Enzyme Complex Research Articles

Recent Advances in Discovery, Structure, Bioactivity, and Biosynthesis of trans-AT Polyketides.

Bacterial trans-acyltransferase polyketide synthases (trans-AT PKSs) are among the most complex enzymes, which are responsible for generating a wide range of natural products, identified as trans-AT polyketides. These polyketides have received significant attention in drug development due to their structural diversity and potent bioactivities. With approximately 300 synthesized molecules discovered so far, trans-AT PKSs are found widespread in bacteria. Their biosynthesis pathways exhibit considerable genetic diversity, leading to the emergence of numerous enzymes with novel mechanisms, serving as a valuable resource for genetic engineering aimed at modifying small molecules' structures and creating new engineered enzymes. Despite the systematic discussions on trans-AT polyketides and their biosynthesis in earlier studies, the continuous advancements in tools, methods, compound identification, and biosynthetic pathways require a fresh update on accumulated knowledge. This review seeks to provide a comprehensive discussion for the 27 types of trans-AT polyketides discovered within the last seven years, detailing their sources, structures, biological activities, and biosynthetic pathways. By reviewing this new knowledge, a more profound understanding of the trans-AT polyketide family can be achieved.

In vitro transcription-based biosensing of glycolate for prototyping of a complex enzyme cascade.

In vitro metabolic systems allow the reconstitution of natural and new-to-nature pathways outside of their cellular context and are of increasing interest in bottom-up synthetic biology, cell-free manufacturing, and metabolic engineering. Yet, the analysis of the activity of such in vitro networks is very often restricted by time- and cost-intensive methods. To overcome these limitations, we sought to develop an in vitro transcription (IVT)-based biosensing workflow that is compatible with the complex conditions of in vitro metabolism, such as the crotonyl-CoA/ethylmalonyl-CoA/hydroxybutyryl-CoA (CETCH) cycle, a 27-component in vitro metabolic system that converts CO2 into glycolate. As proof of concept, we constructed a novel glycolate sensor module that is based on the transcriptional repressor GlcR from Paracoccus denitrificans and established an IVT biosensing workflow that allows us to quantify glycolate from CETCH samples in the micromolar to millimolar range. We investigate the influence of 13 (shared) cofactors between the two in vitro systems to show that Mg2+, adenosine triphosphate , and other phosphorylated metabolites are critical for robust signal output. Our optimized IVT biosensor correlates well with liquid chromatography-mass spectrometry-based glycolate quantification of CETCH samples, with one or multiple components varying (linear correlation 0.94-0.98), but notably at ∼10-fold lowered cost and ∼10 times faster turnover time. Our results demonstrate the potential and challenges of IVT-based systems to quantify and prototype the activity of complex reaction cascades and in vitro metabolic networks.

Dual-functional probe-based multi-signal immunosensor platform for tropane alkaloids: Verification and evaluation

This study aims to realise rapid detecting of tropane alkaloids (TAs) in food. For this purpose, a broad-spectrum single-chain fragment variable was fused with horseradish peroxidase to create an antibody–enzyme complex (AEC) with antigen recognition and catalytic activity. A multi-signal immunosensor platform based on AEC in the direct competitive reaction mode was constructed using 3,3′,5,5′-tetramethylbenzidine and 10-acetyl-3,7-dihydroxyphenoxazine as substrates. The sensitivity of TAs in the immunosensor platform ranged from 0.25 μg/kg to 7912.46 μg/kg. Honey was selected as a representative food sample, and the limit of detection of TAs in honey ranged from 0.02 μg/kg to 409.11 μg/kg, with a recovery rate of 65.7 %–117.1 % and a coefficient of variation less than 21.4 %. Results showed that the immunosensor platform possesses satisfactory accuracy and precision, which highlights its potential for practical applications and its suitability as an ideal tool for rapid screening of TAs in food.

