Lineweaver-Burk Research Articles

Enzymatic properties and clinical associations of serum alpha-galactosidase A in Parkinson's disease.

Recent studies have revealed an association between Parkinson's disease (PD) and Fabry disease, a lysosomal storage disorder; however, the underlying mechanisms remain to be elucidated. This study aimed to investigate the enzymatic properties of serum alpha-galactosidase A (GLA) and compared them with the clinical parameters of PD. The study participants consisted of 66 sporadic PD patients and 52 controls. We measured serum GLA activity and calculated the apparent Michaelis constant (Km ) and maximal velocity (Vmax ) by Lineweaver-Burk plot analysis. Serum GLA protein concentration was measured by enzyme-linked immunosorbent assay. We examined the potential correlations between serum GLA activity and GLA protein concentration and clinical features and the plasma neurofilament light chain (NfL) level. Compared to controls, PD patients showed significantly lower serum GLA activity (P < 0.0001) and apparent Vmax (P = 0.0131), but no change in the apparent Km value. Serum GLA protein concentration was lower in the PD group (P = 0.0168) and was positively associated with GLA activity. Serum GLA activity and GLA protein concentration in the PD group showed a negative correlation with age. Additionally, serum GLA activity was negatively correlated with the motor severity score and the level of plasma NfL, and was positively correlated with the score of frontal assessment battery. This study highlights that the lower serum GLA activity in PD is the result of a quantitative decrement of GLA protein in the serum and that it may serve as a biomarker of disease severity.

Structure identification and inhibitory mechanism evaluation of three novel angiotensin converting enzyme (ACE) inhibitory peptides from small-aroma chicken

Small-aroma chicken with a high protein content, is widely distributed in the mountainous area of Yunnan-Guizhou Plateau, China. In this work, three novel ACE-inhibitory peptides, Leu-Thr-Glu-Lys-Val-Val-Phe (LTQKVVF), Leu-Asp-Asp-His-Phe-Leu (LDDHFL), and Val-Pro-Gly-Pro-Glu-Pro-Lys-Pro (VPGPEPKP), were prepared from the Small-Aroma Chicken breast. The purification and identification protocol employed comprised ion-exchange chromatography, gel filtration chromatography, reverse phase-high performance liquid chromatography (RP-HPLC), and nano liquid chromatography-electrospray ionization–tandem mass (Nano-LC-ESI–MS/MS). Compared with VPGPEPKP and VPGPEPKP, LTQKVVF displayed noticeable ACE inhibitory activity, with IC50 values of 189.34 μM. What’s more, the Lineweaver-Burk plots revealed peptides LTQKVVF and LDDHFL acted as the mixed-competitive inhibitor while VPGPEPKP inhibited ACE in a manner similar to uncompetitive inhibition. The binding free energies of LTQKVVF, LDDHFL, and VPGPEPKP for ACE were −8.1 kcal/mol, −8.6 kcal/mol, and −9.6 kcal/mol, respectively. The strong inhibition of ACE by VPGPEPKP may be attributed to the interaction of hydrogen bonds, alkyl, and pi-alkyl, which formed by crucial residues of ACE.

Selective inhibitory effects of suberosin on CYP1A2 in human liver microsomes.

Suberosin is a natural phytoconstituent isolated from Citropsis articulata, especially employed for its anticoagulant properties. Although metabolic studies assessing suberosin have been conducted, it is possible interactions with drugs and food have not yet been investigated. In the present study, we analyzed the selective inhibitory effects of suberosin on cytochrome P450 (CYP) enzymes using a cocktail probe assay. Various concentrations of suberosin (0-50μM) were incubated with isoform-specific CYP probes in human liver microsomes (HLMs). We found that suberosin significantly inhibited CYP1A2-catalyzed phenacetin O-deethylation, exhibiting IC50 values of 9.39±2.05 and 3.07±0.45μM with and without preincubation in the presence of β-NADPH, respectively. Moreover, suberosin showed concentration-dependent, but not time-dependent, CYP1A2 inhibition in HLMs, indicating that suberosin acts as a substrate and reversible CYP1A2 inhibitor. Using a Lineweaver-Burk plot, we found that suberosin competitively inhibited CYP1A2-catalyzed phenacetin O-deethylation. Furthermore, suberosin showed similar inhibitory effects on recombinant human CYP1A1 and 1A2. In conclusion, suberosin may elicit herb-drug interactions by selectively inhibiting CYP1A2 during the concurrent administration of drugs that act as CYP1A2 substrates.

