Soluble Substrates Research Articles

More is not always better. Multiple carbohydrate-binding domains for efficient starch hydrolysis

Lactobacillus amylovorus α-amylase is an endoenzyme with a peculiar starch-binding domain containing five identical family 26 carbohydrate-binding modules (CBM26). To investigate the impact of CBMs on catalytic activity, C-terminally truncated derivatives were constructed. The catalytic domain alone shows low affinity for the substrate and a very slow reaction rate, highlighting the importance of CBMs in maintaining the enzyme's optimal conformation and dynamics for efficient catalysis. CBMs enhance enzyme performance, as indicated by improved catalytic efficiency (kcat/Km). Interestingly, the enzyme variant LaCD3CBM, with three CBMs, exhibits the best catalytic efficiency on soluble starch, outperforming the wild-type amylase, with five CBMs. In the case of insoluble starch, the catalytic domain alone could not hydrolyze it and even adding a CBM, the release of reducing sugars was very inefficient. However, this efficiency was significantly improved by two orders of magnitude for the three-CBM variant and the wild-type amylase. CBMs also played a crucial role in protein thermostability, contributing to a higher melting temperature of the catalytic domain with just a single CBM. Notably, thermostability did not increase with the number of CBMs. In conclusion, spatial arrangement and interactions between the catalytic domain and CBMs significantly influenced enzymatic efficiency with both soluble and insoluble substrates. These interactions optimize enzymatic activity and improve thermostability.

Modeling microbial growth based on time-dependent kinetic mechanisms of digestion and passage in the ruminoreticulum

The original Cornell Net Carbohydrate and Protein System (CNCPS) model, which was developed in 1992 and revised in 2004, included a rumen submodel with a set of equations that were algebraically programmed in a spreadsheet to predict bacterial N flow to the caudal tract of cattle. In this study, we propose a modification to the original CNCPS rumen submodel based on mathematical concepts that simulate the flow paths of solids and liquid in the rumen, which are known to be essential factors affecting rumen fermentation and microbial growth. This modification allows the quantification of the impact of aging mechanisms on the availability of both soluble and insoluble substrates for microbial growth in the rumen. Additionally, a correction has been proposed on peptide uptake by rumen bacteria. Literature data were used to evaluate the model adequacy of CNCPS versions 1992 and 2004, along with the proposed model, to determine their ability to predict bacterial N flow. The proposed model more accurately predicted the variable of interest, but there was an overall underprediction bias. Despite the rightful critics of the ability of mechanistic models to predict microbial crude protein flow from the rumen, the 1992 and 2004 versions of the CNCPS and the proposed modifications implemented within the CNCPS framework led to predictions that agreed with observational data at a discordance rate of 0.05, tolerance probabilities <0.001 for versions 1992 and 2004, and a tolerance probability equal to 0.13 for the proposed model given a sample size n=39. Therefore, the proposed modifications might improve the ability of CNCPS-based models to predict the bacterial N flow from the rumen, thereby resulting in improved predictive performance compared to the original CNCPS model. Thus, it seems that models built on time-dependent kinetics of food particles and fluid are more fitting than steady-state ones to predict bacterial N flow to the caudal tract.

Production, Characterization, Kinetics, and Thermodynamics Analysis of Amyloglucosidase from Fungal Consortium.

