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Abstract A key player in energy metabolism is phosphofructokinase-1 (PFK1) whose activity and behavior strongly influence glycolysis and thus have implications in many areas. In this research, PFK1 assays were performed to convert F6P and ATP into F-1,6-P and ADP for varied pH and ATP concentrations. PFK1 activity was assessed by evaluating F-1,6-P generation velocity in two ways: (1) directly calculating the time slope from the first two or more datapoints of measured product concentration (the initial-velocity method), and (2) by fitting all the datapoints with a differential equation explicitly representing the effects of ATP and pH (the modeling method). Similar general trends of inhibition were shown by both methods, but the former gives only a qualitative picture while the modeling method yields the degree of inhibition because the model can separate the two simultaneous roles of ATP as both a substrate of reaction and an inhibitor of PFK1. Analysis based on the model suggests that the ATP affinity is much greater to the PFK1 catalytic site than to the inhibitory site, but the inhibited ATP-PFK1-ATP complex is much slower than the uninhibited PFK1-ATP complex in product generation, leading to reduced overall reaction velocity when ATP concentration increases. The initial-velocity method is simple and useful for general observation of enzyme activity while the modeling method has advantages in quantifying the inhibition effects and providing insights into the process.

A key player in energy metabolism is phosphofructokinase-1 (PFK1) whose activity and behavior strongly influence glycolysis and thus have implications in many areas. In this research, PFK1 assays were performed to convert F6P and ATP into F-1,6-P and ADP for varied pH and ATP concentrations. PFK1 activity was assessed by evaluating F-1,6-P generation velocity in two ways: (1) directly calculating the time slope from the first two or more datapoints of measured product concentration (the initial-velocity method), and (2) by fitting all the datapoints with a differential equation explicitly representing the effects of ATP and pH (the modeling method). Similar general trends of inhibition were shown by both methods, but the former gives only a qualitative picture while the modeling method yields the degree of inhibition because the model can separate the two simultaneous roles of ATP as both a substrate of reaction and an inhibitor of PFK1. Analysis based on the model suggests that the ATP affinity is much greater to the PFK1 catalytic site than to the inhibitory site, but the inhibited ATP-PFK1-ATP complex is much slower than the uninhibited PFK1-ATP complex in product generation, leading to reduced overall reaction velocity when ATP concentration increases. The initial-velocity method is simple and useful for general observation of enzyme activity while the modeling method has advantages in quantifying the inhibition effects and providing insights into the process. [ABSTRACT FROM AUTHOR]

Polyethylene terephthalate (PET) is one of the most abundant polyester materials used in daily life and it is also one of the culprits of environmental pollution. ICCG (F243I/D238C/S283C/Y127G) is a quadruple mutant of leaf-branch compost cutinase (LCC) displaying outstanding performance in hydrolyzing PET and holding a great potential in further applications. Substrate concentration is one of the important factors affecting the catalytic degradation efficiency. The conventional fast equilibrium theory holds that the degradation rate reaches the maximum and tends to be stable with the increase of substrate concentration, however, in practice, too much substrate will inhibit the catalytic reaction. In this study, the substrate inhibitory effect of PET plastic particles with different particle sizes on ICCG was evaluated. Combined with kinetic constant analysis, the optimal PET particle size was determined to be 300 μm. Meanwhile, several mutants (Y95K, M166S and H218S) of ICCG were obtained by site-directed mutagenesis. The effect of substrate concentration on mutant was studied under the condition of optimal reaction particle size. This study provides a strategy for obtaining high-efficiency PET degradation mutants and a new possibility of environmentally friendly plastic degradation. [ABSTRACT FROM AUTHOR]

One‐pot cascade reactions of coupled disaccharide phosphorylases enable an efficient transglycosylation via intermediary α‐d‐glucose 1‐phosphate (G1P). Such transformations have promising applications in the production of carbohydrate commodities, including the disaccharide cellobiose for food and feed use. Several studies have shown sucrose and cellobiose phosphorylase for cellobiose synthesis from sucrose, but the boundaries on transformation efficiency that result from kinetic and thermodynamic characteristics of the individual enzyme reactions are not known. Here, we assessed in a step‐by‐step systematic fashion the practical requirements of a kinetic model to describe cellobiose production at industrially relevant substrate concentrations of up to 600 mM sucrose and glucose each. Mechanistic initial‐rate models of the two‐substrate reactions of sucrose phosphorylase (sucrose + phosphate → G1P + fructose) and cellobiose phosphorylase (G1P + glucose → cellobiose + phosphate) were needed and additionally required expansion by terms of glucose inhibition, in particular a distinctive two‐site glucose substrate inhibition of the cellobiose phosphorylase (from Cellulumonas uda). Combined with mass action terms accounting for the approach to equilibrium, the kinetic model gave an excellent fit and a robust prediction of the full reaction time courses for a wide range of enzyme activities as well as substrate concentrations, including the variable substoichiometric concentration of phosphate. The model thus provides the essential engineering tool to disentangle the highly interrelated factors of conversion efficiency in the coupled enzyme reaction; and it establishes the necessary basis of window of operation calculations for targeted optimizations toward different process tasks. [ABSTRACT FROM AUTHOR]

Dark fermentative hydrogen production by Enterobacter aerogenes in a packed bed bioreactor using three immobilization supports was studied. The effects of glucose concentrations (20, 10, 5, 2.5, and 1.5 g/L) and support matrixes (Persian Gulf dead coral, ceramic saddle, and ceramic ball) on the hydrogen yield (HY) and hydrogen production rate (HPR) at low hydraulic retention time (HRT) were investigated. According to the achieved results, glucose concentration, porosity and shape support, and bed voidage affected hydrogen production. The HYs were increased with decreasing glucose concentration and at 2.5 g/L glucose concentration, the maximum HYs of 3.11, 2.86 and 2.79 mol hydrogen/mol glucose were obtained for dead coral, ceramic ball, and ceramic saddle, respectively. The results indicated that at the HRT of 0.6 h, the bioreactor packed with dead coral resulted in higher HY (3.11 mol H 2 /mol glucose) and HPR (173 mL/L.h) representing 8–11%, and 8–15%, enhancement compared to the bioreactor packed with ceramic supports. Besides, based on the acquired results, the HY and HPR of the ceramic ball were higher than the ceramic saddle. • Dead coral was used as support in a packed bed bioreactor for hydrogen production. • Reactor packed with dead coral resulted in higher HY and HPR compared to ceramic supports. • The HY and HPR of the ceramic ball were higher than the ceramic saddle. • Porosity and shape support and bed voidage affect the immobilization process and hydrogen production. [ABSTRACT FROM AUTHOR]

High production cost is one of the major factors that limit the market growth of polyhydroxyalkanoates (PHAs) as a biopolymer. Improving PHA synthesis performance and utilizing low-grade feedstocks are two logical strategies for reducing costs. As an oleaginous yeast, Y. lipolytica has a high carbon flux through acetyl-CoA (the main PHB precursor), which makes it a desired cell factory for PHB biosynthesis. In the current study, two different metabolic pathways (NBC and ABC) were introduced into Y. lipolytica PO1f for synthesizing PHB. Compared to the ABC pathway, the NBC pathway, which includes NphT7 to redirect the lipogenesis pathway and catalyze acetoacetyl-CoA synthesis in a more energy-favored reaction, led to PHB accumulation of up to 11% of cell dry weight (CDW), whereas the ABC pathway resulted in non-detectable accumulations of PHB. Further modifications of the strain with the NBC pathway through peroxisomal compartmentalization and gene dose overexpression reached 41% PHB of CDW and a growth rate of 0.227 h−1. A low growth rate was observed with acetate as the sole source of carbon and energy or glucose as the sole substrate at high concentrations. Using a co-substrate strategy helped overcoming the inhibitory and toxic effects of both substrates. Cultivating the engineered strain in the optimal co-substrate condition predicted by response surface methodology (RSM) led to 83.4 g/L of biomass concentration and 31.7 g/L of PHB. These results offer insight into a more cost-effective production of PHB with engineered Y. lipolytica. [ABSTRACT FROM AUTHOR]