Label-Free Detection of Epinephrine Using Flower-like Biomimetic CuS Antioxidant Nanozymes.

A biosensor comprising crystalline CuS nanoparticles (NPs) was synthesized via a one-step simple coprecipitation route without involvement of a surfactant. The powder X-ray diffraction method has been used to evaluate the crystalline nature and different phases consist of the formation of CuS NPs. Mainly hexagonal unit cells consist of the formation of CuS NP unit cells. Most of the surfaces are covered with rhombohedral microparticles with a smooth exterior and surface clustering, examined by SEM images, and the shape of NPs was spherical, having an average size of 23 nm, as confirmed by TEM analysis. This study has focused on the peroxidase-mimicking activity, superoxide dismutase (SOD)-mimicking activity, and chemosensor-based colorimetric determination and detection of epinephrine (EP) neurotransmitters with excellent selectivity. The CuS NPs catalyzed the oxidation of the oxidase substrate 3, 3-5, 5 tetramethyl benzidine (TMB) with the help of supplementary H2O2 that followed Michaelis-Menten kinetics with excellent Km and Vmax values calculated by the Lineweaver-Burk plot. Taking advantage of the drop in absorbance upon introduction of EP for the CuS NPs-TMB/H2O2 system, a colorimetric route has been developed for selective and real-time detection of EP. The sensitivity of the new colorimetric probe was vibrant, having a linear range of 0-16 μM, and achieved a low limit of detection of 457 nM. Moreover, the present nanosystem exhibited appreciable SOD-mimicking activity which could effectively remove O2•- from commercial cigarette smoke, along with it acting as a potential radical scavenger as well. The new nanosystem effectively scavenged •OH, O2.-, and metal chelation which were investigated calorimetrically.

Antihyperglycemic and anti-type 2 diabetic activity of marine hydroquinone isolated from brown algae (Dictyopteris polypodioides).

Brown algae (Dictyopteris polypodioides) extract (DP) presented high inhibitory potential against α-amylase. The present study aims to isolate, purify and evaluate the antihyperglycemic and anti-type 2 diabetic activities of marine hydroquinone from DP. Marine hydroquinones were isolated using silica gel, HPLC, and NMR spectroscopy was used to identify compound 1 and compound 2 as zonarol and isozonarol, respectively. The anti-hyperglycemic and anti-type 2 diabetic activities of zonarol were investigated by invitro assay (α-amylase, α-glucosidase), Lineweaver-Burk plot and Type 2 diabetes mellitus model (T2DM) mice induced by streptozotocin (STZ). Zonarol had the highest content and the strongest inhibitory activity against α-glucosidase (IC50 value of 6.03mgL-1) and α-amylase (IC50 value of 19.29mgL-1) in a competitive inhibition and mix-type manner, respectively. The maltose and starch loading tests revealed that zonarol significantly reduced postprandial glycemia after 30min loading (9.12 and 8.12mg/dL, respectively), compared to normal (11.37 and 12.37mg/dL, respectively). Zonarol exhibited pancreatic islet cell rejuvenation, as evidenced by increased pancreatic islet mass, and hence helps in the restoration of insulin levels and therefore improves the glucose metabolism in STZ-induced diabetic mice. Zonarol treatment in T2DM elevated abundant levels of main SCFAs (propionate, butyrate, and valeric acid), which are closely related to glucose metabolism homeostasis. Our finding indicates that zonarol could be used as a food supplement to treat hyperglycemia and diabetes.

