Search

Bacterial β-glucuronidase in the intestine is involved in the conversion of 7-ethyl-10- hydroxycamptochecin glucuronide (derived from irinotecan) to 7-ethyl-10-hydroxycamptothecin, which causes intestinal bleeding and diarrhea (side effects of anti-cancer drugs). Twelve compounds (1-12) from Polygala tenuifolia were evaluated in terms of β-glucuronidase inhibition in vitro. 4-O-Benzoyl-3'-O-(O-methylsinapoyl) sucrose (C3) was highly inhibitory at low concentrations. C3 (an uncompetitive inhibitor) exhibited a ki value of 13.4 μM; inhibitory activity increased as the substrate concentration rose. Molecular simulation revealed that C3 bound principally to the Gln158-Tyr160 enzyme loop. Thus, C3 will serve as a lead compound for development of new β- glucuronidase inhibitors.

Malaria is a global health threat that requires immediate attention. Malaria is caused by the protozoan parasite Plasmodium, the most severe form of which is Plasmodium falciparum. The methylerythritol phosphate (MEP) pathway of isoprenoid biosynthesis is essential to the survival of many human pathogens, including P. falciparum, but is absent in humans, and thus shows promise as a new antimalarial drug target. The enzyme 1-deoxy-d-xylulose 5-phosphate reductoisomerase (IspC) catalyzes the first committed step in the MEP pathway. In addition to a divalent cation (Mg2+), the enzyme requires the substrates 1-deoxy-D-xylulose 5-phosphate (DXP) and NADPH to catalyze its reaction. We designed N-alkoxy and N-acyl fosmidomycin analogs to inhibit the activity of P. falciparum IspC in a bisubstrate manner. Enzyme assays reveal that the N-alkoxy fosmidomycin analogs have a competitive mode of inhibition relative to both the DXP- and NADPH-binding sites, confirming a bisubstrate mode of inhibition. In contrast, the N-acyl fosmidomycin analogs demonstrate competitive inhibition with respect to DXP but uncompetitive inhibition with respect to NADPH, indicating monosubstrate inhibitory activity. Our results will have a positive impact on the discovery of novel antimalarial drugs.

Methylenetetrahydrofolate dehydrogenase 2 (MTHFD2) plays an important role in one-carbon metabolism. The MTHFD2 gene is upregulated in various cancers but very low or undetectable in normal proliferating cells, and therefore a potential target for cancer treatment. In this study, we present the structure of MTHFD2 in complex with xanthine derivative 15, which allosterically binds to MTHFD2 and coexists with the substrate analogue. A kinetic study demonstrated the uncompetitive inhibition of MTHFD2 by 15. Allosteric inhibitors often provide good selectivity and, indeed, xanthine derivatives are highly selective for MTHFD2. Moreover, several conformational changes were observed upon the binding of 15, which impeded the binding of the cofactor and phosphate to MTHFD2. To the best of our knowledge, this is the first study to identify allosteric inhibitors targeting the MTHFD family and our results would provide insights on the inhibition mechanism of MTHFD proteins and the development of novel inhibitors.

Aim: To evaluate the dual inhibitory effect of epigallocatechin gallate on Alzheimer’s pathogenesis. Methods: Cholinesterase inhibition studies were performed by kinetic Ellman methods and also the inhibitory effect of molecule on amyloid fibrillation is determined by dye binding thioflavin T method. Results: Epigallocatechin gallate inhibited both types of cholinesterases and amyloid fibrillation. The inhibition of acetylcholinesterase indicated an uncompetitive inhibition whereas butyrylcholinesterase inhibition had an unique pattern. Compound inhibited butyrylcholinesterase with two types of inhibition in a dose related manner. Fibrillation destabilization is also observed by the compound. Conclusion: Our results indicated that epigallocatechin gallate may be accepted as a candidate molecule as the dual effective drugs for neurodegenerative diseases.

A mathematical model of an amperometric biosensor response for uncompetitive inhibition detection is discussed. The model is based on a system of reaction–diffusion equations containing a nonlinear term related to Michaelis–Menten kinetics of the enzymatic reaction. Two highly accurate and easily accessible analytical methods are used to solve the nonlinear differential equations that describe the diffusion coupled with a reaction term. Semi-analytical expressions for the concentrations of substrate, inhibitor, and product in addition to the corresponding current response are derived. The effects of various parameters such as layer thickness, bulk substrate concentration, Michaelis–Menten constant, maximum reaction velocity on current, and the sensitivity and resistance of a biosensor are investigated. A direct comparison with a numerical solution of the governing system is presented to confirm the accuracy of the derived approximate analytical solutions.

This study aimed to produce the cosmetically and nutraceutically protein hydrolysates derived from alkaline-soluble proteins of microalgal Chlorella biomass via Alcalase enzymatic hydrolysis approach. Protein hydrolysates having high degree of hydrolysis and bioactivities were obtained after response surface methodology (RSM) optimization under optimized conditions: pH of 6.5, 60 °C of reaction temperature, 3 h of hydrolysis, and 3% enzyme-to-substrate loading. Under optimum conditions, the Alcalase enzyme exhibited a high efficiency of cleaving peptide bonds within proteins by increased free amino groups in protein hydrolysates > 1.50-fold as compared to the unhydrolyzed protein. Antioxidative properties including DPPH radical scavenging activity and ferric-reducing antioxidant power (FRAP) ability were also increased with the IC50 values of 0.16 and 0.58 mg protein/mL, respectively. Interestingly, protein hydrolysates showed a high-efficiency inhibition effect on tyrosinase activity using L-DOPA and L-tyrosine as substrates in melanogenesis with the IC50 recorded at 0.99 and 0.41 mg protein/mL, respectively. Inhibitory kinetic studies confirmed that these protein hydrolysates demonstrated mixed inhibition of DPPH, FRAP, and tyrosinase enzyme when L-tyrosine was used as substrate and also exhibited an uncompetitive inhibition to tyrosinase enzyme when using L-DOPA substrate. SDS-PAGE proved that the molecular weight of protein hydrolysates was ≤ 2 kDa. More importantly, the presences of essential amino acids (> 62 g/100 g protein) were satisfactory according to amino acid requirements in human nutrients with the WHO/FAO/UNU standard. Therefore, this study highlighted that protein hydrolysates derived enzymatically from microalgal biomass could be potentially used as natural bioactive ingredient in cosmetic and/or nutraceutical applications.

β-glucosidase-catalyzed cellobiose conversion to glucose is the principal rate-limiting step in the deconstruction of lignocellulosic biomass to biofuel production as most β-glucosidases are feedba...