Aldo-keto reductases remain enzymes of interest in biocatalysis due to their ability to reduce carbonyls to alcohols stereospecifically. Based on genomic sequence, we identified aldo-keto reductases of a S. cerevisiae strain extracted from an ancient amber sample. One of the putative enzymes, AKR 163, displays 99% identity with α-amide ketoreductases from the S288C and YJM248 S. cerevisiae strains, which have been investigated for biocatalytic applications. To further investigate AKR 163, we successfully cloned, expressed in E.coli as a glutathione-S-transferase fusion protein, and affinity purified AKR 163. Kinetic studies revealed that AKR 163 experiences strong substrate inhibition by substrates containing halogen atoms or other electron withdrawing groups adjacent to the reactive carbonyl, with K i values ranging from 0.29 to 0.6 mM and K M values ranging from 0.38 to 0.9 mM at pH 8.0. Substrates without electron withdrawing groups do not display substrate inhibition kinetics and possess much larger K M values between 83 and 260 mM under the same conditions. The k cat values ranged from 0.5 to 2.5s−1 for substrates exhibiting substrate inhibition and 0.22 to 0.52s−1 for substrates that do not engage in substrate inhibition. Overall, the results are consistent with rate-limiting dissociation of the NADP+ cofactor after hydride transfer when electron withdrawing groups are present and activating the reduction step. This process leads to a buildup of enzyme-NADP+ complex that is susceptible to binding and inhibition by a second substrate molecule. • The aldo-ketoreductases represent a superfamily of enzymes with applications in biocatalysis. • A new allele of α-amide ketoreductase was discovered, cloned, expressed in E.coli , and purified. • The enzyme exhibits previously unreported substrate inhibition behavior with several β-keto ester substrates. • Substrate inhibition is dependent on the presence of an electron withdrawing group adjacent to the reactive carbonyl. [ABSTRACT FROM AUTHOR]

Pteridine reductase 1 (PTR1) is a folate and pterin pathway enzyme unique for pathogenic trypanosomatids. As a validated drug target, PTR1 has been the focus of recent research efforts aimed at finding more effective treatments against human parasitic diseases such as leishmaniasis or sleeping sickness. Previous PTR1-centered structural studies highlighted the enzyme characteristics, such as flexible regions around the active site, highly conserved structural waters, and species-specific differences in pocket properties and dynamics, which likely impacts the binding of natural substrates and inhibitors. Furthermore, several aspects of the PTR1 function, such as the substrate inhibition phenomenon and the level of ligand binding cooperativity in the enzyme homotetramer, likely related to the global enzyme dynamics, are poorly known at the molecular level. We postulate that future drug design efforts could greatly benefit from a better understanding of these phenomena through studying both the local and global PTR1 dynamics. This review highlights the key aspects of the PTR1 structure and dynamics relevant to structure-based drug design that could be effectively investigated by modeling approaches. Particular emphasis is given to the perspective of molecular dynamics, what has been accomplished in this area to date, and how modeling could impact the PTR1-targeted drug design in the future. [ABSTRACT FROM AUTHOR]

The immobilized glucoamylase kinetic in flow calorimetric nonlinear system is discussed. Approximate closed-form of substrate concentration, effectiveness factor is derived by using AkbariGanji method for all values of parameters. The diffusion associated with three electrodes of various geometries (planar, cylindrical and spherical) is studied using this general reaction diffusion indicator parameter and saturated parameters. The problem is numerically simulated using the Maple / MAT-LAB tool. The outcomes of the analysis are compared to simulated data. There is a good agreement with the simulation results. Also covered were the impacts of various parameters on concentration and effectiveness factor. In addition. the significant contribution of this concentration of substrate and effectiveness factor using experiment values of parameters is represented in graphs and tables. The accuracy errors between numerical simulation with our analytical results are 0.008% and 0.48%. This theoretical investigation may be immediately applied to the field of engineering for the kinetics of immobilised enzymes in a flow calorimeter. [ABSTRACT FROM AUTHOR]

Partial reversible inhibition of enzymes, also called hyperbolic inhibition, is an uncommon mechanism of reversible inhibition, resulting from a productive enzyme–inhibitor complex. This type of inhibition can involve competitive, mixed, non-competitive and uncompetitive inhibitors. While full reversible inhibitors show linear plots for reciprocal enzyme initial velocity versus inhibitor concentration, partial inhibitors produce hyperbolic plots. Similarly, dose–response curves show residual fractional activity of enzymes at high doses. This article reviews the theory and methods of analysis and discusses the significance of this type of reversible enzyme inhibition in metabolic processes, and its implications in pharmacology and toxicology. [ABSTRACT FROM AUTHOR]

Kinetic studies and modeling of production parameters are essential for developing economical biosurfactant production processes. This study will provide a perspective on mechanistic reaction pathways to metabolize Waste Engine Oil (WEO). The results will provide relevant information on (i) WEO concentration above which growth inhibition occurs, (ii) chemical changes in WEO during biodegradation, and (iii) understanding of growth kinetics for the strain utilizing complex substrates. Laboratory scale experiments were conducted to study the kinetics and biodegradation potential of the strain Pseudomonas aeruginosa gi |KP 163922| over a range (0.5–8% (v/v)) of initial WEO concentration for 168 h. The kinetic models, such as Monod, Powell, Edward, Luong, and Haldane, were evaluated by fitting the experimental results in respective model equations. An unprecedented characterization of the substrate before and after degradation is presented, along with biosurfactant characterization. The secretion of biosurfactant during the growth, validated by surface tension reduction (72.07 ± 1.14 to 29.32 ± 1.08 mN/m), facilitated the biodegradation of WEO to less harmful components. The strain showed an increase in maximum specific growth rate (µmax) from 0.0185 to 0.1415 h−1 upto 49.92 mg/L WEO concentration. Maximum WEO degradation was found to be ~ 94% gravimetrically. The Luong model (adj. R2 = 0.97) adapted the experimental data using a non-linear regression method. Biochemical, 1H NMR, and FTIR analysis of the produced biosurfactant revealed a mixture of mono- and di- rhamnolipid. The degradation compounds in WEO were identified using FTIR, 1H NMR, and GC–MS analysis to deduce the mechanism. [ABSTRACT FROM AUTHOR]

The concentration of triclosan in wastewater is expected to rise dramatically as a consequence of the COVID-19 pandemic. A modeling analysis of the growth kinetics of microbial culture during triclosan degradation was necessary in order to establish effective wastewater treatment. The kinetic parameters are used by engineers to aid in the design and process control of biological processes. Studies were conducted using triclosan-acclimated culture to examine biomass growth and associated substrate degradation at different initial substrate concentrations (0.35–4.9 mg L−1) to this end. Substrate inhibition was calculated from experimental growth parameters using unstructured kinetic models. Unlike other model studies, a time-averaged specific bacterial growth rate in the log phase was considered for kinetic models in this study. Overestimation of the conventional log phase calculation for unstructured kinetic model constants was eliminated when the slowdown growth part of the log growth phase was taken into account. The Haldane Model was more accurate to fitting experimental data in an excellent manner. In this case, the time-averaged specific growth rate, saturation constant, and inhibition constant were 0.56 h−1, 12.77 mg L−1, 0.52 mg L−1, respectively. A yield coefficient of 0.404 mgX.mgS−1 was calculated. The critical triclosan concentration was 2.57 mg L−1. Wastewater treatment plants can be more sensitive to the value of the critical triclosan concentration. The value for time-averaged critical specific growth rate was 0.051 h−1. Pre-or post-treatment requirements can be estimated using time-averaged critical growth rate values as a benchmarking tool in biological treatment systems.