N-Methyl Costaricine and Costaricine, Two Potent Butyrylcholinesterase Inhibitors from Alseodaphne pendulifolia Gamb.

Studies have been conducted over the last decade to identify secondary metabolites from plants, in particular those from the class of alkaloids, for the development of new anti-Alzheimer's disease (AD) drugs. The genus Alseodaphne, comprising a wide range of alkaloids, is a promising source for the discovery of new cholinesterase inhibitors, the first-line treatment for AD. With regard to this, a phytochemical investigation of the dichloromethane extract of the bark of A. pendulifolia Gamb. was conducted. Repeated column chromatography and preparative thin-layer chromatography led to the isolation of a new bisbenzylisoquinoline alkaloid, N-methyl costaricine (1), together with costaricine (2), hernagine (3), N-methyl hernagine (4), corydine (5), and oxohernagine (6). Their structures were elucidated by the 1D- and 2D-NMR techniques and LCMS-IT-TOF analysis. Compounds 1 and 2 were more-potent BChE inhibitors than galantamine with IC50 values of 3.51 ± 0.80 µM and 2.90 ± 0.56 µM, respectively. The Lineweaver-Burk plots of compounds 1 and 2 indicated they were mixed-mode inhibitors. Compounds 1 and 2 have the potential to be employed as lead compounds for the development of new drugs or medicinal supplements to treat AD.

Kinetic behaviour of pancreatic lipase inhibition by wine

This study determined the in vitro inhibitory effects of Mukuzani (dry red) wine and its ethyl acetate/aqueous fractions against pancreatic lipase (PL). Also, it characterises the kinetic behaviour of such inhibition. To current knowledge, no studies have been conducted to describe the kinetic behaviour of PL inhibition by wine or the combined effect on the PL activity of some individual phenolic compounds present in wine. Additionally, the influence of different winemaking methods on PL activity is unknown. Lipase activity was determined by the titrimetric assay method. The kinetic analysis was determined by using the graphical method via Lineweaver-Burk plots. The mode of inhibition (MOI) was determined by looking at the interception pattern and crossing linear lines for the reciprocal data of PL activity with and without inhibitors versus olive oil concentration. The mode of inhibition identified for the wine sample was mixed-mode inhibition. In this type of inhibition, the value of Km (244.329±10.214 μmol·mL-1) was higher, and Vmax (80875.4±3489.754 μmol·mL-1·hour-1) was lower compared to the value of Km (170.901±7.544 μmol·mL-1) and Vmax (88735±4036.741 μmol·mL-1·hour-1) for the non-inhibited PL. This inhibitor decreased the PL activity rate and reduced the affinity between the substrate (olive oil) and enzyme (PL). Selected wine showed predominantly competitive inhibition. The value of inhibition constant Ki1 was equal to 40.556±1.932, and Ki2 was equal to 179.361±8.678 μmol·mL-1, meaning that binding affinity to the pancreatic lipase was higher than to the enzyme-substrate complex. The liquid/liquid extraction method was used to fractionate phenolic compounds of wine into aqueous and ethyl acetate fractions. Fractionated phenolic compounds of wine un-competitively inhibited PL activity. They were considered mutually nonexclusive inhibitors, while a synergistic relationship was found among them. The kinetic parameters of apparent Vmax and Km decreased. Due to the uncompetitive inhibition, the same factor decreased these kinetic parameters. The results obtained herein allow one to conclude that red wine made with the Kakhetian winemaking method can inhibit pancreatic lipase in vitro and play a role in body weight management.

Synthesis and hLDH Inhibitory Activity of Analogues to Natural Products with 2,8-Dioxabicyclo[3.3.1]nonane Scaffold.