The current study aimed to produce an amyloglucosidase enzyme from the fungal consortium. The best amylolytic fungal consortia were identified as Alternaria alternata and Aspergillus niger through the 18S rDNA technique. Fermentation kinetics and various nutritional and cultural parameters were analyzed. Maximum production was obtained in M4 media, pH 5.5, 30°C, and 4mL inoculum at 150rpm after 72h of incubation. Along with that, sodium nitrate at 2.5%, maltose, beef extract 1%, zinc sulfate (0.1%), and Tween 80 (0.1%) supported the maximum amyloglucosidase production. Amyloglucosidase was partially purified up to 1.6 purification fold with a specific activity of 1.84 Umg-1 in a stepwise manner by ammonium sulfate purification, dialysis, and ion exchange chromatography. The AMG enzyme also revealed maximum activity at 50°C with 5.0 pH. Upon the kinetic analysis, the specific yield coefficient Yp/x and volumetric rates Qp and Qx were also found to be significant in the above optimized conditions. The Km value 0.33mgmL-1 and Vmax 26.31 U mL-1 were obtained at 1% soluble starch substrate. Thermodynamic parameters for soluble starch hydrolysis were as follows: ΔH = 48.78kJmol-1, (Ea) = - 46.0kJmol-1, and ΔS = - 43.10Jmol-1K-1. This finding indicates the indigenously isolated fungal consortium can be the best candidate for industrial applications.

Reducing sludge formation by enhancing biological decay of biomass: a mathematical model

Abstract We investigate a model for an activated sludge process that contains a chemical reactor unit attached to a bioreactor. Inside the chemical reactor unit, a process takes place which increases the decay coefficient of heterotrophic biomass. This mimics a number of experimental techniques which are used to decrease the mass of sludge. Such techniques are of growing interest as the activated sludge process produces large volumes of sludge; the costs associated with its disposal are significant. Our primary interest is to investigate how the operation of the chemical reactor unit changes the steady-state concentrations of both the total suspended solids within the biological reactor and the chemical oxygen demand in the effluent stream. The operation of a chemical reactor unit always increases the value of chemical oxygen demand in the effluent stream. The behaviour of the total suspended solids is more complicated; in some cases, the operation of the CRU increases the total suspended solids. For a fixed value of the chemical oxygen demand in the influent stream, we show that both the percentage reduction in the total suspended solids and the chemical oxygen demand in the effluent stream are increasing functions of the soluble substrate in the feed. The same trends occur when the influent composition is fixed, and the disintegration rate inside the chemical reactor unit is increased. This leads to dichotomic behaviour, decreasing the total suspended solids increases the chemical oxygen demand in the effluent stream. There are two consequences of this behaviour. Firstly, there are some waste streams that can not be cleaned using the process configuration considered in this paper. Secondly, the imposition of a target value for the chemical oxygen demand in the effluent stream imposes a maximum achievable reduction in the total suspended solids in the biological reactor. Higher reductions can not be achieved without causing the chemical oxygen demand in the effluent stream to exceed the target value.

An efficient approach to produce flavonoid monophosphate by a coupled bienzymatic system

Flavonoids, with therapeutic potential, often encounter challenges due to poor aqueous solubility, which limits their oral bioavailability and efficacy. Phosphorylation presents a promising solution, yet current chemical methods require protective agents to mitigate side product formation. Here, we developed a coupled bienzymatic system using an ATP-dependent dikinase, flavonoid phosphate synthetase (BsFPS), for selective synthesis of flavonoid monophosphates. This system integrates a class III polyphosphate kinase 2 (PPK2-III) for ATP regeneration from AMP, thereby reducing costly ATP consumption and enhancing economic viability. Through PPK2-III screening, ErPPK was identified as a compatible candidate, facilitating a streamlined one-pot system. Structural analysis revealed that the residue at the equivalent site of Arg75ErPPK influences the binding pocket entry distance and flavonoid derivative inhibition propensity on PPK2-III, confirming the compatibility of ErPPK and providing structural insights for identifying PPK2-III with reduced inhibition likelihood. Further optimization strategies, including balancing Mg2+ and polyphosphate ratios, incorporating Tween surfactants for enhanced substrate solubilization without compromising enzyme stability, and increasing the rate-limiting enzyme amount, significantly elevated the conversion rate. Notably, pH control was crucial due to pH decline caused by BsFPS catalysis. pH-stat conversion achieved a 99.0 % conversion of luteolin, the flavonoid target, within 8 h, yielding up to 21.5 mM (7.9 g/L) luteolin monophosphates with up to 95 % reduction in ATP requirement. This enzymatic phosphorylation approach shows promise for advancing polyphenolic monophosphate bioproduction and transforming their functional applications.