One of the main obstacles in biocatalysis is the substrate inhibition (SI) of enzymes that play important roles in biosynthesis and metabolic regulation in organisms. The promiscuous glycosyltransferase UGT72AY1 from Nicotiana benthamiana is strongly substrate-inhibited by hydroxycoumarins (inhibitory constant Ki < 20 µM), but only weakly inhibited when monolignols are glucosylated (Ki > 1000 µM). Apocarotenoid effectors reduce the inherent UDP-glucose glucohydrolase activity of the enzyme and attenuate the SI by scopoletin derivatives, which could also be achieved by mutations. Here, we studied the kinetic profiles of different phenols and used the substrate analog vanillin, which has shown atypical Michaelis–Menten kinetics in previous studies, to examine the effects of different ligands and mutations on the SI of NbUGT72AY1. Coumarins had no effect on enzymatic activity, whereas apocarotenoids and fatty acids strongly affected SI kinetics by increasing the inhibition constant Ki. Only the F87I mutant and a chimeric version of the enzyme showed weak SI with the substrate vanillin, but all mutants exhibited mild SI when sinapaldehyde was used as an acceptor. In contrast, stearic acid reduced the transferase activity of the mutants to varying degrees. The results not only confirm the multi-substrate functionality of NbUGT72AY1, but also reveal that the enzymatic activity of this protein can be fine-tuned by external metabolites such as apocarotenoids and fatty acids that affect SI. Since these signals are generated during plant cell destruction, NbUGT72AY1 likely plays an important role in plant defense by participating in the production of lignin in the cell wall and providing direct protection through the formation of toxic phytoalexins. [ABSTRACT FROM AUTHOR]

Human tryptophan hydroxylase 2 (hTPH2) is the rate-limiting enzyme for serotonin biosynthesis in the brain. A number of naturally-occurring single nucleotide polymorphisms (SNPs) have been reported for hTPH2. We investigated the activity and kinetic characteristics of the most common missense polymorphism rs2887147 (A328 V/E; 0.92% allelic frequency for the two different reported SNPs at the same site) using bacterially expressed hTPH2. The recombinant full-length enzyme A328E had no measurable enzyme activity, but A328V displayed decreased enzyme activity (Vmax). A328V also displayed substrate inhibition and decreased stability compared to the wild-type enzyme. By contrast, in constructs lacking the N-terminal 150 amino acid regulatory domain, the A328V substitution had no effect; that is, there was no substrate inhibition, enzyme stabilities (for wild-type and A328V) were dramatically increased, and Vmax values were not different (while the A328E variant remained inactive). These findings, in combination with molecular modeling, suggest that substitutions at A328 affect catalytic activity by altering the conformational freedom of the regulatory domain. The reduced activity and substrate inhibition resulting from these polymorphisms may ultimately reduce serotonin synthesis and contribute to behavioral perturbations, emotional stress, and eating disorders.

The concentration of triclosan in wastewater is expected to rise dramatically as a consequence of the COVID-19 pandemic. A modeling analysis of the growth kinetics of microbial culture during triclosan degradation was necessary in order to establish effective wastewater treatment. The kinetic parameters are used by engineers to aid in the design and process control of biological processes. Studies were conducted using triclosan-acclimated culture to examine biomass growth and associated substrate degradation at different initial substrate concentrations (0.35–4.9 mg L−1) to this end. Substrate inhibition was calculated from experimental growth parameters using unstructured kinetic models. Unlike other model studies, a time-averaged specific bacterial growth rate in the log phase was considered for kinetic models in this study. Overestimation of the conventional log phase calculation for unstructured kinetic model constants was eliminated when the slowdown growth part of the log growth phase was taken into account. The Haldane Model was more accurate to fitting experimental data in an excellent manner. In this case, the time-averaged specific growth rate, saturation constant, and inhibition constant were 0.56 h−1, 12.77 mg L−1, 0.52 mg L−1, respectively. A yield coefficient of 0.404 mgX.mgS−1 was calculated. The critical triclosan concentration was 2.57 mg L−1. Wastewater treatment plants can be more sensitive to the value of the critical triclosan concentration. The value for time-averaged critical specific growth rate was 0.051 h−1. Pre-or post-treatment requirements can be estimated using time-averaged critical growth rate values as a benchmarking tool in biological treatment systems. [ABSTRACT FROM AUTHOR]

Chemiluminescence (CL) reactions are widely used for the detection and quantification of many types of analytes. Laccase has previously been proposed in CL reactions; however, its light emission behaviour has not been characterized. This study was conducted to characterize the laccase–luminol system, determine its kinetic parameters, and analyze the effects of protein and OHˉ concentration on the CL signal. Laccase from Coriolopsis gallica was combined with different concentrations of luminol (125 nM to 4 mM), and the enzyme kinetics were evaluated using diverse kinetic models. The laccase–luminol system was able to produce CL without an intermediate molecule, but it exhibited substrate‐inhibition behaviour. A two‐site random model was used and suggested that when the first luminol molecule was bound to the active site, laccase affinity for the second luminol molecule was increased. This inhibition effect could be avoided using a low luminol concentration. At 5 μM luminol concentration, 1 mg/ml (0.13 U) laccase is needed to achieve nearly 90% of the maximum CL signal, suggesting that the available luminol could not bind to all active sites. Furthermore, the concentration of NaOH negatively affected the CL signal. The laccase–luminol system represents an alternative to existing CL systems, with potential uses in molecular detection and quantification. [ABSTRACT FROM AUTHOR]

The interest in microorganisms to produce microbial lipids at large-scale processes has increased during the last decades. Rhodosporidium toruloides-1588 could be an efficient option for its ability to simultaneously utilize five- and six-carbon sugars. Nevertheless, one of the most important characteristics that any strain needs to be considered or used at an industrial scale is its capacity to grow in substrates with high sugar concentrations. In this study, the effect of high sugar concentrations and the effect of ammonium sulfate were tested on R. toruloides-1588 and its capacity to grow and accumulate lipids using undetoxified wood hydrolysates. Batch fermentations showed a catabolic repression effect on R. toruloides-1588 growth at sugar concentrations of 120 g/L. The maximum lipid accumulation was 8.2 g/L with palmitic, stearic, oleic, linoleic, and lignoceric acids as predominant fatty acids in the produced lipids. Furthermore, R. toruloides-1588 was able to utilize up to 80% of the total xylose content. Additionally, this study is the first to report the effect of using high xylose concentrations on the growth, sugar utilization, and lipid accumulation by R. toruloides-1588. [ABSTRACT FROM AUTHOR]

High production cost is one of the major factors that limit the market growth of polyhydroxyalkanoates (PHAs) as a biopolymer. Improving PHA synthesis performance and utilizing low-grade feedstocks are two logical strategies for reducing costs. As an oleaginous yeast, Y. lipolytica has a high carbon flux through acetyl-CoA (the main PHB precursor), which makes it a desired cell factory for PHB biosynthesis. In the current study, two different metabolic pathways (NBC and ABC) were introduced into Y. lipolytica PO1f for synthesizing PHB. Compared to the ABC pathway, the NBC pathway, which includes NphT7 to redirect the lipogenesis pathway and catalyze acetoacetyl-CoA synthesis in a more energy-favored reaction, led to PHB accumulation of up to 11% of cell dry weight (CDW), whereas the ABC pathway resulted in non-detectable accumulations of PHB. Further modifications of the strain with the NBC pathway through peroxisomal compartmentalization and gene dose overexpression reached 41% PHB of CDW and a growth rate of 0.227 h−1. A low growth rate was observed with acetate as the sole source of carbon and energy or glucose as the sole substrate at high concentrations. Using a co-substrate strategy helped overcoming the inhibitory and toxic effects of both substrates. Cultivating the engineered strain in the optimal co-substrate condition predicted by response surface methodology (RSM) led to 83.4 g/L of biomass concentration and 31.7 g/L of PHB. These results offer insight into a more cost-effective production of PHB with engineered Y. lipolytica.