Catechol-O-methyltransferase (COMT) methylates both endogenous and exogenous catechol compounds to inactive and safe metabolites. We first optimised conditions for a convenient and sensitive continuous fluorescence-based 6-O-methylation assay of esculetin, which we used for investigating the COMT activity in human, mouse, rat, dog, rabbit, and sheep liver cytosols and microsomes and in ten different rat tissues. Furthermore, we compared the inhibition potencies and mechanisms of two clinically used COMT inhibitors, entacapone and tolcapone, in these species. In most tissues, the COMT activity was at least three times higher in cytosol than in microsomes. In the rat, the highest COMT activity was found in the liver, followed by kidney, ileum, thymus, spleen, lung, pancreas, heart, brain, and finally, skeletal muscle. Entacapone and tolcapone were characterised as highly potent mixed type tight-binding inhibitors. The competitive inhibition type dominated over the uncompetitive inhibition with entacapone, whereas uncompetitive inhibition dominated with tolcapone. Rats, dogs, pigs, and sheep are high COMT activity species, in contrast to humans, mice, and rabbits; COMT activity is highest in the liver. Both entacapone and tolcapone are potent COMT inhibitors, but their inhibition mechanisms differ.

Free ammonia (FA) inhibition has been taken advantage as a strategy to suppress the growth of nitrite-oxidizing bacteria (NOB) in aerobic granules stabilized in a continuous upflow airlift reactor to achieve partial nitritation. However, after nearly 18 months of continuous exposure of aerobic granules to FA in the reactor, the FA inhibition of NOB was proven ineffective, and the partial nitritation gradually shifted to partial nitrification even though the long-term granule structural stability remained excellent under the continuous-flow mode. The extent of NOB resistance to FA inhibition was quantified based on the kinetic response of NOB to various FA concentrations in the form of an uncompetitive inhibition coefficient. It was confirmed that the NOB immobilized in larger granules under longer term exposure to FA tend to become more resistant to FA. Thereby, it was concluded that NOB can develop strong resistance to FA after continuous exposure, and thus, FA inhibition is not a reliable strategy to achieve partial nitritation in mainstream wastewater treatment. PRACTITIONER POINTS: Nitrifying aerobic granules can remain structurally stable in continuous-flow bioreactors. NOB developed free ammonia resistance after 6-month continuous exposure. Larger aerobic granules tended to develop stronger free ammonia resistance. Free ammonia inhibition is not a reliable strategy for mainstream anammox.

The kinetic theory of enzymes that modify insoluble substrates is still underdeveloped, despite the prevalence of this type of reaction both in vivo and industrial applications. Here, we present a steady-state kinetic approach to investigate inhibition occurring at the solid–liquid interface. We propose to conduct experiments under enzyme excess (E0 ≫ S0), i.e. the opposite limit compared with the conventional Michaelis–Menten framework. This inverse condition is practical for insoluble substrates and elucidates how the inhibitor reduces enzyme activity through binding to the substrate. We claim that this type of inhibition is common for interfacial enzyme reactions because substrate accessibility is low, and we show that it can be analyzed by experiments and rate equations that are analogous to the conventional approach, except that the roles of enzyme and substrate have been swapped. To illustrate the approach, we investigated the major cellulases from Trichoderma reesei (Cel6A and Cel7A) acting on insoluble cellulose. As model inhibitors, we used catalytically inactive variants of Cel6A and Cel7A. We made so-called inverse Michaelis–Menten curves at different concentrations of inhibitors and found that a new rate equation accounted well for the data. In most cases, we found a mixed type of surface-site inhibition mechanism, and this probably reflected that the inhibitor both competed with the enzyme for the productive binding-sites (competitive inhibition) and hampered the processive movement on the surface (uncompetitive inhibition). These results give new insights into the complex interplay of Cel7A and Cel6A on cellulose and the approach may be applicable to other heterogeneous enzyme reactions.

Biological nitrogen removal process could be affected due to the presence of heavy metals owing to their toxicity and accumulation in the sludge. In this study, the impact of Cu2+ shock on a long-term nitritation operation was investigated in an air-lift reactor with self-recirculation. Both the dynamics of microbial community and inhibition kinetics under Cu2+ stress were ascertained. The results showed that Cu2+ exerted severe inhibition on nitritation performance of an air-lift reactor (ALR) at 25 mg/L. The corresponding NH4+-N removal efficiency decreased to below 50%, which was mainly due to the variation of microbial community structure, especially the inhibition of nitrifiers like Nitrosomonas (the relative abundance decreased from 30% to 1% after Cu2+ inhibition). Kinetic parameters were obtained and compared after fitting the Haldane model. The long-term Cu2+ stress on the ALR aggravated the ammonium affinity and the resistance to substrate self-inhibition of the nitritation sludge, but reduced the resistance to Cu2+ inhibition. Furthermore, Cu2+ acted as uncompetitive inhibitor on nitritation process. Our results provide new insights into the nitritation characteristics under long-term Cu2+ stress.

An additional reaction of the plant phenylpropanoid pathway is that catalyzed by coniferyl aldehyde dehydrogenase (HCALDH) to form cytosolic ferulic acid from coniferaldehyde. Because ferulic acid anchors lignin in cell wall polysaccharides and causes cell wall recalcitrance, the HCALDH inhibition could be a prominent target to release fermentable sugars from lignocellulose biomass and improve the saccharification efficiency. Here, we evaluated the role of the flavanone glycoside naringin (NRG), prospected in silico, as a selective inhibitor of the HCALDH of Zea mays (ZmaysHCALDH). A homotetrameric structural model in the absence of ligands was used for evaluations. The stability of the ZmaysHCALDH–NRG complex was checked via molecular dynamics simulation of the apo form (Apo–HCALDH) and that bonded to the inhibitor (NRG–HCALDH), both for the tetrameric form. The conformation bound to NRG varied less in relation to the apo form, and thus could be considered more stable. To obtain kinetic parameters, activities of a partially purified ZmaysHCALDH preparation were determined in vitro using high performance liquid chromatography. In comparison to the NRG-free control, Vmax and Km data suggested a kinetic profile of uncompetitive inhibition with respect to the substrate coniferyl aldehyde. In brief, this combinatorial in silico and in vitro approach suggest that NRG-induced inhibition of ZmaysHCALDH may be an attractive strategy to explore new clues regarding the phenylpropanoid pathway, the role of ferulic acid in the cell wall and, and mainly the digestibility of lignocellulosic biomass for biorefinery purposes.