Optimization of quenched fluorescent peptide substrates of SARS-CoV-2 3CLpro main protease (Mpro) from proteomic identification of P6-P6' active site specificity.

SARS-CoV-2 3C-like main protease (3CLpro) is essential for protein excision from the viral polyprotein. 3CLpro inhibitor drug development to block SARS-CoV-2 replication focuses on the catalytic non-prime (P) side for specificity and potency, but the importance of the prime (P') side in substrate specificity and for drug development remains underappreciated. We determined the P6-P6' specificity for 3CLpro from >800 cleavage sites that we identified using Proteomic Identification of Cleavage site Specificity (PICS). Cleavage occurred after the canonical P1-Gln and non-canonical P1-His and P1-Met residues. Moreover, P3 showed a preference for Arg/Lys and P3' for His. Essential H-bonds between the N-terminal Ser1 of protomer-B in 3CLpro dimers form with P1-His, but not with P1-Met. Nonetheless, cleavage occurs at P1-Met456 in native MAP4K5. Elevated reactive oxygen species in SARS-CoV-2 infection oxidize methionines. Molecular simulations revealed P1-MetOX forms an H-bond with Ser1 and notably, strong positive cooperativity between P1-Met with P3'-His was revealed, which enhanced peptide-cleavage rates. The highly plastic S3' subsite accommodates P3'-His that displays stabilizing backbone H-bonds with Thr25 lying central in a "'threonine trio" (Thr24-Thr25-Thr26) in the P'-binding domain I. Molecular docking simulations unveiled structure-activity relationships impacting 3CLpro-substrate interactions, and the role of these structural determinants was confirmed by MALDI-TOF-MS cleavage assays of P1'- and P3'-positional scanning peptide libraries carrying a 2nd optimal cut-site as an internal positive control. These data informed the design of two new and highly soluble 3CLproquenched-fluorescent peptide substrates for improved FRET monitoring of 3CLpro activity with 15× improved sensitivity over current assays.IMPORTANCEFrom global proteomics identification of >800 cleavage sites, we characterized the P6-P6' active site specificity of SARS-CoV-2 3CLpro using proteome-derived peptide library screens, molecular modeling simulations, and focussed positional peptide libraries. In P1', we show that alanine and serine are cleaved 3× faster than glycine and the hydrophobic small amino acids Leu, Ile, or Val prevent cleavage of otherwise optimal non-prime sequences. In characterizing non-canonical non-prime P1 specificity, we explored the unusual P1-Met specificity, discovering enhanced cleavage when in the oxidized state (P1-MetOX). We unveiled unexpected amino acid cooperativity at P1-Met with P3'-His and noncanonical P1-His with P2-Phe, and the importance of the threonine trio (Thr24-Thr25-Thr26) in the prime side binding domain I in defining prime side binding in SARS-CoV-2 3CLpro. From these analyses, we rationally designed quenched-fluorescence natural amino acid peptide substrates with >15× improved sensitivity and high peptide solubility, facilitating handling and application for screening of new antiviral drugs.

Effect of long chain fatty acids on biogas production and biochemical kinetics in anaerobic bioreactors: a review

Long Chain Fatty Acids (LCFAs) are the primary intermediate byproduct of the lipid (fats, oils, and greases) degradation process, and if they accumulate in high concentrations, they can cause failure or reduce the performance of anaerobic bioreactors due to sludge flotation issues, biochemical kinetics problems for soluble substrates, inhibition of microbial activity, and inefficient biogas recovery. Understanding the biochemical kinetics of anaerobic bioreactors requires taking into account the entire process, including microbe growth, substrate degradation, and product synthesis. Biochemical kinetics of anaerobic treatment is the study of polymer biodegradation rates of insoluble organic matter in wastewater, which is the mechanism of bond breaking and bond formation in biochemical reactions. As a result, biochemical kinetics allow for the design of both desired and undesirable reaction phases. The kinetic parameters acquired are utilized to design, operate, and optimize anaerobic bioreactors for wastewater treatment on a technical scale.