Human lactate dehydrogenase (hLDH) is a tetrameric enzyme present in almost all tissues. Among its five different isoforms, hLDHA and hLDHB are the predominant ones. In the last few years, hLDHA has emerged as a therapeutic target for the treatment of several kinds of disorders, including cancer and primary hyperoxaluria. hLDHA inhibition has been clinically validated as a safe therapeutic method and clinical trials using biotechnological approaches are currently being evaluated. Despite the well-known advantages of pharmacological treatments based on small-molecule drugs, few compounds are currently in preclinical stage. We have recently reported the detection of some 2,8-dioxabicyclo[3.3.1]nonane core derivatives as new hLDHA inhibitors. Here, we extended our work synthesizing a large number of derivatives (42-70) by reaction between flavylium salts (27-35) and several nucleophiles (36-41). Nine 2,8-dioxabicyclo[3.3.1]nonane derivatives showed IC50 values lower than 10 µM against hLDHA and better activity than our previously reported compound 2. In order to know the selectivity of the synthesized compounds against hLDHA, their hLDHB inhibitory activities were also measured. In particular, compounds 58, 62a, 65b, and 68a have shown the lowest IC50 values against hLDHA (3.6-12.0 µM) and the highest selectivity rate (>25). Structure-activity relationships have been deduced. Kinetic studies using a Lineweaver-Burk double-reciprocal plot have indicated that both enantiomers of 68a and 68b behave as noncompetitive inhibitors on hLDHA enzyme.

Inhibitory Potential of Synthetic Amino Acid Derivatives against Digestive Enzymes as Promising Hypoglycemic and Anti-Obesity Agents

Over the last decades, the increased incidence of metabolic disorders, such as type two diabetes and obesity, has motivated researchers to investigate new enzyme inhibitors. In this study, the inhibitory effects of synthetic amino acid derivatives (PPC80, PPC82, PPC84, PPC89, and PPC101) on the activity of digestive enzymes were assessed using in vitro assays. The inhibitory effect was determined by the inhibition percentage and the 50% inhibitory concentration (IC50), and the mechanism of action was investigated using kinetic parameters and Lineweaver–Burk plots. PPC80, PPC82, and PPC84 inhibited pancreatic lipase (IC50 of 167–1023 µM) via competitive or mixed mechanisms. The activity of pancreatic α-amylase was suppressed by PPC80, PPC82, PPC84, PPC89, and PPC101 (IC50 of 162–519 µM), which acted as competitive or mixed inhibitors. Finally, PPC84, PPC89, and PPC101 also showed potent inhibitory effects on α-glucosidase (IC50 of 51–353 µM) as competitive inhibitors. The results suggest that these synthetic amino acid derivatives have inhibitory potential against digestive enzymes and may be used as therapeutic agents to control metabolic disorders.

Thermally Stable and Reusable Silica and Nano-Fructosome Encapsulated CalB Enzyme Particles for Rapid Enzymatic Hydrolysis and Acylation.

This study reports the preparation of silica-coated and nano-fructosome encapsulated Candida antarctica lipase B particles (CalB@NF@SiO2) and a demonstration of their enzymatic hydrolysis and acylation. CalB@NF@SiO2 particles were prepared as a function of TEOS concentration (3-100 mM). Their mean particle size was 185 nm by TEM. Enzymatic hydrolysis was performed to compare catalytic efficiencies of CalB@NF and CalB@NF@SiO2. The catalytic constants (Km, Vmax, and Kcat) of CalB@NF and CalB@NF@SiO2 were calculated using the Michaelis-Menten equation and Lineweaver-Burk plot. Optimal stability of CalB@NF@SiO2 was found at pH 8 and a temperature of 35 °C. Moreover, CalB@NF@SiO2 particles were reused for seven cycles to evaluate their reusability. In addition, enzymatic synthesis of benzyl benzoate was demonstrated via an acylation reaction with benzoic anhydride. The efficiency of CalB@NF@SiO2 for converting benzoic anhydride to benzyl benzoate by the acylation reaction was 97%, indicating that benzoic anhydride was almost completely converted to benzyl benzoate. Consequently, CalB@NF@SiO2 particles are better than CalB@NF particles for enzymatic synthesis. In addition, they are reusable with high stability at optimal pH and temperature.