PSXI-10 Effects of feeding doses of enzyme complex and corn ethanol coproduct on performance and carcass traits of finishing beef cattle

Abstract The use of enzyme complex and the inclusion of corn ethanol co-products are strategies that can increase feed efficiency and carcass weight of feedlot beef cattle. Crossbred bulls (n = 389) were assigned to a randomized completed block design, according to their initial shrunk body weight (BW; 314 ± 40 kg; 10 pen/treatment). The treatment arrangement was a 2 × 3 factorial, with Factor 1 defined by the inclusion of corn dried bran/fiber plus distillers solubles (15 or 45% of DFS; FS Ouro from FS Bioenergy) and factor 2 defined by the increasing doses of multicarbohydrase complex (Rovabio Advance from Adisseo Company; 0, 0.75 or 1.0 g for every 10 kg of diet DM)). The diets contained 8.5% sugar cane bagasse, 44 or 73% ground flint corn, 15 or 45% DFS, 0.5 or 1.5% urea and 2% mineral mix plus monensin (30 ppm in the diet) with the respective enzyme complex doses. On d -1 and 122, individual BW were recorded after a 16-h of fasting (feed and water). The dry matter (DM) intake (DMI) was determined by the difference of feed offer and orts samples after dried at 105°C for 24 h to determine DM. The pen average daily gain (ADG) and DMI were used to calculate feed efficiency (ADG/DMI). On d 122, subcutaneous fat thickness (FT) and the rib eye area (REA) were obtained by means of an ultrasound image, and hot carcass weight (HCW) was obtained upon slaughter on the next day. Data were analyzed using the MIXED procedure of SAS. There was no interaction between the inclusion of DFS and enzyme doses for the variables evaluated (P > 0.05). The use of enzyme complex had no effect on cattle DMI, ADG and FE (P > 0.05). No effects of DFS levels were observed on daily DMI (P ≥ 0.45) or final BW (P ≥ 0.33). Nonetheless, cattle fed 45% DFS had greater ADG (1.37 vs. 1.28 kg; P = 0.02) and FE (153 vs. 145 g/ kg; P = 0.03) than cattle fed 15% DFS. There was no effect of the inclusion of DFS or the doses of the enzyme complex on the carcass parameters evaluated. In summary, including 45% DFS in finishing diets improved feedlot cattle performance compared with 15%. The use of the enzyme complex did not affect animal performance.

PPP2R5E: New gene potentially involved in specific learning disorders and myopathy

Protein phosphatase 2A (PP2A) is a family of multifunctional enzymatic complexes crucial for cellular signalling, playing a pivotal role in brain function and development. Mutations in specific genes encoding PP2A complexes have been associated with neurodevelopmental disorders with hypotonia and high risk of seizures. In the current work, we present an individual with specific learning problems, motor coordination disorders, hypotonia and behavioural issues. Although whole exome sequencing (WES) did not unveil pathogenic variants in known genes related to these symptoms, a de novo heterozygous variant Glu191Lys was identified within PPP2R5E, encoding the PP2A regulatory subunit B56ε. The novel variant was not observed in the four healthy brothers and was not detected as parental somatic mosaicism. The mutation predicted a change of charge of the mutated amino acid within a conserved LFDSEDPRER motif common to all PPP2R5 B-subunits. Biochemical assays demonstrated a decreased interaction with the PP2A A and C subunits, leading to disturbances in holoenzyme formation, and thus likely, function. For the first time, we report a potential causal link between the observed variant within the PPP2R5E gene and the symptoms manifested in the subject, spanning specific learning problems and motor coordination disorders potentially associated with myopathy.

Coupling of the catalytic reactions from formate dehydrogenase and hydrogenase in solution: Insights from computations and in situ IR spectroscopy

Sophisticated enzymatic systems have evolved in nature to efficiently couple distinct biochemical reactions in form of cascades. As such they serve as reference models to understand the indirect interactions of catalytic centers. Herein, we studied, in solution, the coupling of the reactions from membrane‐bound [NiFe] hydrogenase (MBH) from Cupriavidus necator (reversible H2 splitting into H+ and e‐) and the molybdenum‐dependent formate dehydrogenase from Rhodobacter capsulatus (reversible formate to CO2 interconversion). To follow their interplay via the characteristic absorptions from the MBH's active site or the respective substrate and product bands of FDH, we utilized in situ IR spectrocopy and GC(‐MS), in the absence or presence of soluble redox mediators. Coarse grained molecular dynamics (cgMD) computations revealed the lack of productive enzyme complexes for direct electron transfer (ET). Thus, the observed minor amounts of H2 or formate were produced from transient interactions between the two enzymes. On the contrary, the significantly increased product formation in the presence of methylene viologen can be related to the putative multiple interaction sites of the redox mediator with FDH identified by cgMD. Our study represents a proof‐of‐concept approach that can be used in future to develop novel coupled biocatalytic systems by identifying potential ET pathways.