Exploring the pH dependence of an improved PETase

Enzymatic recycling of plastic and especially of polyethylene terephthalate (PET) has shown great potential to reduce its negative impact on our society. PET hydrolases (PETases) have been optimized using rational design and machine learning, but the mechanistic details of the PET depolymerization process remain unclear. Belonging to the carboxylic-ester hydrolase family with a canonical Ser-His-Asp catalytic triad, their observed alkaline pH optimum is generally thought to be related to the protonation state of the catalytic His. Here, we explore this aspect in the context of LCCICCG, an optimized PETase, derived from the leaf-branch compost cutinase enzyme. We use NMR to identify the dominant tautomeric structure of the six histidines. Five show surprisingly low pKa values below 4.0, whereas the catalytic H242 in the active enzyme displays a pKa value that varies from 4.9 to 4.7 when temperatures increase from 30°C to 50°C. Whereas the hydrolytic activity of the enzyme toward a soluble substrate can be modeled by the corresponding protonation/deprotonation curve, an important discrepancy is found when the substrate is the solid plastic. This opens the way to further mechanistic understanding of the PETase activity and underscores the importance of studying the enzyme at the liquid-solid interface.

Identification and recombinant expression of a cutinase from Papiliotrema laurentii that hydrolyzes natural and synthetic polyesters.

Given the multitude of extracellular enzymes at their disposal, many of which are designed to degrade nature's polymers (lignin, cutin, cellulose, etc.), fungi are adept at targeting synthetic polyesters with similar chemical composition. Microbial-influenced deterioration of xenobiotic polymeric surfaces is an area of interest for material scientists as these are important for the conservation of the underlying structural materials. Here, we describe the isolation and characterization of the Papiliotrema laurentii 5307AH (P. laurentii) cutinase, Plcut1. P. laurentii is basidiomycete yeast with the ability to disperse Impranil-DLN (Impranil), a colloidal polyester polyurethane, in agar plates. To test whether the fungal factor involved in this clearing was a secreted enzyme, we screened the ability of P. laurentii culture supernatants to disperse Impranil. Using size exclusion chromatography (SEC), we isolated fractions that contained Impranil-clearing activity. These fractions harbored a single ~22 kD band, which was excised and subjected to peptide sequencing. Homology searches using the peptide sequences identified, revealed that the protein Papla1 543643 (Plcut1) displays similarities to serine esterase and cutinase family of proteins. Biochemical assays using recombinant Plcut1 confirmed that this enzyme has the capability to hydrolyze Impranil, soluble esterase substrates, and apple cutin. Finally, we confirmed the presence of the Plcut1 in culture supernatants using a custom antibody that specifically recognizes this protein. The work shown here supports a major role for the Plcut1 in the fungal degradation of natural polyesters and xenobiotic polymer surfaces.IMPORTANCEFungi play a vital role in the execution of a broad range of biological processes that drive ecosystem function through production of a diverse arsenal of enzymes. However, the universal reactivity of these enzymes is a current problem for the built environment and the undesired degradation of polymeric materials in protective coatings. Here, we report the identification and characterization of a hydrolase from Papiliotrema laurentii 5307AH, an aircraft-derived fungal isolate found colonizing a biodeteriorated polymer-coated surface. We show that P. laurentii secretes a cutinase capable of hydrolyzing soluble esters as well as ester-based compounds forming solid surface coatings. These findings indicate that this fungus plays a significant role in biodeterioration through the production of a cutinase adept at degrading ester-based polymers, some of which form the backbone of protective surface coatings. The work shown here provides insights into the mechanisms employed by fungi to degrade xenobiotic polymers.