A volumetric dispersion model (VDM) with inhibitory kinetics was proposed to describe the biogas production from the acetolastic methanogenesis of the acetate medium process in modeling aspects of the operating capacity and stability. The influence of axial mixing promoted by ultrasonic disruption had been investigated during the methane conversion under a progressive inhibition of substrate in the up-flow anaerobic sludge blanket (UASB) reactor. Increasing the organic loading rates was conducted for the model calibration and prediction to indicate when the process instability occurs due to a potent inhibition and suggest a proper axial mixing. For this simplified substrate characteristics investigation with controlled hydraulic retention time, the results showed that the flow and transfer system with a Peclet number of 1.254 could prevent the system failure. The process instability due to a potent inhibition could be manipulated by using suitable axial mixing to eliminate the inhibitory effect within the reactor.

Xanthine oxidase is an oxidase that has a molybdopterin structure with substrate inhibition. Here, we show that a single point mutation (Q201) in the Acinetobacter baumannii xanthine oxidase (AbXOD) obtained mutant Q201E (kcat =799.44 s−1, no inhibition) with high enzyme activity and decrease of substrate inhibition in 5 mmol/L high substrate model, and which cause two loops structure change at active center, characterized by complete loss of substrate inhibition without reduction of enzymatic activity. Molecular docking results showed that the change of flexible loop increased the affinity between substrate and enzyme, and the formation of a π-π bond and two hydrogen bonds made the substrate more stable in the active center. Ultimately, Q201E can still maintain better enzyme activity under high purine content (an approximately 7-fold improvement over the wild-type), indicating a broader application prospect in the manufacture of low-purine food.

Methanogenic archaea have received attention due to their potential use in biotechnological applications such as methane production, so their metabolism and regulation are topics of special interest. When growing in a nutrient‐rich medium, these organisms exhibit gluconeogenic metabolism; however, under starvation conditions, they turn to glycolytic metabolism. To date, no regulatory mechanism has been described for this gluconeogenic/glycolytic metabolic switch. Here, we report that adenosine monophosphate (AMP) activates both enzymatic activities of the bifunctional adenosine diphosphate (ADP)‐dependent phosphofructokinase/glucokinase from Methanococcus maripaludis (MmPFK/GK). To understand this phenomenon, we performed a comprehensive kinetic characterisation, including determination of the kinetics, substrate inhibition and AMP activation mechanism of this enzyme. We determined that MmPFK/GK has an ordered‐sequential mechanism, in which MgADP is the first substrate to bind and AMP is the last product released. The enzyme also displays substrate inhibition by both sugar substrates; we determined that this inhibition occurs through the formation of catalytically nonproductive enzyme complexes caused by sugar binding. For both activities, the AMP activation mechanism occurs primarily through incremental changes in the affinity for the sugar substrate, with this effect being higher in the GK than in the PFK activity. Interestingly, due to the increase in the sugar substrate affinity caused by AMP, an enhancement in the sugar substrate inhibition effect was also observed for both activities, which can be explained by an increase in sugar binding leading to the formation of dead‐end complexes. These results shed light on the regulatory mechanisms of methanogenic archaeal sugar metabolism, a phenomenon that has been largely unexplored. [ABSTRACT FROM AUTHOR]

l‐Lactate oxidase (LOx) is a flavin mononucleotide (FMN)‐dependent triose phosphate isomerase (TIM) barrel fold enzyme that catalyzes the oxidation of l‐lactate using oxygen as a primary electron acceptor. Although reductive half‐reaction mechanism of LOx has been studied by structure‐based kinetic studies, oxidative half‐reaction and substrate/product‐inhibition mechanisms were yet to be elucidated. In this study, the structure and enzymatic properties of wild‐type and mutant LOxs from Enterococcus hirae (EhLOx) were investigated. EhLOx structure showed the common TIM‐barrel fold with flexible loop region. Noteworthy observations were that the EhLOx crystal structures prepared by co‐crystallization with product, pyruvate, revealed the complex structures with "d‐lactate form ligand," which was covalently bonded with a Tyr211 side chain. This observation provided direct evidence to suggest the product‐inhibition mode of EhLOx. Moreover, this structure also revealed a flip motion of Met207 side chain, which is located on the flexible loop region as well as Tyr211. Through a saturation mutagenesis study of Met207, one of the mutants Met207Leu showed the drastically decreased oxidase activity but maintained dye‐mediated dehydrogenase activity. The structure analysis of EhLOx Met207Leu revealed the absence of flipping in the vicinity of FMN, unlike the wild‐type Met207 side chain. Together with the simulation of the oxygen‐accessible channel prediction, Met207 may play as an oxygen gatekeeper residue, which contributes oxygen uptake from external enzyme to FMN. Three clades of LOxs are proposed based on the difference of the Met207 position and they have different oxygen migration pathway from external enzyme to active center FMN. PDB Code(s): 6M73 and 6M74; [ABSTRACT FROM AUTHOR]

The myrosinase enzyme hydrolyzes glucosinolates, among which is glucoraphanin, the precursor of the anticancer isothiocyanate sulforaphane (SFN). The main source of glucoraphanin is Brassicaceae; however, its natural concentration is relatively low, limiting the availability of SFN. An option to obtain SFN is its exogenous production, through enzymatic processes and under controlled conditions, allowing complete conversion of glucoraphanin to SFN. We characterized the kinetics of wild-type (BMYR) and recombinant broccoli myrosinases produced in E. coli (EMYR) and S. cerevisiae (SMYR) in terms of the reaction conditions. Kinetics was adjusted using empirical and mechanistic models that describe reaction rate as a function of substrate concentration, temperature, and pH, resulting in R2 values higher than 90%. EMYR kinetics differed significantly from those of BMYR and SMYR probably due to the absence of glycosylations in the enzyme produced in E. coli. BMYR and SMYR were subjected to substrate inhibition but followed different kinetic mechanisms attributed to different glycosylation patterns. EMYR (inactivation Ea = 76.1 kJ/mol) was more thermolabile than BMYR and SMYR. BMYR showed the highest thermostability (inactivation Ea = 52.8 kJ/mol). BMYR and EMYR showed similar behavior regarding pH, with similar pK1 (3.4 and 3.1, respectively) and pK2 (5.4 and 5.0, respectively), but differed considerably from SMYR. [ABSTRACT FROM AUTHOR]

A dynamic-kinetic model for gas fermentation of C. carboxidivorans is presented. The model described the acid/alcohol production in gas-fed stirred tank reactors (GFSTR) and in batch pressurized reactors. The model took into account gas-liquid mass transfer rate, carbon monoxide uptake rate and three conversion paths: cell growth associated with acid/alcohol production, alcohol production by acid conversion and direct alcohol production not associated to cell growth. The assessment of kinetic parameters/yields was carried out by regression of experimental data retrieved from the literature. The model was then used to study the effects of several process conditions on the fermentation performances. The sensitivity was analysed with respect to three operating variables (agitation speed, gas feeding flow-rate and bioreactor filling ratio) and also to the kinetic parameters/yields. The proposed model successfully reproduced the experimental data: the R² of the investigated variable concentrations ranged between 0.769 and 0.885. Sensitivity analysis pointed out that: i) the model was significantly affected by parameters related to the growth kinetic whereas it was not impacted by parameters related to direct alcohol production; ii) fermentation performances could be improved tuning the operating conditions, in particular, the decrease of the mass transfer rate could increase the cell/metabolite production. [ABSTRACT FROM AUTHOR]