Glucose Oxidase-Based Glucose Biosensing with a Simple Dual Ag/AgCl Probe Conductivity Readout.

We describe a conductometric assay of the enzymatic conversion of glucose to gluconic acid by dissolved glucose oxidase (GOx), using the generation of proton and gluconate from the reaction product dissociation for glucose detection. Simple basics of ionic conductivity, a silver/silver chloride wire pair, and a small applied potential translate glucose-dependent GOx activity into a scalable cell current. Enzyme immobilization and complex sensor design, involving extra nanomaterials or microfabrication of electrode structures, are entirely avoided, in contrast to all modern electrochemical glucose biosensors. Assay calibration showed a response linearity up to 500 μM, with a sensitivity of about 1.3 nA/μM. Selectivity tests excluded signals from sugars other than glucose, and glucose quantifications with recovery rates close to 100% were reached with a model sample and a beverage. Easy use of elementary physicochemical phenomena and a satisfactory performance are assets of the proposed non-amperometric glucose biosensing strategy. Assay integration into a planar dual electrode platform, with or without microfluidic application option, is feasible because of the simplicity of the sensor readout and suggests a route to affordable glucose analysis in beverage, food, and body fluid samples.

EPOP Restricts PRC2.1 Targeting to Chromatin by Directly Modulating Enzyme Complex Dimerization.

Polycomb repressive complex 2 (PRC2) mediates developmental gene repression as two classes of holocomplexes, PRC2.1 and PRC2.2. EPOP is an accessory subunit specific to PRC2.1, which also contains PCL proteins. Unlike other accessory subunits that collectively facilitate PRC2 targeting, EPOP was implicated in an enigmatic inhibitory role, together with its interactor Elongin BC. We report an unusual molecular mechanism whereby EPOP regulates PRC2.1 by directly modulating its oligomerization state. EPOP disrupts the PRC2.1 dimer and weakens its chromatin association, likely by disabling the avidity effect conferred by the dimeric complex. Congruently, an EPOP mutant specifically defective in PRC2 binding enhances genome-wide enrichments of MTF2 and H3K27me3 in mouse epiblast-like cells. Elongin BC is largely dispensable for the EPOP-mediated inhibition of PRC2.1. EPOP defines a distinct subclass of PRC2.1, which uniquely maintains an epigenetic program by preventing the over-repression of key gene regulators along the continuum of early differentiation.

Biodegradation and Detoxification of Some Dyes by Crude Lignin Peroxidase Complex Produced by Escherichia coli Accession No: LR0250096.1 and Pseudomonas aeruginosa Accession No: CP031449.2

Synthetic and untreated dyes discharged in wastewater effluents are a threat to an ecosystem. This study investigated dye degradation and detoxification efficiency of crude lignin peroxidase separately obtained from the cultures of Escherichia coli (LR0250096.1) and Pseudomonas aeruginosa (CP031449.2). The ability of the crude lignin peroxidase to degrade Malachite Green (MG), Remazol Brilliant Blue R (RBBR), Congo Red (CR), and Azure B (AZ) was evaluated at different operating conditions (enzyme, dye, and hydrogen peroxide concentrations; pH; temperature; and contact time). The ability of the degraded dyes to support the growth of bacteria was also investigated. The observed optimum operating conditions for lignin peroxidase extracts of the Escherichia coli on AZ were 20 mg/mL enzyme concentration, 50 mg/L dye, pH 7.0, temperature 50 °C, and 1.5 mM hydrogen peroxide within 20–50 min of incubation time and on MG were 20 mg/mL, 50 mg/L, 9.0, 30 °C, 0.1 mM, and 20 min, respectively. The enzyme extract from Pseudomonas aeruginosa on AZ demonstrated optimum operation conditions of 20 mg/mL, 50 mg/L, pH 9.0, 40 °C, 1.5 mM, and 50 min, respectively and on MG, they were 20 mg/mL, 50 mg/L, 6.0, 30 °C, 1.0 mM, and 20 min, respectively). The prepared enzyme showed an appreciable degradative effect on CR and RBBR compared with commercial lignin peroxidase. The degraded dyes were able to support the growth of two Gram-positive (Bacillus cereus and Staphylococcus aureus), and two Gram-negative (Proteus mirabilis and Escherichia coli) bacteria, indicating the efficiency and the potential use of the enzyme complexes in the clean-up of industrial dyes’ waste.

Identification of Potential Tryptase Inhibitors from FDA-Approved Drugs Using Machine Learning, Molecular Docking, and Experimental Validation.