Pathways of inhibition of filamentous sludge bulking by slowly biodegradable organic compounds

The organic compound composition of wastewater, serves as a crucial indicator for the operational performance of activated sludge processes and has a major influence on the development of filamentous bulking in activated sludge. This study focused on the impact of typical soluble and slowly-biodegradable organic compounds, investigating the pathways through which these substrates affect the occurrence of filamentous bulking in systems operated under both high- and low-oxygen conditions. Results showed that slowly-biodegradable organic compounds lead to a concentrated distribution of microorganisms within flocs, with inward growth of filamentous bacteria. Both Tween-80 and granular starch treated systems exhibited a significant increase in protein content. The glucose system, utilizing soluble substrates, exhibited a markedly higher total polysaccharide content. Microbial communities in the Tween-80 and granular starch treated systems were characterized by a higher abundance of bacteria known to enhance sludge flocculation and settling, such as Competibacter, Xanthomonadaceae and Zoogloea. These findings are of high significance for controlling the operational performance and stability of activated sludge systems, deepening our understanding and providing a novel perspective for the improvement of wastewater treatment processes.

A Thiourea Derivative of 2-[(1R)-1-Aminoethyl]phenol as a Chiral Sensor for the Determination of the Absolute Configuration of N-3,5-Dinitrobenzoyl Derivatives of Amino Acids.

In the exploration of chiral solvating agents (CSAs) for nuclear magnetic resonance (NMR) spectroscopy designed for the chiral analysis of amino acid derivatives, notable advancements have been made with thiourea-CSAs. 1-TU, derived from 2-[(1R)-1-aminoethyl]phenol and benzoyl isothiocyanate, is effective in the enantiodifferentiation of N-3,5-dinitrobenzoyl (N-DNB) amino acids. In order to broaden the application of 1-TU for configurational assignment, enantiomerically enriched N-DNB amino acids were analyzed via NMR. A robust correlation was established between the relative position of specific 1H and 13C NMR resonances of the enantiomers in the presence of 1-TU. 1,4-Diazabicyclo[2.2.2]octane (DABCO) was selected for the complete solubilization of amino acid substrates. Notably, the para and ortho protons of the N-DNB moiety displayed higher frequency shifts for the (R)-enantiomers as opposed to the (S)-enantiomers. This trend was consistently observed in the 13C NMR spectra for quaternary carbons bonded to NO2 groups. Conversely, an inverse correlation was noted for quaternary carbon resonances of the carboxyl moiety, amide carbonyl, and methine carbon at the chiral center. This observed trend aligns with the interaction mechanism previously reported for the same chiral auxiliary. The configurational correlation can be effectively exploited under conditions of high dilution or, significantly, under sub-stoichiometric conditions.

Enhancing short-chain fatty acids production via acidogenic fermentation of municipal sewage sludge: Effect of sludge characteristics and peroxydisulfate pre-oxidation.

This study first employed a combined pretreatment of low-dose peroxy-disulfate (PDS) and initial pH 10 to promote short-chain fatty acids (SCFAs) production via acidogenic fermentation using different types of sewage sludge as substrates. The experimental results showed that the yield of maximal SCFAs and acetate proportion after the combined pretreatment were 1513.82±28.25mg chemical oxygen demand (COD)/L and 53.64%, and promoted by 1.28 and 1.56 times higher, respectively, compared to the sole initial pH 10 pretreatment. Furthermore, in terms of the disintegration degree of sewage sludge, it increased by more than 18% with the combined pretreatment compared to the pretreatment of sole initial pH 10. Waste-activated sludge (WAS) from A2/O and Bardenpho processes were more biodegradable, explained by the 1.47- and 1.35-times higher disintegration rate than those from oxidation ditch and they favored acetate dominant fermentation. Correlation analysis revealed a strong correlation (p ≤ 0.01) between SCFAs production and soluble COD, total proteins, proteins in soluble-extracellular polymeric substances (SEPS), total polysaccharides, and polysaccharides in SEPS. Mechanism explorations showed that preoxidation with PDS enhanced the solubilization and biodegradability of complex substrates, and altered the microbial community structure during the fermentation process. Firmicutes and Tetrasphaera were proven to play a key role in improving SCFA production, especially in promoting acetate production by converting additional SCFAs into acetate. Additionally, the addition of PDS greatly promoted sulfur and iron-related metabolic activities. Finally, the combined pretreatment was estimated to be a cost-effective solution for reutilizing and treating Fe-sludge.