Manufacturing processes in semiconductor and photonics industries involve the use of a significant amount of organic solvents. Recycle and reuse of these solvents produce distillate residues and require treatment before being discharged. This study aimed to evaluate the performance of the biological treatment system in a full-scale wastewater treatment plant that treats wastewater containing distillate residues from the recycling of electronic chemicals. Batch experiments were conducted to investigate the optimal operational conditions for the full-scale wastewater treatment plant. To achieve good nitrogen removal efficiency with effluent ammonia and nitrate concentrations below 20 mg N/L and 50 mg N/L, respectively, it was suggested to control the ammonia concentration and pH of the influent below 500 mg N/L and 8.0, respectively. In addition, the biodegradability of N-methylpyrrolidone, diethylene glycol monobutyl ether, and cyclopentanone distillate residues from the electronic chemicals manufacturing process were evaluated under aerobic, anoxic, and anaerobic conditions. N-methylpyrrolidone and cyclopentanone distillate residues were suggested to be treated under anoxic condition. However, substrate inhibition occurred when using cyclopentanone distillate residue as a carbon source with chemical oxygen demand (COD) levels higher than 866 mg/L and nitrate levels higher than 415 mg N/L. Under aerobic condition, the COD from both N-methylpyrrolidone and cyclopentanone distillate residues could be easily degraded. Nevertheless, a negative effect on nitrification was observed, with a prolonged lag time for ammonia oxidation as the initial COD concentration increased. The specific ammonia oxidation rate and nitrate production rate decreased under high COD concentration contributed by N-methylpyrrolidone and cyclopentanone distillate residues. Furthermore, the biodegradability of diethylene glycol monobutyl ether distillate residue was found to be low under aerobic, anoxic, and anaerobic conditions. With respect to the abundance of nitrogen removal microorganisms in the wastewater treatment plant, results showed that Comammox may have an advantage over ammonia oxidizing bacteria under high pH conditions. In addition, Comammox may have higher resistance to environmental changes. Dominance of Comammox over ammonia oxidizing bacteria under high ammonia condition was first reported in this study. [Display omitted] • Optimizing influent ammonia and pH for high ammonia wastewater treatment in a full-scale WWTP. • Reversible inhibition of NMP and CPN distillate residues on nitrification was observed. • Treatment of NMP and CPN distillate residues was suggested under anoxic condition in the A/O process. • Dominance of Comammox over AOB under high ammonia condition was first reported. [ABSTRACT FROM AUTHOR]

A dual immobilized lipase and oxovanadium co-catalyzed dynamic kinetic resolution (DKR) system was established and successfully applied to synthesize (R)-1-(3-Methoxyphenyl) acetate in batch, resulting in a key chiral intermediate of (S)-rivastigmine with highly yield (97.6 %), e.e (99.01 %) and selectivity Sel (99.4 %). Herein, oxovanadium was immobilized onto diatomite via grinding immobilization for the first time to access a novel immobilized oxovanadium (V-D) used to catalyze an in situ racemization of undesired enantiomer substrate. The racemization rate of hydrated vanadyl sulfate was found for the first time to be much higher than that of vanadyl sulfate without crystalline water because water molecules in the former may form ion clusters of solvation membranes to disperses the charge density of positive carbon ions and promote their stability. This grinding immobilization approach may become a promising alternative for the immobilization of metals, especially precious metals due to its low cost, high efficiency, easily separated and large reaction interface. The kinetic model of the kinetic resolution catalyzed by LA-MR was developed, which may obey the sequential mechanism with both substrate and product inhibition, while the in-situ racemization reaction catalyzed by V-D may comply with power law. • A novel dual immobilized lipase and oxovanadium co-catalyzed DKR system. • The grinding immobilization of oxovanadium onto diatomite was first employed. • The enzymatic kinetic resolution may obey the sequential mechanism. • The yield, e.e and selectivity Sel are greater than 99 % in continuous flow DKR. [ABSTRACT FROM AUTHOR]

Perchlorate is an important ion on both Earth and Mars. Perchlorate reductase (PcrAB), a specialized member of the dimethylsulfoxide reductase superfamily, catalyzes the first step of microbial perchlorate respiration, but little is known about the biochemistry, specificity, structure, and mechanism of PcrAB. Here we characterize the biophysics and phylogeny of this enzyme and report the 1.86-Å resolution PcrAB complex crystal structure. Biochemical analysis revealed a relatively high perchlorate affinity (Km = 6 μm) and a characteristic substrate inhibition compared with the highly similar respiratory nitrate reductase NarGHI, which has a relatively much lower affinity for perchlorate (Km = 1.1 mm) and no substrate inhibition. Structural analysis of oxidized and reduced PcrAB with and without the substrate analog SeO3 (2-) bound to the active site identified key residues in the positively charged and funnel-shaped substrate access tunnel that gated substrate entrance and product release while trapping transiently produced chlorate. The structures suggest gating was associated with shifts of a Phe residue between open and closed conformations plus an Asp residue carboxylate shift between monodentate and bidentate coordination to the active site molybdenum atom. Taken together, structural and mutational analyses of gate residues suggest key roles of these gate residues for substrate entrance and product release. Our combined results provide the first detailed structural insight into the mechanism of biological perchlorate reduction, a critical component of the chlorine redox cycle on Earth.

Malate dehydrogenase (MDH) catalyzes the reduction of oxaloacetate to L-malate. Geobacillus stearothermophilus MDH (gs-MDH) is used as a diagnostic reagent; however, gs-MDH is robustly inhibited at high substrate concentrations, which limits its reaction rate. Here, we reduced substrate inhibition of gs-MDH by deleting its C-terminal residues. Computational analysis showed that C-terminal residues regulate the position of the active site loop. C-terminal deletions of gs-MDH successfully increased K i values by 5- to 8-fold with maintained thermal stability (>90% of the wild-type enzyme), although k cat/ K m values were decreased by <2-fold. The structure of the mutant showed a shift in the location of the active site loop and a decrease in its volume, suggesting that substrate inhibition was reduced by eliminating the putative substrate binding site causing inhibition. Our results provide an effective method to reduce substrate inhibition of the enzyme without loss of other parameters, including binding and stability constants. [ABSTRACT FROM AUTHOR]

Bacteria in biological wastewater treatment process play an important role in the removal of substrate concentration. When bacteria removes the substrate, they continuously growth until such time when the substrate concentration is depleted. This paper aims to review the performance of kinetic growth rate models that describe specific bacteria growth rate. The kinetic growth rate models reviewed were: Monod, Blackman, Haldane, Teissier, Moser, Contois, Logarithmic, Powell, Han and Levenspiel, Logistic, Luong, Webb, Yano and Koga, and Aiba-Edwards. The performance review of the models were based on coefficient of determination (R²). Another statistical measure of performance was root mean square error (RMSE). All kinetic growth rate models performed well when subjected to both toxic and non-toxic substrate concentration. The most notable kinetic growth rate model was the Haldane model which was well represented in the literature.

Ribonuclease A (RNase A) is an endonuclease that plays a significant role in antimicrobial activity by the cleavage and hydrolysis of ssRNA. In this study, the interactions between RNase A and cCMP have been investigated, through molecular docking and a 200 ns molecular dynamics simulation. The enzyme kinetic properties were analyzed using saturation curve, Eadie-Hofstee, and Hill plots. The docking results indicate that the cCMP-RNase A complexes are stabilized through hydrophobic interaction, hydrogen bonding, and π-π stacking interaction. The most binding site is observed in the catalytic cleft, especially at residue His12 and His119. Enzyme-ligand docking study indicates that cCMP binds to residues located in the internal cavity of the catalytic site and shows three phases of binding modes. The analysis of MD simulations shows that RMSD, Rg, and RMSF reach equilibrium. The ΔG binding of the cCMP-RNase A was negative (-619.673 kJ/mol), The comparison between the results pre and post MD simulation showed that ΔG binding after MD simulation causes to more stable the structure than before simulation. Experimental methods such as saturation, Eadie-Hofstee, and Hill plots confirm theoretical data and show three phases of binding modes as well. Two significant events are demonstrated in the interaction between RNase A and cCMP: substrate activation and substrate inhibition are observed in concentrations below and above 0.8 mM, respectively, for cCMP. Choosing the appropriate concentration of cCMP is very important in investigation of ribonuclease A's catalytic behavour, especially for exploration of the effects of some drugs on biological behaviours related to ribonuclease A.Communicated by Ramaswamy H. Sarma.