This study explores the innovative use of machine learning (ML) to identify novel tryptase inhibitors from a library of FDA-approved drugs, with subsequent confirmation via molecular docking and experimental validation. Tryptase, a significant mediator in inflammatory and allergic responses, presents a therapeutic target for various inflammatory diseases. However, the development of effective tryptase inhibitors has been challenging due to the enzyme's complex activation and regulation mechanisms. Utilizing a machine learning model, we screened an extensive FDA-approved drug library to identify potential tryptase inhibitors. The predicted compounds were then subjected to molecular docking to assess their binding affinity and conformation within the tryptase active site. Experimental validation was performed using RBL-2H3 cells, a rat basophilic leukemia cell line, where the efficacy of these compounds was evaluated based on their ability to inhibit tryptase activity and suppress β-hexosaminidase activity and histamine release. Our results demonstrated that several FDA-approved drugs, including landiolol, laninamivir, and cidofovir, significantly inhibited tryptase activity. Their efficacy was comparable to that of the FDA-approved mast cell stabilizer nedocromil and the investigational agent APC-366. These findings not only underscore the potential of ML in accelerating drug repurposing but also highlight the feasibility of this approach in identifying effective tryptase inhibitors. This research contributes to the field of drug discovery, offering a novel pathway to expedite the development of therapeutics for tryptase-related pathologies.

Genes of Streptomyces globisporus 1912-4Crt Encoding Chitin Catabolism Enzymes

Polysaccharide chitin is one of the most common biopolymers in nature. Chitin (when used as the sole source of energy, carbon, and nitrogen) has been shown to be a substrate sufficient to enable the growth and synthesis of secondary metabolites by the S. griseus NCIMB 8136 strain and some other streptomycetes. The species Streptomyces globisporus and S. griseus belong to the same lower hierarchical taxon (S. griseus clade). The aim was to find in the S. globisporus 1912-4Crt genome genes encoding proteins that are necessary for chitin fermentation and transmembrane transport of resulting products. Methods. The object of the study was a sequence of the S. globisporus 1912-4Crt genome (reference NZ_QWFA01000000.1, GenBank) on the server of NCBI (The National Center for Biotechnology Information). Streptomycete S. globisporus 1912 and its variants are producers of antibiotic landomycin E and carotenoids. Search for and analysis of nucleotide and amino acid sequences were performed using programs BLAST (Basic Local Alignment Search Tool) on the aforementioned server. Results. A set of genes that determine enzymes of chitin catabolism and Ngc-transporter proteins were identified in the S. globisporus 1912-4Crt genome. Genes encoding 10 endo-acting chitinases (from the GH18 and GH19 families) and 2 exo-acting hydrolases (GH20) were identified in the S. globisporus 1912-4Crt sequence. Several genes determining deacetylases that deacetylate both chitin and oligosaccharides were found in the genome of the strain. One gene that determines endo-acting chitosanase from the GH75 family was discovered there. The ability of the wild-type strain S. globisporus 1912 and a number of its variants (including S. globisporus 1912-4Crt) to ferment chitin was proven in vitro. Conclusions. A set of genes sufficient for chitin assimilation was established in the S. globisporus 1912-4Crt genome sequence. Streptomycetes from different clades (for example, strains of S. coelicolor (S. albidoflavus group) and S. griseus, S. globisporus (S. griseus group)) containing different complexes of chitinolytic enzymes could be suggested.

Flexible 2,4-diaminopyrimidine bearing a butyrolactone as Plasmodium falciparum dihydrofolate reductase inhibitors

Recently, P218, a new flexible antifolate targeting Plasmodium falciparum dihydrofolate reductase (PfDHFR), has entered its clinical trial with good safety profile and effective Pf infection prevention. However, it carries a free carboxyl terminal, which is hydrophilic and prone to metabolic glucuronidation. Here, a new series of P218 analogues carrying butyrolactone has been synthesized with the purpose of enhancing lipophilicity and minimizing metabolic instability. The inhibition constants against the mutant PfDHFR enzymes are in sub-nanomolar level and the antimalarial activity against antifolate-resistant parasites are in the low micromolar range. The crystal structure of the most potent analogue LA1 bound enzyme complex indicates interaction with multiple residues, including Arg122 and Phe116 in the active site. In vitro log D7.4 and kinetic solubility confirmed a higher lipophilicity of this butyrolactone series as compared to P218. These outcomes suggest the possibility to further develop butyrolactone derivatives as non-carboxyl antiplasmodial antifolates.