Triaging of α-helical proteins to the mitochondrial outer membrane by distinct chaperone machinery based on substrate topology

Mitochondrial outer membrane ⍺-helical proteins play critical roles in mitochondrial-cytoplasmic communication, but the rules governing the targeting and insertion of these biophysically diverse proteins remain unknown. Here, we first defined the complement of required mammalian biogenesis machinery through genome-wide CRISPRi screens using topologically distinct membrane proteins. Systematic analysis of nine identified factors across 21 diverse ⍺-helical substrates reveals that these components are organized into distinct targeting pathways that act on substrates based on their topology. NAC is required for the efficient targeting of polytopic proteins, whereas signal-anchored proteins require TTC1, a cytosolic chaperone that physically engages substrates. Biochemical and mutational studies reveal that TTC1 employs a conserved TPR domain and a hydrophobic groove in its C-terminal domain to support substrate solubilization and insertion into mitochondria. Thus, the targeting of diverse mitochondrial membrane proteins is achieved through topological triaging in the cytosol using principles with similarities to ER membrane protein biogenesis systems.

Enzymatic Knoevenagel condensation in dual‐functionalized water‐mimicking ionic liquids and tertiary amide solvents

AbstractBACKGROUNDKnoevenagel condensation is an important tool for building carbon–carbon (CC) bonds, especially when catalyzed by enzymes to enable a potentially high chemo‐, regio‐ and/or stereoselectivity. Although many Knoevenagel condensation reactions are carried out in aqueous solutions, insoluble hydrophobic substrates often lead to poor catalytic efficiencies. The use of water‐miscible organic solvents improves the substrate solubilization, but usually induces activity suppression or inactivation of enzymes. There is a great need to develop alternative solvents for both substrate dissolution and enzyme compatibility in CC bond formation reactions.RESULTSOur group previously developed dual‐functionalized water‐mimicking ionic liquids (ILs) for the activation and stabilization of hydrolases (e.g. lipase and protease). In the present study, we evaluated the Knoevenagel condensation of 4‐chlorobenzaldehyde with acetylacetone, and found that porcine pancreas lipase in water‐mimicking ILs carrying ammonium, imidazolium and benzimidazolium cations enabled higher reaction rates (up to 3.22 μmol min−1 g−1 lipase) and better yields than tert‐butanol, glymes and [BMIM][Tf2N]. Interestingly, tertiary amide solvents such as N‐methyl‐2‐pyrrolidone (NMP), N,N‐dimethylformamide (DMF) and N,N‐dimethylacetamide (DMAc) led to 8.2‐ to 11.1‐fold increases in the initial rate (up to 35.66 μmol min−1 g−1 lipase) when compared with dual‐functionalized ILs, which is likely due to some synergistic effect of these tertiary amides with the lipase.CONCLUSIONDual‐functionalized ILs based on ammonium, imidazolium and benzimidazolium cations improved Knoevenagel condensation reaction rates and yields when compared with tert‐butanol and glymes. Tertiary amides (NMP, DMF and DMAc) significantly increased the reaction rate. © 2024 The Authors. Journal of Chemical Technology and Biotechnology published by John Wiley &amp; Sons Ltd on behalf of Society of Chemical Industry (SCI).