Investigation of improved uricase release and kinetic parameters through dual affected responsive nanopolymers

Uricase suppresses the harmful effects of uric acid accumulation by the degradation of uric acid to allantoin. Maintaining enzyme stability is a major challenge. The stability issue can be avoided when using enzymes loaded into polymeric structures. For this purpose, uricase loaded into responsive nanopolymers (UO-RNPs) was synthesized. The characterizations such as SEM, FTIR, and zeta potential of UO-RNPs were performed. Maximum uricase loading to RNPs was investigated and the release performance of UO-RNPs also under variable conditions such as pH and temperature. On the other hand, loading yield (LY) and loading efficiency (LE) was founded 73.22 ± 3.62% and 71.24 ± 4.61%, while storage stability and reusability of the UO-RNPs were found to be about 68% and 51% of the original activity after 4 weeks and 10 cycles, respectively. From Lineweaver-Burk plot the Km was founded 0.192 and 0.327 mM for loaded and free uricase. While V max was founded 0.131 and 0.0714 μM/min for free uricase and UO-RNPs, respectively. UO-RNPs showed promising matrices for high catalytic efficiency and enhanced stability. Up until now, as far as we know, the present study of the loaded uricase into responsive nanopolymers is the first attempt of uricase enzyme in such an investigation.

Purification, Identification, and Inhibitory Mechanisms of a Novel ACE Inhibitory Peptide from Torreya grandis.

Torreya grandis meal has a high protein content and an appropriate amino acid ratio, making it an excellent protein source for producing ACE inhibitory peptides. To promote its application in food, medicine, and other fields, an alkaline protease hydrolysate of Torreya grandis was used in this study to isolate and identify a novel angiotensin-converting enzyme inhibitory peptide, VNDYLNW (VW-7), using ultrafiltration, gel chromatography purification, LC-MS/MS, and in silico prediction. The results show that the IC50 value of VW-7 was 205.98 µM. The Lineweaver-Burk plot showed that VW-7 had a mixed-type inhibitory effect on ACE. Meanwhile, according to the results of molecular docking, VW-7 demonstrated a strong affinity for ACE (binding energy -10 kcal/mol). VW-7 was bound to ACE through multiple binding sites. In addition, VW-7 could remain active during gastrointestinal digestion in vitro. Nitric oxide (NO) generation in human endothelial cells could rise after receiving a pretreatment with VW-7. These results indicated that Torreya grandis meal protein can be developed into products with antihypertensive function, and VW-7 has broad application prospects in the field of antihypertensive.

Synthesis and biological evaluation of novel zinc (II) and nickel (II) phthalocyanines as cholinesterase inhibitors

Alzheimer's disease (AD) is one of the irreversible neurodegenerative disorders among older people and causes behavior, memory and thinking problems. In this study, peripheral tetra-[(6-(3-(diethylaminophenoxy)hexyl]oxy) substituted zinc (II) and nickel (II) phthalocyanines and their water soluble derivatives were prepared for the first time. To determine the potential of novel synthesized compounds to be used in Alzheimer's disease (AD), in vitro and in silico AChE/BuChE inhibitory and cytotoxic effects were investigated. The docking scores and ∆G values clearly indicated a stronger affinity between DE-C6-CN and AChE than BuChE. In addition, DE-C6-ZnQ and DE-C6-NiQ had higher inhibitory effects than galantamine and both compounds were competitive inhibitors on AChE and BuChE according to Lineweaver-Burk plots. DE-C6-NiQ had a more potent cytotoxicity effect than DE-C6-ZnQ in SH-SY5Y cells. All these results indicated that DE-C6-ZnQ had the potential to be used in the treatment of AD.