In this work, 11 novel compounds based on vaniline and benzylidenehydrazine structure were synthesized with various substituents on phenyl aromatic ring of the molecule and evaluated as tyrosinase inhibitors. These new derivatives showed significant anti-tyrosinase activities, among which 4i demonstrated to be the most potent compound, with IC50 values of 1.58 µM . The structure–activity relationship study of the novel constructed analogs was fully discussed. Kinetic study of compound 4i showed uncompetitive inhibition towards tyrosinase. Furthermore, the high potency of 4i was supported theoretically by molecular docking evaluations.

Succinic acid (SA), a dicarboxylic acid of industrial importance, can be efficiently produced by metabolically engineered Mannheimia succiniciproducens. Malate dehydrogenase (MDH) is one of the key enzymes for SA production, but has not been well characterized. Here we report biochemical and structural analyses of various MDHs and development of hyper-SA producing M. succiniciproducens by introducing the best MDH. Corynebacterium glutamicum MDH (CgMDH) shows the highest specific activity and least substrate inhibition, whereas M. succiniciproducens MDH (MsMDH) shows low specific activity at physiological pH and strong uncompetitive inhibition toward oxaloacetate (ki of 67.4 and 588.9 μM for MsMDH and CgMDH, respectively). Structural comparison of the two MDHs reveals a key residue influencing the specific activity and susceptibility to substrate inhibition. A high-inoculum fed-batch fermentation of the final strain expressing cgmdh produces 134.25 g L−1 of SA with the maximum productivity of 21.3 g L−1 h−1, demonstrating the importance of enzyme optimization in strain development., Malate dehydrogenase (MDH) is one of the key enzymes for succinic acid (SA) bioproduction. Here, the authors report biochemical and structural analyses of various MDHs to reveal amino acids influencing the specific activity and susceptibility to substrate inhibition, and achieve industrial-level SA production.

Lipasa pancreática es una enzima clave en el metabolismo de lípidos. Los flavonoides son compuestos bioactivos de gran relevancia debido a sus interacciones con enzimas digestivas. Se evaluó la actividad de lipasa pancreática en presencia de flavonoides. Mediante espectroscopía UVVisible se determinó que el mejor inhibidor fue quercetina, seguido de rutina > luteolina > catequina > hesperetina, con valores de IC50 de 10.30, 13.50, 14.70, 28.50 y 30.50 μM, respectivamente. Todos los flavonoides mostraron una inhibición mixta, excepto catequina que mostró una inhibición acompetitiva. La capacidad inhibitoria de los flavonoides se relacionó con propiedades estructurales compartidas entre los distintos flavonoides, como la hidroxilación en las posiciones C5, C7 (anillo A), C2’ y C3’ (anillo B), y el doble enlace entre C2 y C3 (anillo C). Los resultados de inhibición coincidieron con el análisis de la fluorescencia extrínseca. Los estudios de docking molecular indicaron que la interacción entre lipasa pancreática y los flavonoides fue principalmente mediante interacciones hidrofóbicas (pi-stacking). Las interacciones de todos los flavonoides, excepto rutina, se dieron en el mismo sitio (subsitio 1) de la enzima. La insaturación entre C2 y C3 fue determinante para el acomodo de los flavonoides con la enzima, principalmente por interacciones de pi-stacking.ABSTRACTPancreatic lipase is a key enzyme in lipid metabolism. Flavonoids are bioactive compounds obtained from vegetables with big relevance, due to their intrinsic interaction with digestive enzymes. Pancreatic lipase activity was evaluated in the presence of flavonoids, through UV-Vis spectroscopy. All tested flavonoids showed a mixed-type inhibition, except catechin, which showed a uncompetitive inhibition. The best inhibitor was quercetin followed by rutin > luteolin > catechin > hesperetin, with IC50 values of 10.30, 13.50, 14.70, 28.50 and 30.50 μM, respectively. The flavonoids inhibitory capacity was related to structural properties shared between the different flavonoids, such as the hydroxylation at C5, C7 (ring A), C2’ and C3’ (ring B), and the double bond between C2 and C3 (ring C). The inhibition results are in agreement with the extrinsic fluorescence analysis. Molecular docking studies indicated that the interaction between pancreatic lipase and flavonoids was mainly through hydrophobic interactions (pi-stacking). The interactions of all flavonoids, except rutin, occurred at the same enzyme site (subsite 1). Instauration between C2 and C3 was decisive for the arrangement of flavonoids with the enzyme, mainly due to pi-stacking interactions.

The lipopolysaccharide biosynthesis pathway is considered an attractive drug target against the rising threat of multi-drug-resistant Gram-negative bacteria. Here, we report two novel small-molecule inhibitors (compounds 1 and 2) of the acyltransferase LpxA, the first enzyme in the lipopolysaccharide biosynthesis pathway. We show genetically that the antibacterial activities of the compounds against efflux-deficient Escherichia coli are mediated by LpxA inhibition. Consistently, the compounds inhibited the LpxA enzymatic reaction in vitro. Intriguingly, using biochemical, biophysical, and structural characterization, we reveal two distinct mechanisms of LpxA inhibition; compound 1 is a substrate-competitive inhibitor targeting apo LpxA, and compound 2 is an uncompetitive inhibitor targeting the LpxA/product complex. Compound 2 exhibited more favorable biological and physicochemical properties than compound 1 and was optimized using structural information to achieve improved antibacterial activity against wild-type E. coli. These results show that LpxA is a promising antibacterial target and imply the advantages of targeting enzyme/product complexes in drug discovery.

Background: The purpose of this study was to evaluate α-amylase inhibitory effects of hydroethanolic extracts of bark from Daniella oliveri, Sclerocarya birrea, Maranthes polyandra, and Pteleopsis suberosa to fight type-II diabetes. Methods: Compound extractions were performed by hydroethanol maceration followed by liquid-liquid fractionation with solvents. TLC profiling was carried out with different fractions. The inhibitory effects of plant extracts on α-amylase activity were determined using rice starch as a substrate. Results: TLC profiling of different fractions showed different phytochemical compounds. The hydroethanolic plant extracts exhibited dose-dependent inhibition of α-amylase. D. oliveri displayed competitive inhibition, M. polyandra and S. birrea showed uncompetitive inhibition and Pteleopsis suberosa exerted mixed-inhibition. M. polyandra extract exerted the highest inhibitory effect (IC50 = 0.5 mg/mL). Conclusions: The barks of M. polyandra exhibit a remarkable α-amylase inhibitory effect which can be a novel source of antidiabetic molecules.