Enhancing Single- and Two-Stage Anaerobic Digestion of Thickened Waste-Activated Sludge through FNA-Heat Pretreatment

This study aimed to investigate the effect of combined Free Nitrous Acid (FNA)-Heat (i.e., FNH) pretreatment on single- and two-stage anaerobic digestion (AD) of thickened waste-activated sludge (TWAS). Single-stage AD was conducted in batches, while two-stage AD involved acidogenic fermentation under semi-continuous flow followed by batch methanogenesis. FNH pretreatment was applied before the acidogenic stage, using 1.4 mg HNO2-N/L FNA concentration at 25 °C, 37 °C, and 60 °C for 24 h. Among the scenarios, the most promising results were observed with two-stage AD fed with FNH-pretreated TWAS at 60 °C, showing higher COD solubilization and a reduction in volatile solids. Combined FNA-Heat pretreatment in two-stage AD yielded elevated methane production (363–415 mL CH4/g VS added) compared to single-stage digestion. Methane yields from FNA-Heat pretreated single-stage ranged from 332 to 347 mL CH4/g VS added, contrasting with 212 mL CH4/g VS added for untreated TWAS. Methane generation commenced early in both untreated and pretreated samples, attributed to soluble substrate abundance.

Plant root associated chitinases: structures and functions.

Chitinases degrade chitin, a linear homopolymer of β-1,4-linked N-acetyl-D-glucosamine (GlcNAc) residues found in the cell walls of fungi and the exoskeletons of arthropods. They are secreted by the roots into the rhizosphere, a complex and dynamic environment where intense nutrient exchange occurs between plants and microbes. Here we modeled, expressed, purified, and characterized Zea mays and Oryza sativa root chitinases, and the chitinase of a symbiotic bacterium, Chitinophaga oryzae 1303 for their activities with chitin, di-, tri-, and tetra-saccharides and Aspergillus niger, with the goal of determining their role(s) in the rhizosphere and better understanding the molecular mechanisms underlying plant-microbe interactions. We show that Zea mays basic endochitinase (ZmChi19A) and Oryza sativa chitinase (OsChi19A) are from the GH19 chitinase family. The Chitinophaga oryzae 1303 chitinase (CspCh18A) belongs to the GH18 family. The three enzymes have similar apparent K M values of (20-40 µM) for the substrate 4-MU-GlcNAc3. They vary in their pH and temperature optima with OsChi19A activity optimal between pH 5-7 and 30-40°C while ZmChi19A and CspCh18A activities were optimal at pH 7-9 and 50-60°C. Modeling and site-directed mutation of ZmChi19A identified the catalytic cleft and the active residues E147 and E169 strategically positioned at ~8.6Å from each other in the folded protein. Cleavage of 4-MU-GlcNAc3 was unaffected by the absence of the CBD but diminished in the absence of the flexible C-terminal domain. However, unlike for the soluble substrate, the CBD and the newly identified flexible C-terminal domain were vital for inhibiting Aspergillus niger growth. The results are consistent with the involvement of the plant chitinases in defense against pathogens like fungi that have chitin exoskeletons. In summary, we have characterized the functional features and structural domains necessary for the activity of two plant root chitinases that are believed to be involved in plant defense and a bacterial chitinase that, along with the plant chitinases, may participate in nutrient recycling in the rhizosphere.

Electrochemical Characterization of Two Gut Microbial Strains Cooperatively Promoting Multiple Sclerosis Pathogenesis.

In this study, we explored the extracellular electron transfer (EET) capabilities of two bacterial strains, OTU0001 and OTU0002, which are demonstrated in biofilm formation in mouse gut and the induction of autoimmune diseases like multiple sclerosis. OTU0002 displayed significant electrogenic behaviour, producing microbial current on an indium tin-doped oxide electrode surface, particularly in the presence of glucose, with a current density of 60 nA/cm2. The presence of cell-surface redox substrate potentially mediating EET was revealed by the redox-based staining method and electrochemical voltammetry assay. However, medium swapping analyses and the addition of flavins, a model redox mediator, suggest that the current production is dominated by soluble endogenous redox substrates in OTU0002. Given redox substrates were detected at the cell surface, the secreted redox molecule may interact with the cellular surface of OTU0002. In contrast to OTU0002, OTU0001 did not exhibit notable electrochemical activity, lacking cell-surface redox molecules. Further, the mixture of the two strains did not increase the current production from OTU0001, suggesting that OTU0001 does not support the EET mechanism of OTU0002. The present work revealed the coexistence of EET and non-EET capable pathogens in multi-species biofilm.