Development of tetravalent antibody–enzyme complexes employing a lactate oxidase and the application to electrochemical immunosensors

Antibody–enzyme complexes (AECs) are ideal for immunosensing. Although AECs using antibody fragments can be produced by bacterial hosts, their low affinity limits their sensing applications. We have improved the affinity of AECs by combining two antibodies using Catcher/Tag systems; however, it requires multiple antibodies and an enzyme production process. In this study, to realize the production of AECs harboring multiple antibody fragments in a single production process, we report a versatile development method of unique AECs based on a multimeric enzyme structure. Using the homotetrameric enzyme, lactate oxidase (LOx), as a labeling enzyme, tetravalent AECs were developed as an electrochemical immunosensor. Homogeneous tetravalent AECs were successfully fabricated by fusing the anti-epidermal growth factor receptor (EGFR) variable domain of a heavy chain of heavy chain antibody to the N-terminus of LOx. The prepared AECs bound to EGFR, maintain their enzyme activity, and worked well as sensing elements in electrochemical sandwich enzyme-linked immunosorbent assay. Moreover, tetravalent AECs exhibited higher sensitivity than monovalent AECs because of their avidity. The fabricated AECs were successfully used in a wash-free homogeneous electrochemical detection system combined with magnetic separation. Our findings offer new insights into the applications of the LOx tetrameric enzyme for the fabrication of AECs with tetravalent antibodies, which may serve as scaffolds for immunosensors.

Evaluation of Dietary Supplementation of a Multi-Carbohydrase Enzyme Complex on Growth Performance and Carcass Traits of Pekin Ducks Fed Corn–Soy Based Diets

The present study investigated the effect of supplementing a multi-carbohydrase enzyme complex (MCE) in corn–soy-based diets of Pekin ducks. The treatments were as follows: positive control (PC, 2980 and 3120 kcal/kg for starter and grower, respectively); negative control (NC, −132 kcal/kg energy reduction to PC achieved by reduction of fat and wheat middlings as filler); NC + MCE at 75 ppm (E75); 100 ppm (E100); and 125 ppm (E125) randomly distributed in 10 replicate pens with 25 birds each. Performance was evaluated after 14 and 35 d. On day 36 of the trial, five ducks/pen were processed to evaluate carcass traits. During days 1–14, the PC had a lower (p < 0.01) feed intake compared to all other treatments. At 14 and 35 d of age the NC decreased (p < 0.001) the body weight (BW) of the ducks compared to the PC by −8.3% and −5.3%, respectively. The NC BW was lower (p < 0.001) compared to all MCE-supplemented treatments at 14 and 35 d. The BW of E75, E100, and E125 treatments was not different (p > 0.05) from the PC at both evaluation periods. Cumulatively (1–35 d), the NC resulted in a weight-adjusted FCR increase (p = 0.001) of 9.9% compared to the PC, and the FCR of E75, E100, and E125 were able to recover 72.3%, 66.4%, and 63.5%, respectively, compared to the PC. The carcass and breast weights were lower (p < 0.001) in the NC compared to all other treatments, and no differences (p > 0.05) were observed between the MCE-supplemented treatments and the PC. In conclusion, these results suggest that the MCE supplementation can maintain duck growth performance with no negative effects on carcass traits in energy-reduced corn–soybean meal-based diets. From the results of the trial, 75 ppm MCE delivered the best performance recovery and 125 ppm MCE supplementation had the best % breast yield.

Insights into bacterial interactions: Comparing fluorine-containing 1,2,4-triazoles to antibiotics using molecular docking and molecular dynamics approaches

Understanding the interactions between drugs and enzymes is crucial for designing effective therapeutics. This study employed a combination of molecular docking and molecular dynamics (MD) simulations to evaluate the binding affinity, stability, and dynamic behavior of two new compounds (compound 1 and compound 2) compared to vancomycin and meropenem against Staphylococcus aureus and Serratia marcescens bacterial enzymes. Molecular docking studies provided insights into the binding interactions and affinities of these compounds, revealing that both compound 1 and compound 2 exhibit promising binding profiles. In particular, compound 1 demonstrated lower binding energies with key enzymes from Staphylococcus aureus compared to vancomycin, suggesting enhanced potential. MD simulations further elucidated the dynamic stability of these complexes. Results indicated that compound 1 maintains consistent binding modes with low RMSD and RMSF values, implying stable interactions. In contrast, vancomycin exhibited high RMSD and RMSF values in some enzyme complexes, reflecting potential instability. Compound 2 showed competitive stability and binding behavior compared to meropenem, with comparable RMSD and RMSF values across various enzyme targets. These findings highlight the potential of compound 1 and compound 2 as viable candidates for further development, offering insights into their stability and efficacy as new therapeutic agents.