Substrate competition for human acetylcholinesterase (AChE) and human butyrylcholinesterase (BChE) was studies under steady-state conditions using wide range of substrate concentrations. Competing couples of substates were acetyl-(thio)esters. Phenyl acetate (PhA) was the reporter substrate and competitor were either acetylcholine (ACh) or acetylthiocholine (ATC). The common point between investigated substrates is that the acyl moiety is acetate, i.e. same deacylation rate constant for reporter and competitor substrate. Steady-state kinetics of cholinesterase-catalyzed hydrolysis of PhA in the presence of ACh or ATC revealed 3 phases of inhibition as concentration of competitor increased: a) competitive inhibition, b) partially mixed inhibition, c) partially uncompetitive inhibition for AChE and partially uncompetitive activation for BChE. This sequence reflects binding of competitor in the active centrer at low concentration and on the peripheral anionic site (PAS) at high concentration. In particular, it showed that binding of a competing ligand on PAS may affect the catalytic behavior of AChE and BChE in an opposite way, i.e. inhibition of AChE and activation of BChE, regardless the nature of the reporter substrate. For both enzymes, progress curves for hydrolysis of PhA at very low concentration (≪Km) in the presence of increasing concentration of ATC showed that: a) the competing substrate and the reporter substrate are hydrolyzed at the same time, b) complete hydrolysis of PhA cannot be reached above 1 mM competing substrate. This likely results from accumulation of hydrolysis products (P) of competing substrate and/or accumulation of acetylated enzyme·P complex that inhibit hydrolysis of the reporter substrate.

Alzheimer’s disease (AD) is considered one of the greatest global public burdens. Pathophysiology of AD is proposed to be associated with reduced levels of the neurotransmitter acetylcholine (ACh). Cholinesterase enzymes, namely acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) cleave ACh via hydrolysis. Cholinesterase inhibitors (ChEIs) are the main group of drugs currently used for the treatment of AD. Novel chromone-2-carboxamido-alkylamines (7–18) were designed, synthesized, and evaluated for cholinesterase inhibitory activity. The compounds exhibited potent AChE inhibitory activities at micromolar range (IC50 0.09–9.16 µM) and demonstrated weak BChE inhibitory activities (IC50 12.09–44.56 µM). Compound 14 (IC50 0.09 ± 0.02 µM) was the most potent AChEI in this series; it showed higher activity than the clinical used drug tacrine. Enzyme kinetic study suggested that 14 was an uncompetitive inhibitor. Molecular docking study revealed that 14 was a dual-binding site inhibitor. Compound 14 did not induce any concentration-related cytotoxic effect against SH-SY5Y cells. It also showed neuroprotective effect in the cell line. Chromone-2-carboxamido-alkylamines can be promising lead compounds for development of anti-Alzheimer’s agents.

The studies on natural compounds to diabetes mellitus treatment have been increasing in recent years. Research suggests that natural components can inhibit alpha-glucosidase activities, an important strategy in the management of blood glucose levels. In this work, for the first time in the literature, the compounds produced by Ganoderma lipsiense extracts were identified and evaluated on the inhibitory effect of these on alpha-glucosidase activity. Four phenolic compounds were identified by high-performance liquid chromatography-electrospray ionization tandem mass spectrometry (HPLC-ESI-MS/MS) to crude extract from G. lipsiense grown in red rice medium (RCE) and synthetic medium (SCE), being syringic acid identified in both extracts. Gas chromatography-mass spectrometry (GC-MS) analysis showed fatty acids and their derivatives, terpene, steroid, niacin, and nitrogen compounds to SCE, while RCE was rich in fatty acids and their derivatives. Both extracts demonstrated alpha-glucosidase inhibition (RCE IC50 = 0.269 ± 8.25 mg mL−1; SCE IC50 = 0.218 ± 9.67 mg mL−1), and the purified hexane fraction of RCE (RHEX) demonstrated the highest inhibition of enzyme (81.1%). Studies on kinetic inhibition showed competitive inhibition mode to RCE, while SCE showed uncompetitive inhibition mode. Although the inhibitory effects of RCE and SCE were satisfactory, the present findings identified some unpublished compounds to G. lipsiense in the literature with important therapeutic properties.

In Brazilian fish farms, trichlorfon has been widely used to control acanthocephalan infections in Colossoma macropomum. Toxicity tests were conducted to estimate the median lethal concentration (LC50–96 h) and evaluate the effects of trichlorfon on acetylcholinesterase (AChE) and glutathione S-transferase (GST) from different tissues of C. macropomum. The LC50–96 h of trichlorfon was estimated to be 0.87 mg L−1. In the sublethal toxicity tests, concentrations of 0.26 mg L−1 (30% of LC50–96 h) and 0.43 mg L−1 (50% of LC50–96 h) were used. AChE and GST activities were measured in the brain, muscle, intestine, and liver. In vitro studies were conducted to estimate the kinetic properties and half-maximal inhibitory concentration (IC50) values of AChE in the brain and muscle for trichlorfon. In the sublethal toxicity experiments, inhibition of more than 90% of AChE in the brain, muscle, and intestine was observed. However, the activity of GST did not vary in any of the tissues studied. This finding suggests that trichlorfon is not metabolised by this enzyme. The in vitro assay results suggest that trichlorfon tends to be a classic uncompetitive inhibitor of AChE in both the brain and muscle, since Km and Vmax values decrease, while the slope remains unchanged. The IC50 values ​of muscle AChE are lower than those of the brain. All these results show that C. macropomum has low tolerance to this pesticide and suggest that brain AChE can be used as a biochemical biomarker, while muscle AChE may be used as an indicator of mortality in toxicological studies.

In this study, the inhibition potential of 3- and 4-arylcoumarin derivatives on Theileria annulata enolase (TaENO) was assessed for the first time in the literature. Firstly, protein stabilization analyses of TaENO were performed and it was found that the enzyme remains stable with the addition of 6 M ethylene glycol at + 4 °C. Inhibitor screening analyses were carried out using 25 coumarin derivatives on highly purified TaENO (> 95%), and four coumarin derivatives [4-(3,4-dimethoxyphenyl)-6,7-dihydroxy-2H-chromen-2-one (C8); 4-(3,4-dihydroxyphenyl)-7,8 dihydroxy-2H-chromen-2-one (C9); 4-(3,4-dihydroxyphenyl)-6,7-dihydroxy-2H-chromen-2 one (C21); and 3-(3,4-dihydroxyphenyl)-7,8-dihydroxy-2H-chromen-2-one (C23)] showed the highest inhibitory effects with the IC50 values of 10.450, 13.170, 8.871 and 10.863 µM, respectively. The kinetic results indicated that these compounds inhibited the enzyme by uncompetitive inhibition. In addition, the successful binding of the most potent inhibitor (C21) into TaENO was confirmed by using MALDI-TOF mass spectrophotometry. Molecular docking analyses have predicted that C8 and C21 coumarin derivatives which showed high inhibitory effects on TaENO were interacted with high affinity to the potential regions out of the active site. Taken together, these coumarin derivatives (C8, C9, C21 and C23) are first known potent, nonsubstrate, uncompetitive inhibitors of TaENO and these results will facilitate further in vitro and in vivo analysis toward structure-based drug design studies.