The interest in microorganisms to produce microbial lipids at large-scale processes has increased during the last decades. Rhodosporidium toruloides-1588 could be an efficient option for its ability to simultaneously utilize five- and six-carbon sugars. Nevertheless, one of the most important characteristics that any strain needs to be considered or used at an industrial scale is its capacity to grow in substrates with high sugar concentrations. In this study, the effect of high sugar concentrations and the effect of ammonium sulfate were tested on R. toruloides-1588 and its capacity to grow and accumulate lipids using undetoxified wood hydrolysates. Batch fermentations showed a catabolic repression effect on R. toruloides-1588 growth at sugar concentrations of 120 g/L. The maximum lipid accumulation was 8.2 g/L with palmitic, stearic, oleic, linoleic, and lignoceric acids as predominant fatty acids in the produced lipids. Furthermore, R. toruloides-1588 was able to utilize up to 80% of the total xylose content. Additionally, this study is the first to report the effect of using high xylose concentrations on the growth, sugar utilization, and lipid accumulation by R. toruloides-1588.

Esomeprazole with chiral sulfoxides structure is used to treat gastric ulcer disease. Soybean pod shell peroxidase (SPSP) is a peroxidase extracted from soybean pods shells which are one of the most abundant natural resources in the world. In the production of chiral sulfoxides catalyzed by SPSP, it is very important to establish the reaction kinetic model and explore the reaction mechanism for the development of the process, however, there is no report on the establishment of the model. Asymmetric sulfoxidation reactions catalyzed by SPSP in water‐in‐oil microemulsions were carried out, and the King‐Altman approach was used to establish a kinetic model. A yield of 91% and e.e. value of 96% for esomeprazole were obtained at the activity of SPSP of 3200 U ml−1 and 50 °C for 5 h. The mechanism with a two‐electron reduction of SPSP‐I is accompanied with a single‐electron transfer to SPSP‐I and nonenzymatic reactions, indicating that three concomitant sub‐mechanisms contribute to the asymmetric oxidation involving five enzymatic and two nonenzymatic reactions, which can represent the asymmetric sulfoxidation of organic sulfides to form enantiopure sulfoxides. With 5.44% of the average relative deviation, a kinetic model fitting experimental data was developed. The enzymatic reactions may follow ping‐pong mechanism with substrate inhibition of H2O2 and product inhibition of esomeprazole, while nonenzymatic reactions follow a power law. Those results indicate that SPSP with a lower cost and higher thermal stability may be used as an effective substitute for horseradish peroxidase. [ABSTRACT FROM AUTHOR]

A rigorous knowledge of the bacterial growth kinetics is essential for the scaling-up and optimization of biodegradation process conditions in a bioreactor. Although a great deal of literature is available on the modeling of bacterial growth kinetics considering the inhibition at high substrate-loading, the inhibition caused by toxic metabolic byproducts was not accounted in the bacterial growth kinetics. This work primarily aimed at developing a parametric bacterial growth model to account for metabolite inhibition, indicated by a decelerating log-phase growth, which was rarely discussed in the previous studies. An efficient azo-dye degrading bacterium (Bacillus subtilis MN372379) was isolated from the sludge-waste nearby a carpet-dyeing unit. The isolated bacterial strain was used to decolorize the simulated wastewater containing Congo red dye. This study proposed a computational approach to calculate specific bacterial growth rate time-averaged over the entire sigmoidal log phase (including the decelerating phase) for incorporating the effect of metabolite-inhibition, in contrast to the conventional studies where only the initial part (accelerating) of log phase was considered. The nature of metabolite inhibition was also determined and found to be non-competitive. Next, the computed time-averaged specific bacterial growth rate was incorporated into three substrate inhibition models to account for both, the metabolite and substrate inhibitions, and subsequently their kinetic parameters were also determined. Finally, the initial dye concentration and inoculum size were optimized to yield maximum dye utilization rate. This study paves the way for predicting bacterial growth kinetic with improved accuracy to enable a better optimization of bioreactors at the industrial scale. [ABSTRACT FROM AUTHOR]

Extremophillic microalga Chlorella sorokiniana is grown mixotrophically in a 5 L externally-illuminated tubular bubble-column photobioreactor with an external light intensity of 10,500 lx (≈195 μmol-photons.m−2.s−1) at atmospheric (0.04 %), 1 %, 2 %, 3 %, 4 %, and 5 % CO 2 concentrations, with acetic acid and urea as the organic carbon and nitrogen sources, respectively, for 6–8 days. Two different setups are employed: one permits axial air flow, and another promotes radial mixing of algal particles, dissolved CO 2 and nutrients along with axial sparging. We show that radial mixing increases biomass yields by reducing both photo-limitation and photo-inhibition up to CO 2 concentrations <3 %, above which substrate (CO 2) inhibition necessitates lower reactor mixing. Radial mixing increases the lipid yield from 1.06 and 1.61 g/L to 1.27 and 1.78 g/L at 2 % and 3 % CO 2 , respectively, in nitrogen-limited cases, resulting in a 10–20 % increase. We show that complete reactor mixing, facilitated by the dual effects of branched (radial) and axial sparger, is preferred only in the absence of substrate (CO 2) inhibition. Subsequently, we stratify the tubular photobioreactor design strategy into two regimes based on CO 2 inlet concentrations. In one regime (for inlet CO 2 < 3 %), we recommend the deployment of branched sparger to facilitate both radial and axial mixing simultaneously, thereby creating a well-mixed reactor at lower CO 2 concentrations. In the other regime (for inlet CO 2 > 3 %), axial sparging alone is recommended since complete mixing of CO 2 increases substrate inhibition by reducing the reactor pH below 7.5. [Display omitted] • Radial mixing effects on multiscale algal growth and molecular synthesis • Biomass and lipid yields increase by 7.5–20 % at optimal inlet CO 2 (2–3 %). • Substrate inhibition at >3 % CO 2 reduces extracellular pH < 7.5. • Two different reactor mixing strategies are suggested based on inlet CO 2 %. • Recommended design strategy: radial mixing with axial sparging at CO 2 < 3 %(v /v); only axial sparging at CO 2 > 3 % [ABSTRACT FROM AUTHOR]

Dye-decolorizing peroxidases (DyPs) have been intensively investigated for the purpose of industrial dye decolourization and lignin degradation. Unfortunately, the characterization of these peroxidases is hampered by their non-Michaelis-Menten kinetics, exemplified by substrate inhibition and/or positive cooperativity. Although often observed, the underlying mechanisms behind the unusual kinetics of DyPs are poorly understood. Here we studied the kinetics of the oxidation of 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), hydroquinones, and anthraquinone dyes by DyP from the bacterium Thermobifida halotolerans (Th DyP) and solved its crystal structure. We also provide rate equations for different kinetic mechanisms explaining the complex kinetics of heme peroxidases. Kinetic studies along with the analysis of the structure of Th DyP suggest that the substrate inhibition is caused by the non-productive binding of ABTS to the enzyme resting state. Strong irreversible inactivation of Th DyP by H 2 O 2 in the absence of ABTS suggests that the substrate inhibition by H 2 O 2 may be caused by the non-productive binding of H 2 O 2 to compound I. Positive cooperativity was observed only with the oxidation of ABTS but not with the two electron-donating substrates. Although the conventional mechanism of cooperativity cannot be excluded, we propose that the oxidation of ABTS assumes the simultaneous binding of two ABTS molecules to reduce compound I to the enzyme resting state, and this causes the apparent positive cooperativity. [Display omitted] • Th DyP shows positive cooperativity with the single electron donating substrate. • Th DyP is substrate inhibited by both the reducing substrate and H 2 O 2. • Without the reducing substrate Th DyP is irreversibly inactivated by H 2 O 2. • 5 molecules of H 2 O 2 is sufficient for inactivation of one molecule of Th DyP. [ABSTRACT FROM AUTHOR]