Enhanced emulsion capacity and gel properties of bighead carp myofibrillar protein through interaction with fresh maize bran arabinoxylan extracted by complex enzymes

SummaryBighead carp has the largest production amount among freshwater fish in China, but its physicochemical properties change rapidly with the denaturation of their myofibril protein (MP). The protein–polysaccharide complexes have shown interesting physicochemical properties as stabilisers and emulsifiers. In this study, arabinoxylans (AXs) were prepared and mixed with bighead carp MP to form a composite gel. The effect of AXs on the thermal stability, emulsification and emulsion stability of the composite gel was investigated. The results showed that fraction precipitated by 60% ethanol (F60) and by 90% (F90) improved the denaturation temperature of bighead carp MP from 65.99 °C to 67.39 °C and 68.22 °C, respectively. The emulsifying capacity of F60 (0.54 m2 g−1) was similar to that of sucrose (0.52 m2 g−1). The effect of F90 on the emulsion stability of bighead carp MP was better than that of F60. The elasticity of bighead carp MP was improved by F60 and F90. The hydroxyl group interactions, hydrophobic interactions and electrostatic forces exist between the macromolecules. AXs of fresh maize bran could prevent protein denaturation and improve the emulsion capacity and gel properties of bighead carp MP.

Development of a method for inactivating lipoxygenases in linseed using chemical reagents

The object of the study is the oxidative stability of the lipid component of linseed treated with a citric acid and sodium chloride solution. The rational composition of chemical reagents for inactivating linseed lipoxygenases was determined in the work. The obtained results make it possible to develop an effective linseed treatment method for increasing stability to oxidative spoilage. The proposed composition of the linseed treatment solution (citric acid – 1.0...1.3 %; sodium chloride – 0.6...0.8 %) significantly reduces the peroxide and anisidine numbers of the lipid component. This helps reduce oxidative spoilage during accelerated oxidation and storage under normal conditions. Rational treatment conditions were determined based on the approximate dependency of these indicators on the concentrations of chemical reagents. The data obtained in the work are explained by chemical interactions between the solution components and the enzyme complex of linseed, leading to enzyme denaturation and, accordingly, increased oxidative stability of the lipid component. A feature of the obtained results is the competitiveness of treated linseed, characterized by increased nutritional value due to improved technological properties. The results of the study allow minimizing the loss of nutritional value and increasing the shelf life of linseed products. The results are important for developing new oilseed processing technologies. This makes it possible not only to increase the stability of products against oxidative spoilage, but also to preserve their high nutritional value. Further research in this area will contribute to improving oilseed processing technologies, in particular linseed, which is an important contribution to the development of the food industry

Computational design of serine hydrolases.

Enzymes that proceed through multistep reaction mechanisms often utilize complex, polar active sites positioned with sub-angstrom precision to mediate distinct chemical steps, which makes their de novo construction extremely challenging. We sought to overcome this challenge using the classic catalytic triad and oxyanion hole of serine hydrolases as a model system. We used RFdiffusion1 to generate proteins housing catalytic sites of increasing complexity and varying geometry, and a newly developed ensemble generation method called ChemNet to assess active site geometry and preorganization at each step of the reaction. Experimental characterization revealed novel serine hydrolases that catalyze ester hydrolysis with catalytic efficiencies (k cat /K m ) up to 3.8 x 103 M-1 s-1, closely match the design models (Cα RMSDs < 1 Å), and have folds distinct from natural serine hydrolases. In silico selection of designs based on active site preorganization across the reaction coordinate considerably increased success rates, enabling identification of new catalysts in screens of as few as 20 designs. Our de novo buildup approach provides insight into the geometric determinants of catalysis that complements what can be obtained from structural and mutational studies of native enzymes (in which catalytic group geometry and active site makeup cannot be so systematically varied), and provides a roadmap for the design of industrially relevant serine hydrolases and, more generally, for designing complex enzymes that catalyze multi-step transformations.