Human salivary aldehyde dehydrogenase (hsALDH) protects us from the toxic effect of aldehydes. It has both diagnostic and therapeutic importance. Citral possesses many biological and pharmacological properties. The aim of this work was to investigate the inhibitory effect and the mechanism of inhibition of citral on hsALDH. Citral inhibits the dehydrogenase activity of hsALDH. It decreased the substrate affinity and to a lesser extent, the catalytic efficiency of hsALDH. Citral showed linear mixed-type inhibition with a higher tendency of competitive behavior with little, but significant, non-competitive inhibition. The nucleophilicity of active site Cys residue is not a significant contributing factor in the inhibition process. Citral shows uncompetitive inhibition towards the co-enzyme (NAD+). α-helix and β-sheet content of the enzyme were changed in presence of citral. Biophysical studies showed that citral quenches the intrinsic fluorescence of hsALDH in a static manner by forming complex with the enzyme. Molecular docking study showed that both the isomers of citral bind to the catalytic site of hsALDH interacting with few evolutionary preserved amino acid residues through multiple non-covalent interactions. Ligand efficiency metrics values indicate that citral is an efficient ligand for the enzyme in terms of its physicochemical and pharmacokinetic properties.

Lipoxygenases (LOXs) are dioxygenases that catalyze the oxygenation of polyunsaturated fatty acids to hydroperoxyl derivates. These products are precursors for different lipid mediators which are associated with pathogenesis of various diseases such as asthma, atherosclerosis and cancer. Several LOXs suffer from substrate inhibition, a potential regulatory mechanism, yet it is unclear what is the cause of this phenomenon. One such enzyme is the coral 11R-LOX which displays a significant decrease in turnover rate at arachidonic acid concentrations above 30 μM. In this report, site-directed mutagenesis and inhibition assays were employed to shed light on the mechanism of substrate inhibition in 11R-LOX. We found that introduction of a positive charge to the active site entrance with Gly188Arg substitution completely eliminates the slow-down at higher substrate concentrations. Inhibition of 11R-LOX by its catalysis product, 11(R)-hydroperoxyeicosatetraenoic acid, suggests an uncompetitive mechanism. We reason that substrate inhibition in 11R-LOX is due to additional fatty acid binding by the enzyme:substrate complex at an allosteric site situated in the very vicinity of the active site entrance.

Infectious diseases are serious public health problems, affecting a large portion of the world's population. A molecule that plays a key role in pathogenic organisms is trehalose and recently has been an interest in the metabolism of this molecule for drug development. The trehalose-6-phosphate synthase (TPS1) is an enzyme responsible for the biosynthesis of trehalose-6-phosphate (T6P) in the TPS1/TPS2 pathway, which results in the formation of trehalose. Studies carried out by our group demonstrated the inhibitory capacity of T6P in the TPS1 enzyme from Saccharomyces cerevisiae, preventing the synthesis of trehalose. By in silico techniques, we compiled sequences and experimentally determined structures of TPS1. Sequence alignments and molecular modeling were performed. The generated structures were submitted in validation of algorithms, aligned structurally and analyzed evolutionarily. Molecular docking methodology was applied to analyze the interaction between T6P and TPS1 and ADMET properties of T6P were analyzed. The results demonstrated the models created presented sequence and structural similarities with experimentally determined structures. With the molecular docking, a cavity in the protein surface was identified and the molecule T6P was interacting with the residues TYR-40, ALA-41, MET-42, and PHE-372, indicating the possible uncompetitive inhibition mechanism provided by this ligand, which can be useful in directing the molecular design of inhibitors. In ADMET analyses, T6P had acceptable risk values compared with other compounds from World Drug Index. Therefore, these results may present a promising strategy to explore to develop a broad-spectrum antibiotic of this specific target with selectivity, potency, and reduced side effects, leading to a new way to treat infectious diseases like tuberculosis and candidiasis.

The properties of the mitochondrial F1 FO -ATPase catalytic site, which can bind Mg2+ , Mn2+ , or Ca2+ and hydrolyze ATP, were explored by inhibition kinetic analyses to cast light on the Ca2+ -activated F1 FO -ATPase connection with the permeability transition pore (PTP) that initiates cascade events leading to cell death. While the natural cofactor Mg2+ activates the F1 FO -ATPase in competition with Mn2+ , Ca2+ is a noncompetitive inhibitor in the presence of Mg2+ . Selective F1 inhibitors (Is-F1 ), namely NBD-Cl, piceatannol, resveratrol, and quercetin, exerted different mechanisms (mixed and uncompetitive inhibition) on either Ca2+ - or Mg2+ -activated F1 FO -ATPase, consistent with the conclusion that the catalytic mechanism changes when Mg2+ is replaced by Ca2+ . In a partially purified F1 domain preparation, Ca2+ -activated F1 -ATPase maintained Is-F1 sensitivity, and enzyme inhibition was accompanied by the maintenance of the mitochondrial calcium retention capacity and membrane potential. The data strengthen the structural relationship between Ca2+ -activated F1 FO -ATPase and the PTP, and, in turn, on consequences, such as physiopathological cellular changes.

The sweet potato respiration rate versus gas composition was mathematically modeled, as required to design an effective modified atmosphere packaging (MAP) system. Storage tests of sweet potato were conducted at 15–30 °C. The O2 and CO2 concentrations were measured over time in a closed system. The respiration rate was estimated to be a derivative of the quadratic function of gas concentration over time and decreased with decreasing O2 and increasing CO2. The model of the uncompetitive inhibition enzyme reaction rate fitted well with the experimental results. The temperature dependency of the equation parameters (Vm, Km, and Ki) followed the Arrhenius relationships. The use of the proposed models to simulate the respiration rates as a function of temperature revealed less temperature dependence in low O2 and high CO2 concentrations. This gas composition, more desirable in practice, also agreed with the typical gas composition of MAP.