Boundary-layer approach to the linear karren instability

The present paper concerns the linear fate of transverse perturbations in a gravity-driven, thin-film flow over a soluble substrate. We propose a reduced-order model, based on a boundary-layer treatment of the solute transport and a depth-integration of the Stokes equations, using two extended lubrication methodologies found in the literature. We obtain a closed-form dispersion relation, which we compare to a previous, fully resolved analytical investigation (Bertagni and Camporeale, J. Fluid Mech., vol. 913, 2021, A34). The results allow us to distil the essential physical mechanisms behind the instability.

Differential Impact of the Biodegradation Sunflower Oil, Particulate Substrate, Caused by the Presence of Saccharose, Soluble Substrate, on Activated Sludge Treatment

This research studies the biodegradation of sunflower-type vegetative oil in two proposed activated sludge systems, the first one to biologically treat an influent containing only vegetative oil and the second one to treat a mixture of vegetable oil plus saccharose. The purpose of these analyses is to evaluate the differential impact caused by the soluble substrate saccharose on the removal of vegetative oil. Vegetative oil biodegradation in both systems was studied and quantified via integral mass balance, and relevant operating parameters were monitored. This experimentation based on the mass balance estimation of biodegraded vegetative oil serves as a reference to understand the effect of soluble substrates present in mixed wastewater on oil biodegradation. Information was generated on the performance of the two activated sludge treatment systems. Both influents were pre-stirred before they entered the bench-scale activated sludge plants. The working range for sunflower oil concentration was 120 to 520 mg/L for the influent with sunflower oil and 180 to 750 mg/L for the influent with sunflower oil and saccharose. Biodegradation was in the order of 56 to 72% and 47 to 67%, respectively. The removal of sunflower oil in biodegradation and flotation was in the order of 90% in both scenarios.

Nitrite acts as the key “switch” governing the free nitrous acid pretreatment in anaerobic digestion: A comprehensive mechanism of abiotic, bioinformatics and bioenergetics effects

Free nitrous acid (FNA) pretreatment has been an effective and environmentally-friendly strategy to improve anaerobic digestion (AD) performance, and the mechanism was generally ascribed to the abiotic role of enhancing substrate solubilization. However, a comprehensive understanding of FNA is lack considering the different biotic roles on various steps, microbes and functional genes, as well as the variation of thermodynamic conditions. Herein, FNA pretreatment significantly enhanced the AD performance of food waste (FW), and the maximum increment of methane yield (36.45 %) was observed for FNA level of 1.42 mg HNO2 − N/L. Nevertheless, FNA exhibited an inhibition-to-enhancement effects as the AD process proceeds, and the introduced nitrite was the key “switch”. Initially, the abiotic effects of FNA reduced the molecular weight of components in FW, benefiting the solubilization and formation of available organics (glucose and amino acid). The individual steps experiment confirmed that residual nitrite severely inhibited methanogenesis rather than hydrolysis and acidogenesis, which resulted in the decrement of methane yield and accumulation of VFAs. Meanwhile, the methanogens (Methanothrix and Methanobacterium) and genes involved in methanogenesis metabolisms (aceticlastic and hydrogenotrophic pathways) were downregulated. However, with the elimination of nitrite, the microbial activities and gene expressions restored and were even enhanced, and Gibbs-energy-based analysis suggested that accumulated VFAs provided favorable thermodynamic conditions for subsequent methanogenesis. This study firstly proved that the abiotic breakdown of macromolecule components, selective and reversible inhibition on microbes and functional genes induced by nitrite and varying thermodynamic conditions together governed the FNA affecting the AD process of FW.