Toward the Use of Methyl-Coenzyme M Reductase for Methane Bioconversion Applications.

ConspectusAs the main component of natural gas and renewable biogas, methane is an abundant, affordable fuel. Thus, there is interest in converting these methane reserves into liquid fuels and commodity chemicals, which would contribute toward mitigating climate change, as well as provide potentially sustainable routes to chemical production. Unfortunately, specific activation of methane for conversion into other molecules is a difficult process due to the unreactive nature of methane C-H bonds. The use of methane activating enzymes, such as methyl-coenzyme M reductase (MCR), may offer a solution. MCR catalyzes the methane-forming step of methanogenesis in methanogenic archaea (methanogens), as well as the initial methane oxidation step during the anaerobic oxidation of methane (AOM) in anaerobic methanotrophic archaea (ANME). In this Account, we highlight our contributions toward understanding MCR catalysis and structure, focusing on features that may tune the catalytic activity. Additionally, we discuss some key considerations for biomanufacturing approaches to MCR-based production of useful compounds.MCR is a complex enzyme consisting of a dimer of heterotrimers with several post-translational modifications, as well as the nickel-hydrocorphin prosthetic group, known as coenzyme F430. Since MCR is difficult to study in vitro, little information is available regarding which MCRs have ideal catalytic properties. To investigate the role of the MCR active site electronic environment in promoting methane synthesis, we performed electric field calculations based on molecular dynamics simulations with a MCR from Methanosarcina acetivorans and an ANME-1 MCR. Interestingly, the ANME-1 MCR active site better optimizes the electric field with methane formation substrates, indicating that it may have enhanced catalytic efficiency. Our lab has also worked toward understanding the structures and functions of modified F430 coenzymes, some of which we have discovered in methanogens. We found that methanogens produce modified F430s under specific growth conditions, and we hypothesize that these modifications serve to fine-tune the activity of MCR.Due to the complexity of MCR, a methanogen host is likely the best near-term option for biomanufacturing platforms using methane as a C1 feedstock. M. acetivorans has well-established genetic tools and has already been used in pilot methane oxidation studies. To make methane oxidation energetically favorable, extracellular electron acceptors are employed. This electron transfer can be facilitated by carbon-based materials. Interestingly, our analyses of AOM enrichment cultures and pure methanogen cultures revealed the biogenic production of an amorphous carbon material with similar characteristics to activated carbon, thus highlighting the potential use of such materials as conductive elements to enhance extracellular electron transfer.In summary, the possibilities for sustainable MCR-based methane conversions are exciting, but there are still some challenges to tackle toward understanding and utilizing this complex enzyme in efficient methane oxidation biomanufacturing processes. Additionally, further work is necessary to optimize bioengineered MCR-containing host organisms to produce large quantities of desired chemicals.

Short-chain fructooligosaccharides synthesis using a commercial enzyme complex from Aspergillus sp. and anti-cancer activity on HCT116 and HT-29 cell lines

Over the last years, short-chain fructooligosaccharides (FOS) have gained significant attention as valuable food and dietary supplement components. Apart from implications such as dietary fibres, sweeteners, and humectants, FOS are hailed for their prebiotic properties. Moreover, much research is dedicated to their role as anti-cancer agents. Despite the ongoing interest, both technical aspects, challenges of FOS production, and their anti-cancer role necessitate more in-depth investigations to understand their healthcare role better. This study aimed to optimise critical FOS synthesis parameters using a commercial enzyme, Viscozyme® L, to obtain a preparation with a high FOS content. A central composite design and response surface methodology evaluated the effect of pH, temperature, synthesis time, substrate, and enzyme load on multiple responses, chosen as independent variables, were utilised to define optimal conditions. Under the optimal conditions (temperature 50 °C, pH 5.5, sucrose concentration 352 g/L, enzyme concentration 2.5% v/v, and 5.5 h of reaction), a preparation with a total FOS content of 58.8% was obtained. The FOS preparation showed minimal in vitro antioxidant capacity in the ORAC, CUPRAC, and ABTS*+assays. In addition, the cytotoxicity of FOS against tested cell lines was evaluated. FOS did not reduce HT-29 cell viability even at the concentration of 2.5 mg/mL, whereas FOS inhibited the proliferation of HCT116 cells by 50% at 2.35 mg/mL. Finally, the sensitisation effect of FOS on HCT116 cell treatment with doxorubicin has been revealed.