Background The present study was aimed to evaluate the molecular level anticaries effect of different medicinal plants against Streptococcus mutans (S.mutans) glucosyltransferases (gtf). Methods A total of six natural sources named as Terminalia chebula (T.chebula), Psidium guajava (P.guajava), Azadirachta indica (A.indica) and Pongamia pinnata (P.pinnata); two essential oils, clove (Syzygium aromaticum) and peppermint oil (Mentha piperita) were selected as test samples. Hydroalcoholic plant extracts and essential oils were examined for their inhibitory potential on gtf isolated from S.mutans. Polyherbal mouth wash was prepared and its effect on gtf activity was compared with commercial chlorhexidine mouth wash (5%w/v). Enzyme kinetic study was carried out in order to explore the molecular mechanism of enzyme action. Results Out of six natural sources tested, A.indica has shown maximum inhibitory effect of 91.647% on gtf and T.chebula has shown IC50 of 1.091 mg/ml which is significant when compared to standard chlorhexidine. From the final result of kinetic analysis it was found that T.chebula, P.guajava and P.pinnata have show uncompetitive inhibition where as A.indica has shown non-competitive inhibition. Surprisingly, both essential oils have shown allosteric inhibition (sigmoidal response). The polyherbal moutwash has shown significant inhibitory potential on gtf (95.936%) when compared to commercial chlorhexidine mouthwash (p

The respiration rate and the shelf-life were studied on Crotalaria longirostrata, an edible leaf cultivated on the Central American region, locally known as chipilin. Besides being a native plant in El Salvador, chipilin is a commercialized food product. In order to obtain experimental data of respiration rates, a closed chamber approach was employed at temperatures of 6, 20 and 30 °C. The experimental data obtained was fit to a two-parameter non-exponential equation to obtain the respiration rates in terms of CO2 and O2 changes over time. The gas concentration results were also fit to an enzyme kinetics respiration model of the Michaelis–Menten type. Furthermore, the respiration rates from the enzyme kinetics model were used to determine the temperature effect on the respiration. This study also provides results of the shelf-life of the unpacked and packed chipilin with different materials, low density polyethylene (LDPE) and polypropylene (PP), by using a sensory panel and fitting data to the Weibull’s probabilistic distribution. Overall results show that the enzyme kinetics model, based on uncompetitive inhibition of CO2, applies very well to chipilin. The shelf life study results indicated that packaging in LDPE and PP bags increased chipilin shelf life by 3.7 and 2.5 times respectively, compared to storage without packaging.

Glucocorticoid hormone might cause intrauterine growth restriction. The glucocorticoid-metabolizing enzyme 11β-hydroxysteroid dehydrogenase 2 (HSD11B2) in the placenta eliminates excess levels of glucocorticoids during pregnancy. The aim of the current study was to define the effects of diethylstilbestrol (DES) on HSD11B2 activity in the mammalian placentas and identify its mode of action. Rat and human placental microsomal HSD11B2 were incubated with different concentrations of DES, and IC50 values were determined. The mode of action was analyzed by incubation of DES together with substrates, glucocorticoid and NAD+. DES suppressed rat and human HSD11B2 with IC50 values of 5.33 and 12.62 μM, respectively. DES was a competitive inhibitor of rat and human HSD11B2 when steroid substrates were added, while it was an uncompetitive inhibitor when cofactor NAD+ was exposed. Oral administration of DES (0.5 mg/kg) to the rat delayed the cortisol metabolism in adult female Sprague–Dawley rats, as indicated by the increases in cortisol’s elimination half-life, maximum concentration and area under the curve. In conclusion, DES is a potent HSD11B2 inhibitor, possibly contributing to the intrauterine growth restriction.

Indoleamine 2,3-dioxygenase 1 (IDO1) is emerging as a promising therapeutic target for the treatment of malignant tumors characterized by dysregulated tryptophan metabolism. However, the antitumor efficacy of existing small-molecule IDO1 inhibitors is still unsatisfactory, and the underlying mechanism remains largely undefined. To identify novel IDO1 inhibitors, an in-house natural product library of 2000 natural products was screened for inhibitory activity against recombinant human IDO1. High-throughput fluorescence-based screening identified 79 compounds with inhibitory activity > 30% at 20 μM. Nine natural products were further confirmed to inhibit IDO1 activity by > 30% using Ehrlich’s reagent reaction. Compounds 2, 7, and 8 were demonstrated to inhibit IDO1 activity in a cellular context. Compounds 2 and 7 were more potent against IDO1 than TDO2 in the enzymatic assay. The kinetic studies showed that compound 2 exhibited noncompetitive inhibition, whereas compounds 7 and 8 were graphically well matched with uncompetitive inhibition. Compounds 7 and 8 were found to bind to the ferric-IDO1 enzyme. Docking stimulations showed that the naphthalene ring of compound 8 formed “T-shaped” π–π interactions with Phe-163 and that the 6-methyl-naphthalene group formed additional hydrophobic interactions with IDO1. Compound 8 was identified as a derivative of tanshinone, and preliminary SAR analysis indicated that tanshinone derivatives may be promising hits for the development of IDO1 inhibitors. This study provides new clues for the discovery of IDO1/TDO2 inhibitors with novel scaffolds.

The objective of this study was to investigate the porcine pancreatic α-amylase (PPA) inhibitory activity of botanical food proteins, represented by soy protein isolate (SPI) and wheat gluten protein (WGP). The half maximal inhibitory concentration (IC50) was determined, and the kinetics of inhibition were investigated using Dixon, Cornish-Bowden, and Lineweaver-Burk plots. The results showed WGP functioned as mixed-type inhibitors with both competitive and uncompetitive inhibitory characteristics, while SPI showed competitive inhibitory effects on α-amylase. The competitive inhibition constants (Kic) of WGP and SPI were 5.753 and 30.212 mg/mL, respectively, and the uncompetitive inhibition constants (Kiu) of WGP was 45.110 mg/mL, respectively. The IC50 values of WGP and SPI were 3.086 and 33.899 mg/mL, respectively. For WGP, the lower Kic vs. Kiu for mixed-type inhibitors suggested that they bound more tightly to free PPA than the PPA-starch complex. Compared with SPI, WGP displayed a stronger inhibitory effect on α-amylase. These results indicated that SPI and WGP may delay the digestion of starchy foods by inhibiting starch hydrolytic enzymes, which may be of relevance in vivo during gastrointestinal digestion. The findings are also of important practical value for the development of carbohydrate-restricted diet and protein-based functional foods.

