treating municipal wastewater in temperate climates

Particulate matter hydrolysis is the bottleneck in anaerobic treatment of municipal wastewater in temperate climates. Low temperatures theoretically slow enzyme-substrate interactions, hindering utilization kinetics, but this remains poorly understood. β-glucosidase, protease, and lipase activities were evaluated in two pilot-scale upflow anaerobic sludge blanket (UASB) reactors, inoculated with different sludges and later converted to anaerobic membrane bioreactors (AnMBRs). Despite similar methane production and solids hydrolysis rates, significant differences emerged. Specific activity peaked at 37 °C, excluding the predominance of psychrophilic enzymes. Nevertheless, the Michaelis-Menten constant (Km) indicated high enzyme-substrate affinity at the operational temperature of 15–20 °C, notably greater in AnMBRs. It is shown, for the first time, that different seed sludges can equally adapt, as hydrolytic enzymatic affinity to the substrate reached similar values in the two reactors at the operational temperature and identified that membrane ultrafiltration impacted hydrolysis by a favourable enzyme Michaelis-Menten constant.