Isolation and Characterization of Extracellular Chitinase Produced by Chitinolytic Bacteria Isolated from Soil Samples

Chitin is one of the most common biopolymers found in both marine and terrestrial habitats. Chitinase enzyme has gained popularity due to its diverse biotechnological applications, particularly in agriculture for the biocontrol of phytopathogenic fungi and harmful insects. In this work bacteria producing chitinase enzyme were isolated from soil and water samples. Three bacterial isolates C2, C3, and C4 were found to be Gram-negative coccobacilli while bacterial isolate C9 was found to be Gram-positive cocci. The optimum pH and temperature for all the isolates were studied. The effect of pH, temperature, substrate variation, and incubation period on enzyme activity was studied. The activity of the chitinase enzyme for all the bacterial isolates increased with an increase in temperature up to 37°C. However, a further rise in reaction temperature caused a loss of chitinase activity. Michaelis-Menten plot and Line weaver Burk plot were constructed to calculate the Km and Vmax values. Partial Purification of the chitinase enzyme was carried out and the enzyme activity, protein content, specific activity, and purification fold were estimated. It was observed that the enzyme activity of the crude sample was less than the fractionated sample. The highest enzyme activity was found to be at a 40% fraction. Of the four isolates, C2 was the best in its enzyme activity. The future prospect is the production of chitinases at an industrial scale for various biotechnological applications. Chitinase-producing isolates have the ability to produce chitinase between temperatures 22ºC and 40ºC which is the field temperature for the cultivation of most the crop, so it may be applicable to field conditions against plant pathogenic fungi which is the major problem for agricultural food production. The management of seafood waste businesses may potentially benefit from the usage of this enzyme.

Understanding the Inhibitory Mechanism of Exogenous Protein on Enzymatic Preparation of Resistant Starch

AbstractEnzymatic synthesis of resistant starch strongly depends on the food composition and their interaction with exogenous/endogenous proteins during preparation process. Therefore, the present study systematically investigates the inhibitory effect of egg albumin on pancreatic‐α‐amylase for synthesis of resistant starch from maize flour. The maximum inhibitory concentration of egg albumin is 5 mg mL−1 with % inhibition of 95.15% for pancreatic‐α‐amylase. Lineweaver burk plot reveals that Vmax is constant with and without addition of egg albumin. Whereas Km is increased in presence of albumin to achieve the same Vmax. Increase in the substrate concentration (Km) from 5 to 25 mg mL−1 leads to a decrease in enzyme inhibition. Moreover, the resistant starch content of enzymatically prepared resistant starch with and without albumin as compared to gelatinized maize starch (control) is increased. Scanning electron micrograph reveals interaction of egg albumin with maize starch as thread like aggregation. FTIR and XRD analysis reveal the increase in crystallinity of enzymatically treated maize starch against control. Moreover, egg albumin is negatively associated with enzymatic hydrolysis by preventing its access to maize starch granule and act as a competitive inhibitor. As a result of this interaction decrease in resistant starch yield from maize starch is investigated by enzymatic treatment.

A facile green synthesis of 3,4-dihydropyrimidin-2(1H)-ones using cysteine as a bio-organic catalyst: Potent urease inhibitors, in vitro evaluation, kinetic mechanism, and molecular docking studies

The aim of the present research was to develop a one-pot, three-component method for the synthesis of some 3,4-dihydro pyrimidine-2-(1H)-one (DHPM) derivatives using aldehydes, urea, and ethyl acetoacetate in the presence of cysteine as a green, bio-organic catalyst. This procedure has several key advantages, including a facile and convenient method, green and low-cost catalyst, high yield, short reaction time, and avoidance of the use of environmentally harmful solvents. It is important to note that the synthesized compounds (4a–j) were evaluated for in vitro urease inhibitory activity, and all were found to be more effective than the standard thiourea (IC50 = 4.745 ± 0.054 µM), with urease inhibitory potentials between IC50 = 0.058 ± 0.006 µM to 0.297 ± 0.066 µM. Lineweaver-Burk plots were used to study the kinetics of the most potent compound (4e), and the results showed that the compound non-competitively inhibited urease by forming an enzyme-inhibitor complex. In addition, HT1080 human fibroblast cell lines were used to test the toxicity of the synthesized compounds on cell viability using the MTT assay method, and all compounds except 4g, 4i, and 4j exhibited no significant toxic effects on cells even at high concentrations. Moreover, molecular docking was used to determine the binding interactions between the molecules (ligands) and the active site of the urease enzyme, and the ligands indicated acceptable binding energy values.