Background: The main characteristic of Diabetes type II is the impaired activation of intracellular mechanisms triggered by the action of insulin. PTP1b is a Protein Tyrosine Phosphatase that dephosphorylates insulin receptor causing its desensitization. Since inhibition of PTP1b may prolong insulin receptor activity, PTP1b has become a drug target for the treatment of Diabetes II. Although a number of inhibitors have been synthesized during the last decades, the research still continues for the development of more effective and selective compounds. Moreover, several constituents of plants and edible algae with PTP1b inhibitory action have been found, adding this extra activity at the pallet of properties of the specific natural products. Objective: Sideritis L. (Lamiaceae) is a herbal plant growing around the Mediterranean sea which is included in the Mediterranean diet for centuries. The present study is the continuation of a previous work where the antioxidant and anti-inflammatory activities of the components of Sideritis L. were evaluated and aimed to investigate the potential of some sideritis’s components to act as PTP1b inhibitors, thus exhibiting the beneficial effect in the treatment of diabetes II. Methods: Docking analysis was done to predict PTP1b inhibitory action. Human recombinant PTP1b enzyme was used for the evaluation of the PTP1b inhibitory action, while inhibition of the human LAR and human T-cell PTP was tested for the estimation of the selectivity of the compounds. Conclusion: Docking analysis effectively predicted inhibition and mode of inhibitory action. According to the experimental results, four of the components exhibited PTP1b inhibitory action. The most active ones were acetoside, which acted as a competitive inhibitor, with an IC50 of 4 µM and lavandufolioside, which acted as an uncompetitive inhibitor, with an IC50 of 9.3 µM. All four compounds exhibited increased selectivity against PTP1b.

Hyaluronidase (hyase) is a hyaluronic acid (HA) depolymerizing enzyme produced by many pathogenic bacteria as a virulence factor to establish and spread infections. Present studies established that a steroidal fraction (SF) isolated from leaves of Carissa carandas act as a strong hyase inhibitor. The kinetic parameters involved in the inhibition of hyase by purified SF were studied and compared with standard hyase inhibitor quercetin. The purified SF showed the highest inhibition with an IC50 of 5.19 mM in comparison with a standard inhibitor, quercetin (IC50 8.63 mM). The inhibition constant (Ki) of purified SF determined by Dixon plot was 8.32 mM, which was significantly lower than that of quercetin standard. The kinetic behavior of enzyme hyase revealed to be more complex than classical competitive and uncompetitive inhibition where inhibitor affects both Km and Vmax. The inhibitor (I) favored the binding to the enzyme-substrate (ES) complex where Km value appeared to decrease (Kmapp < Km). The inhibitor also leads to decrease in the apparent maximum velocity of the enzyme-substrate reaction (Vmaxapp < Vmax). These results signpost toward mixed nature of inhibition of enzyme hyase by purified SF. Anti-hyaluronidase activity by a bioactive metabolite from C. carandas has not been reported so far and has high therapeutic potential against spread of pathogen and its toxins in the host.

Introduction: Collagenase plays an important role in the degradation of dermal matrix proteins leading to wrinkle formation. The objectives of this study were to evaluate the inhibitory effect of water-pepper (Polygonum hydropiper L.) sprout extract on the activity of collagenase and to identify the inhibitory compounds.Methods: Collagenase inhibitory activity was measured by spectrophotometric assay. Activity-guided fractionation was performed using liquid-liquid extraction of water and n-butanol and Diaion HP-20 column chromatography, followed by high-performance liquid chromatography (HPLC) fraction collection.Results: A methanolic extract of water-pepper sprout inhibited collagenase activity in a concentration-dependent manner with an IC50 value of 156.7 μg/mL. Collagenase inhibitory activity (IC50 = 23.5 μg/mL) was found in 50% methanol eluate from the HP-20 column chromatography of the n-butanol soluble fraction. The active compound (IC50 = 1.9 μg/mL) in the eluate was isolated by HPLC and identified as quercetin-3-O-galactoside (hyperoside) from comparing retention time, UV-Vis absorption, and mass spectra with those of the standard. Lineweaver-Burk plots revealed that hyperoside was an uncompetitive inhibitor against collagenase. Hyperoside was also the most abundant flavonoid present in the methanolic extract.Conclusion: These results suggest that water-pepper sprouts could be beneficial as a natural source of collagenase inhibitor which might be used for the treatment of skin aging.

The stabilization effects on a novel commercial β-glucosidase preparation from Aspergillus fumigatus during saccharification of ethanol-water pretreated wheat straw were analysed in comparison to this enzyme stability during cellobiose hydrolysis. For this purpose, the kinetics of β-glucosidase residual activity during cellobiose hydrolysis from 40 till 70 °C were studied, resulting in the fitting of a first-order partial deactivation model. Furthermore, a subsequent fitting of a kinetic model including this first-order deactivation equation and a Michaelis-Menten equation with double competitive inhibition by glucose and uncompetitive inhibition by cellobiose to released glucose was successful. Finally, global enzyme deactivation and prospective deactivation of enzyme remaining in the liquid phase were evaluated during wheat straw hydrolysis at 50 °C as a relevant saccharification process. Results suggest that the presence of a solid substrate dramatically reduces the global deactivation rate of the enzyme and, in addition, there is no loss the stability of the enzyme in the liquid phase along the saccharification process, even for 72 h.

Bellamya bengalensis muscle meat is known for ethnopharmacological benefits. The present study focuses on the identification of ACE inhibitory peptides from the proteolytic digests of muscle protein of Bellamya bengalensis and its underlying mechanism. After ultrafiltration of 120 min alcalase hydrolysates (BBPHA120) to isolate the small peptide fraction (&lt, 3 kDa), in vitro ACE inhibitory activity was analyzed. The IC50 value of the 120 min hydrolysate ultrafiltered fraction was 86.74 ± 0.575 µg/mL, while the IC50 of lisinopril was 0.31 ± 0.07 µg/mL This fraction was assessed in a MALDI-ToF mass spectrometer and five peptides were identified from the mass spectrum based on their intensity (&gt, 1 × 104 A.U.). These peptides were sequenced via de novo sequencing. Based on the apparent hydrophobicity (%), the IIAPTPVPAAH peptide was selected for further analysis. The sequence was commercially synthesized by solid-phase standard Fmoc chemistry (purity 95–99.9%, by HPLC). The synthetic peptide (IC50 value 8.52 ± 0.779 µg/mL) was used to understand the thermodynamics of the inhibition by checking the binding affinity of the peptide to ACE by isothermal titration calorimetry compared with lisinopril, and the results were further substantiated by in silico site-specific molecular docking analysis. The results demonstrate that this peptide sequence (IIAPTPVPAAH) can be used as a nutraceutical with potent ACE inhibition.