Tyrosinase is a bifunctional enzyme mediating the o-hydroxylation and two-electron oxidation of monophenols to o-quinones. The monophenolase activity of tyrosinase is much desired for the industrial synthesis of catechols. However, the generally low ratio of monophenolase/diphenolase activity of tyrosinase limited its utilization in the industry. In this study, a novel tyrosinase from Armillaria ostoyae strain C18/9 (AoTyr) was characterized, and the results showed that the enzyme has an optimal temperature of 25°C and an optimal pH of 6. The enzyme has comparable monophenolase and diphenolase activities and exhibits substrate inhibition in both of the activities. In silico analysis and mutagenesis experiments showed that residues 262 and 266 play important roles in modulating the substrate inhibition and enzymatic activities of AoTyr, and the replacement of D262 with asparagine significantly increased the monophenolase/diphenolase catalytic efficiencies (kcat/Km ratios) (1.63-fold) of the enzyme. The results from this study indicated that this novel tyrosinase could be a potential candidate for the industrial biosynthesis of catechols. [ABSTRACT FROM AUTHOR]

Rhodopseudomonas palustris is a model microorganism for studying the anaerobic metabolism of aromatic compounds. While it is well documented which aromatics can serve as sole organic carbon sources, co-metabolism of other aromatics is poorly understood. This study used kinetic modeling to analyze the simultaneous degradation of aromatic compounds present in corn stover hydrolysates and model the co-metabolism of aromatics not known to support growth of R. palustris as sole organic substrates. The simulation predicted that p-coumaroyl amide and feruloyl amide were hydrolyzed to p-coumaric acid and ferulic acid, respectively, and further transformed via p-coumaroyl-CoA and feruloyl-CoA. The modeling also suggested that metabolism of p-hydroxyphenyl aromatics was slowed by substrate inhibition, whereas the transformation of guaiacyl aromatics was inhibited by their p-hydroxyphenyl counterparts. It also predicted that substrate channeling may occur during degradation of p-coumaroyl-CoA and feruloyl-CoA, resulting in no detectable accumulation of p-hydroxybenzaldehyde and vanillin, during the transformation of these CoA ligated compounds to p-hydroxybenzoic acid and vanillic acid, respectively. While the simulation correctly represented the known transformation of p-hydroxybenzoic acid via the benzoyl-CoA pathway, it also suggested co-metabolism of vanillic acid and syringic acid, which are known not to serve as photoheterotrophic growth substrate for R. palustris. [ABSTRACT FROM AUTHOR]

A differential biotrickling filter (DBTF) was developed as a research tool to minimize radial and longitudinal gradients, which hinder analysis in integral (column) reactors. The main design modifications were a very high gas recycle rate and a low, uniform liquid addition rate via an aerosol generator. The elimination capacity (EC), uniformity of biofilm formation, and long-term reliability of the reactor were evaluated. The maximum toluene elimination capacity was approximately 430 g/m3 h , which was higher than the EC in most previous reports. The high EC potentially was due to the thin liquid film over the biofilm generated by low liquid trickling rates. Moreover, the high gas recycle rate (2.5–100 times the feed flow rate) allowed uniform substrate and nutrients distribution throughout the bed hence promoting favorable growth and performance of the microbes. These findings can serve as a guide in improving performance of industrial biotrickling filters. Substrate inhibition was observed at loading rates (LRs) higher than 513±27 g/m3 h. Despite operational issues that affected its long-term reliability, such as (1) unwanted growth of microbes on the pipes and in the aerosol reservoir, (2) a decline in the performance of the aerosol generator, and (3) limited fan capacity, the DBTF is a promising tool that can improve biofiltration research. [ABSTRACT FROM AUTHOR]

Substrate and product inhibitions have a significant effect on dark fermentative hydrogen gas production. Particularly, rapid formation of volatile fatty acids leads to fast pH decreases shifting the metabolic pathway. Therefore, controlling volatile fatty acid accumulation has great importance in maintaining effective hydrogen production. In this context, a dilution strategy was applied to regulate volatile fatty acids levels within the desired concentration range. A three-factor Box-Behnken statistical experiment design was established to assess the effects of dilution time, dilution percentage and initial COD concentration on hydrogen formation yield and rate. Highest hydrogen yield (7.7 mL H 2 /mL reactor) and rate (21. 47 mL H 2 /h or 9.38 mmol/L reactor.h) were achieved when 85 gCOD/L containing fermentation media was diluted with a percentage of 130 of the initial working volume at the 3rd hour of the fermentation period. Moreover, this strategy enabled to start fermentation with 55 g glucose/L. Image 1 • Hydrogen production was improved with a proper dilution of dark fermentation media. • Dilution conditions were assessed by Box-Behnken statistical experiment design. • Negative effects of substrate and product inhibitions were reduced significantly. • Dilution strategy enabled to ferment high sugar-containing molasses. [ABSTRACT FROM AUTHOR]

γ-Butyrobetaine hydroxylase (γ-BBH) is the last limiting enzyme of the L-carnitine biosynthesis pathway and plays an important role in catalyzing the hydroxylation of γ-butyrobetaine (γ-BB) to L-carnitine. To study the developmental effect of substrate concentration on the enzyme’s specific activity, kinetics of γ-BBH were measured in liver and kidney from newborn and 1-, 7-, 21-, 35-, 56-, and 210-day-old domestic pigs. Fresh tissue homogenates were assayed under nine concentrations of γ-BB from 0 to 1.5 mM. Substrate inhibition associated with age was observed at γ0.6 mM of γ-BB. Hepatic activity was low at birth but increased after 1 day. By 21 days, the activity rose by 6.6-fold (P < 0.05) and remained constant after 56 days. Renal activity was higher than in liver at birth but remained constant through 35 days. By 56 days, the velocity increased by 44% over the activity at birth (P < 0.05). The apparent Km for γ-BB at birth on average was 2.8-fold higher than at 1 day. The Km value was 60% higher in kidney than liver during development but showed no difference in adult pigs. The total organ enzyme activity increased by 130-fold for liver and 18-fold for kidney as organ weight increased from birth to 56 days. In conclusion, age and substrate affect γ-BBH specific activity and Km for γ-BB in liver and kidney. Whereas the predominant organ for carnitine synthesis is likely the kidney at birth, the liver appears to predominate after the pig exceeds 7 days of age. [ABSTRACT FROM AUTHOR]

The enzyme galacturonate oxidoreductase PcGOR from Penicillium camemberti reduces the C-1 carbon of D-glucuronate and C-4 epimer D-galacturonate to their corresponding aldonic acids, important reactions in both pectin catabolism and ascorbate biosynthesis. PcGOR was active on both glucuronic acid and galacturonic acid, with similar substrate specificities (k cat / K m) using the preferred co-substrate NADPH. Substrate acceptance extended to lactone congeners, and D-glucurono-3,6-lactone was converted to L-gulono-1,4-lactone, an immediate precursor of ascorbate. Reaction with glucuronate showed only minor substrate inhibition, and the product L-gulonate and L-gulono-1,4-lactone were both found to be competitive inhibitors with K i in the low mM range. In contrast, reaction with C-4 epimer galacturonate displayed marked substrate inhibition. Moreover, the product L-galactonate and L-galactono-1,4-lactone were observed to mitigate substrate inhibition by galacturonate, with the lactone having a greater effect than the acid. [ABSTRACT FROM AUTHOR]