Background: Elephantorrhiza elephantina (Burch.) Skeels is a medicinal plant used in folkloric medicine for the management of several metabolic and infectious diseases. Aim: This aim of this research study was to investigate the antioxidant and antidiabetic effects of extracts and partitioned fractions in order to validate its folkloric use. Setting: The plant material purchased from herb sellers in Qwaqwa township, authenticated at Department of Plant Science Qwaqwa herbarium, was evaluated in the same unit of the University of the Free State. Methods: The antioxidative and antidiabetic activities of extracts and fractions were assessed with 1,1-diphenyl-2-picrylhydrazyl, 2,2-azino-bis(3-ethylbenzothiazoline-6)-sulphonic acid, hydroxyl radicals, metal chelating agents, and α-amylase, as well as α-glucosidase inhibitions based on standard methods. The subfractions with considerable yields from the partitioned n-hexane fraction of the crude extract were subjected to gas chromatography-mass spectrometry analysis or profiling for possible compound identification. Results: The aqueous extract showed the most effective 1,1-diphenyl-2-picrylhydrazyl, hydroxyl radical and metal chelating activities judging by half-maximal inhibitory concentrations (IC 50 : 0.573, 0.059 and 1.937 mg/mL, respectively), whilst the ethanol extract revealed maximum activity (0.017 mg/mL) against 2,2-azino-bis(3-ethylbenzothiazoline-6)-sulphonic acid. However, the ethanol extract displayed the most potent alpha-amylase (0.346 mg/mL) inhibition, whilst the aqueous extract (0.363 mg/mL) was best against alpha-glucosidase. The modes of enzymes inhibition revealed that the aqueous extract displayed near-competitive inhibition against alpha-amylase and uncompetitive inhibition against alpha-glucosidase. Additionally, good antioxidative and antihyperglycaemic effects were established by the n-hexane fraction when compared with standards (gallic acid and acarbose). The GC-MS chromatogram of subfractions (4 and 9) from the n-hexane fraction afforded compounds, such as 2,4-bis (1, 1-dimethylethyl)-phenol, 9-octadecenoic acid (Z)-, methyl ester, dodecanoic acid and 1-methylethyl ester already established in the literature with potential pharmacological activities (antioxidant, anti-inflammatory, etc.). Conclusion: The research study provides evidence on the folkloric use and insights on the prospect of the plant as natural antioxidative and antidiabetic agents.

Tyrosinase (TYR) is a type III copper oxidase present in fungi, plants and animals. The inhibitor of human TYR plays a vital role in pharmaceutical and cosmetic fields by preventing synthesis of melanin in the skin. To search for an effective TYR inhibitor from various plant extracts, a kinetic study of TYR inhibition was performed with mushroom TYR. Among Panax ginseng, Alpinia galanga, Vitis vinifera and Moringa oleifera, the extracts of V. vinifera seed, A. galanga rhizome and M. oleifera leaf reversibly inhibited TYR diphenolase activity with IC50 values of 94.8 ± 0.2 µg/mL, 105.4 ± 0.2 µg/mL and 121.3 ± 0.4 µg/mL, respectively. Under the same conditions, the IC50 values of the representative TYR inhibitors of ascorbic acid and kojic acid were found at 235.7 ± 1.0 and 192.3 ± 0.4 µg/mL, respectively. An inhibition kinetics study demonstrated mixed-type inhibition of TYR diphenolase by A. galanga and V. vinifera, whereas a rare uncompetitive inhibition pattern was found from M. oleifera with an inhibition constant of Kii 73 µg/mL. Phytochemical investigation by HPLC-MS proposed luteolin as a specific TYR diphenolase ES complex inhibitor, which was confirmed by the inhibition kinetics of luteolin. The results clearly showed that studying TYR inhibition kinetics with plant extract mixtures can be utilized for the screening of specific TYR inhibitors.

The cytoplasm of a cell is extremely crowded, with 20-30% being large biomolecules. This crowding enforces a significant amount of the physical and chemical barrier around biomolecules, so understanding any biomolecular event within the cellular system is challenging. Unsurprisingly, enzymes show a diverse kind of catalytic behavior inside a crowded environment and thus have remained an area of active interest in the last few decades. The situation can become even more complex and exciting in the case of understanding the behavior of a membrane-bound enzyme (almost 25-30% of enzymes are membrane-bound) in such a crowded environment that until now has remained unexplored. Herein, we have particularly investigated how a membrane-bound enzyme (using liposome-bound alkaline phosphatase) can behave in a crowded environment comprising polymer molecule-like poly(ethylene glycol) (PEG) of different weights (PEG400, PEG4000, and PEG9000) and Ficoll 400. We have compared the activity using a polymer microbead conjugated enzyme and have found that liposome-bound alkaline phosphatase had much higher activity in crowded environments, showing the importance and superiority of soft-deformable particles (i.e., vesicles) over hard spheres in macro-molecularly crowded media. Interstingly, we have found a paradoxical behavior of inhibitors in terms of both their extent and pathway of inhibitory action. For instance, phosphates, known as competitive inhibitors in buffer, behave as uncompetitive inhibitors in liposome-bound enzymes in crowded media with an ∼5-fold less inhibitory effect, whereas phenyl alanine (an uncompetitive inhibitor in buffer) did not show any inhibitory potential when the enzyme was membrane-bound and in crowded media containing PEG9000 (30 wt %). Overall, this demonstration elucidates aspects of membrane-bound enzymes in crowded media in terms of both catalytic behavior and inhibitory actions and can lead to further studies of the understanding of enzymatic behavior in such complex crowded environments having a dampening effect in regular diffusive transport.

Inorganic salt pretreatment of lignocellulosic biomass has proven to be an efficient way to increase the efficiency of enzymatic saccharification. However, it is not clear that this improvement is the result of modification of the lignocellulosic substrate after pretreatment, or removal of inhibitor, or enhancement of cellulase or a combination of these events. Therefore, this study aimed to analyze the effects of inorganic salts on kinetics of cellulase enzymes (celluclast 1.5L and accellerase 1500). Two substrates rich in cellulose content [carboxymethylcellulose (CMC), avicel (AV)] and lignocellulose substrate [sugarcane bagasse (SB)] were considered. The enzymatic saccharification was carried with and without the addition of inorganic salts (NaCl and KCl) at 0.5 M and 1.0 M concentration. The kinetic parameters, Km and Vm, were determined to mechanically understand the pattern of inhibition and enhancement of inorganic salts on enzymatic saccharification. The kinetics parameters of celluclast 1.5L and accellerase 1500 for hydrolysis of CMC and AV with NaCl showed uncompetitive inhibition. Whereas, influences of KCl on both cellulase were differentiated to function in inhibition or enhancement modes when challenged with different substrates. On the other hand, enzymatic hydrolysis efficiencies of SB using both cellulases were enhanced under addition of NaCl and KCl, by increasing Vm of celluclast 1.5L from 0.303 to 0.635 mg/mL min (0.5 M KCl) and accellerase 1500 from 0.383 to 0.719 mg/mL min (1.0 M NaCl). The details of kinetic analysis in this work revealed the mechanism of inorganic salts on cellulase kinetics to be involved in substrate modification and removal of inhibitor.