The biodesulfurization 4S pathway can specifically desulfurize an aromatic S heterocyclic compound (which is difficult to desulfurize by hydrodesulfurization) and maintain the integrity of its combustion value. The four Dsz enzymes in the pathway convert the model compound dibenzothiophene (DBT) into the sulfur-free compound 2-hydroxybiphenyl (HBP). DszC is the first enzyme in the 4S pathway and is subject to feedback inhibition and substrate inhibition. This study is the first attempt to further modify the DszC mutant AKWC to improve its tolerance to DBT. Alanine scanning was performed on the dimeric surface of the DszC mutant AKWC, and the HBP yield of the BAD (AKWCP413A) strain was increased compared to the BAD (AKWC) strain. Site-directed saturation mutagenesis was performed on the 413th amino acid of AKWC, and the substrate inhibition parameter KI value of the mutant AKWCPI was 5.6 times higher than that of AKWC. When the DBT concentration was 0.25 mM, the HBP production of the recombinant strain overexpressing AKWCPI was increased by approximately 1.4-fold compared to the BL21(DE3)/BADC*+C* strain. The protein engineering of DszC further improved the substrate tolerance after overcoming the feedback inhibition, which provided a reference for the analysis of the inhibition mechanism of DszC substrate. Overexpression of DszC-beneficial mutants also greatly improved the efficiency of desulfurization. [ABSTRACT FROM AUTHOR]

A low-cost Kodo millet bran residue was utilized as feedstock for the production of D (−) lactic acid (DLA) using Lactobacillus delbrueckii NBRC3202 under anaerobic condition. Data culled from a series of batch fermentation processes with different initial Kodo millet bran residue hydrolysate (KMBRH) and DLA concentrations were used for kinetic model development. Both simulated and experimental data were in good agreement for cell growth, KMBRH utilization, and DLA formation. The values of kinetic constants specific growth rate, (μm = 0.17 h−1); growth (αP = 0.96 g.g−1) and non-growth (βP = 1.19 g.g−1.h−1) associated constant for DLA production and the maximum specific KMBRH utilization rate, (qG, max = 1.18 g.g−1.h−1) were in good agreement with the literature reports. Kinetic analysis elucidated that L. delbrueckii growth was predominantly influenced by KMBRH limitation and highly sensitive to DLA inhibition. Fed-batch fermentation studies demonstrated the existence of substrate and product inhibition paving the scope for process intensification. [ABSTRACT FROM AUTHOR]

• A crystal structure of a reductase Ss CR in complex with NADP+ was solved. • Three residues on loops 80 and 200 were engineered by crystal structure analyses. • Mutant L211H exhibited the 16-fold substrate inhibition constant K i of wild-type. • Mutant L211H enhanced asymmetric reduction of a ketone substrate efficiently. • The balance between attenuating substrate inhibition and maintaining catalytic efficiency was achieved. Substrate inhibition of enzymes is one of the main obstacles encountered frequently in industrial biocatalysis. Haloketone reductase Ss CR was seriously inhibited by substrate 2,2′,4′-trichloroacetophenone. In this study, two essential loops were found that have a relationship with substrate binding by conducting X-ray crystal structure analysis. Three key residues were selected from the tips of the loops and substituted with amino acids with lower hydrophobicity to weaken the hydrophobic interactions that bridge the two loops, resulting in a remarkable reduction of substrate inhibition. Among these variants, L211H showed a significant attenuation of substrate inhibition, with a K i of 16 mM, which was 16 times that of the native enzyme. The kinetic parameter k cat / K m of L211H was 3.1 × 103 s−1 mM−1, showing the comparable catalytic efficiency to that of the wild-type enzyme (WT). At the substrate loading of 100 mM, the space time yield of variant L211H in asymmetric reduction of the haloketone was 3-fold higher than that of the WT. [ABSTRACT FROM AUTHOR]

We provide initial rate data from enzymatic reaction experiments and tis processing to estimate the kinetic parameters from the substrate uncompetitive inhibition equation using the median method published by Eisenthal and Cornish-Bowden (Cornish-Bowden and Eisenthal, 1974; Eisenthal and Cornish-Bowden, 1974). The method was denominated the direct linear plot and consists in the calculation of the median from a dataset of kinetic parameters Vmax and Km from the Michaelis–Menten equation. In this opportunity we present the procedure to applicate the direct linear plot to the substrate uncompetitive inhibition equation; a three-parameter equation. The median method is characterized for its robustness and its insensibility to outlier. The calculations are presented in an Excel datasheet and a computational algorithm was developed in the free software Python. The kinetic parameters of the substrate uncompetitive inhibition equation Vmax, Km and Ks were calculated using three experimental points from the dataset formed by 13 experimental points. All the 286 combinations were calculated. The dataset of kinetic parameters resulting from this combinatorial was used to calculate the median which corresponds to the statistic estimator of the real kinetic parameters. A comparative statistical analyses between the median method and the least squares was published in Valencia et al. [3].

The amides 4-trifluoromethylnicotinamide, acrylamide, and benzamide are widely used in agriculture and industry, posing hazards to the environment and animals. Immobilized bacteria are preferred in wastewater treatment, but degradation of these amides by immobilized engineered bacteria has not been explored. Here, engineered Pseudomonas putida KT2440 pLSJ15-amiA was constructed by introducing a new amidase gene expression vector into environmentally safe P. putida KT2440. P. putida KT2440 pLSJ15-amiA had high amidase activity, even at 80 °C. P. putida KT2440 pLSJ15-amiA immobilized with calcium alginate exhibited a greater environmental tolerance than free cells. The amides were rapidly degraded by the immobilized cells, but the activity was inhibited by high concentrations of substrates. The substrate inhibition model revealed that the optimum initial concentrations of 4-trifluoromethylnicotinamide, acrylamide, and benzamide for degradation by immobilized cells were 197.65, 350.76, and 249.40 μmol/L, respectively. This study develops a novel and excellent immobilized biocatalyst for remediation of wastewater containing hazardous amides.

Endoglucanase E (EngE) is a cellulosomal enzyme of the glycoside hydrolase family 5 generated by the cellulosome-producing bacterium Clostridium cellulovorans 743B. Although its basic activities and properties have been characterized, its substrate specificity, product range, and steady-state kinetics remain unclear. The current study prepared recombinant EngE (rEngE) and analyzed its substrate specificity and product range using thin layer chromatography. When carboxymethyl cellulose (CMC) or phosphoric acid swollen cellulose was used as a substrate, disaccharides and trisaccharides were the main products. However, no product was detected with microcrystalline cellulose as the substrate. This indicated that rEngE is a cellulase that hydrolyzes low-crystallinity cellulose. Furthermore, products were detected when glucomannan, lichenan, or β-glucan was used, but no product was obtained with xylan. These results suggested that rEngE hydrolyzes the β-1,4 glycosidic bond between glucose residues of the substrate. In the kinetic analysis, at CMC concentrations of ≥3 mg/mL, the reaction rate decreased. Application of the above data to three substrate inhibition models generated a better fit to a model that generates products not only from the enzyme–substrate complex but also from enzyme–substrate–substrate (ESS) complexes, in which two substrates are bound to the enzymes. In addition, it was found that a carbohydrate-binding module (CBM) contained in EngE binds to cellulose. Therefore, substrate inhibition likely occurred because the binding site of CBM may correspond to one of the substrate-binding sites in the ESS complex. [ABSTRACT FROM AUTHOR]