Design, synthesis, biochemical and in silico characterization of novel naphthalene-thiourea conjugates as potential and selective inhibitors of alkaline phosphatase.

Naphthalene ring is present in a number of FDA-approved, commercially available medications, including naphyrone, terbinafine, propranolol, naproxen, duloxetine, lasofoxetine, and bedaquiline. By reacting newly obtained 1-naphthoyl isothiocyanate with properly modified anilines, a library of ten novel naphthalene-thiourea conjugates (5a-5j) were produced with good to exceptional yields and high purity. The newly synthesized compounds were observed for their potential to inhibit alkaline phosphatase (ALP) and scavenge free radicals. All of the investigated compounds displayed a more powerful inhibitory profile than the reference agent, KH2PO4 particularly compound 5h and 5a exhibited strong inhibitory potential against ALP with IC50 value of 0.365 ± 0.011 and 0.436 ± 0.057 µM respectively. In addition, Lineweaver-Burk plots revealed the non-competitive inhibition mode of the most powerful derivative i.e., 5h (ki value 0.5 µM). To investigate the putative binding mode of selective inhibitor interactions, molecular docking was performed. It is recommended that future research will focus on developing selective alkaline phosphatase inhibitors by modifying the structure of the 5h derivative.

ERRATUM: Evaluation of the Anti-inflammatory, Antioxidant, and Protease Inhibitory Activity of the Crude Methanol Extract of Portulaca oleracea

Protease inhibitors have considerable applications in biomedicine, biotechnology, and therapy. The aim of the study was to evaluate the methanol crude extracts of aerial portions of Portulaca oleracea (P. oleracea) for anti-inflammatory, antioxidant, and protease inhibitor activities, and to evaluate their inhibitory action against various therapeutically significant proteases. A modified colorimetric assay was used to analyze the protease inhibitors on trypsin and other therapeutic enzymes. P. oleracea crude leaf extract exhibited 79.92% antioxidant and 75% anti-inflammatory activity. Analysis of the effects of crude P. oleracea extract on the values of enzyme kinetics (Km, Vmax) was determined by Lineweaver-Burk reciprocal plot. It was found that the extract exhibited a potent inhibitory effect on the Vmax (µmole/min) of the chymotrypsin 26.0, trypsin 18.0, papain 8.33, and elastase 10.0, when compared to control 41.0, 73.0, 11.11, and 20.3, respectively. While no effect on Km except for elastase which was decreased from 0.026 mM for the control to 0.01 mM. It was found that this effect showed a powerful inhibition in a different way. There is a mixed non-competitive inhibition on elastase as well as pure non-competitive inhibition on chymotrypsin, trypsin, and papain. The crude extract contained 53.3%, 55.7%, 14.9%, and 26.9%, respectively, of protease inhibitors for the therapeutic protease enzymes trypsin, papain, chymotrypsin, and elastase. Therefore, trypsin, papain, chymotrypsin, and elastase have been inhibited by the crude extract of P. oleracea. Characterization of the crude extract indicated potent anti-inflammatory and antioxidant effects.

A new approach for the direct acylation of bio-oil enriched with levoglucosan: Kinetic study and lipase thermostability

Lignocellulosic biomass can be valorized by fast pyrolysis, generating a bio-oil with levoglucosan (BoE), anhydrous sugar applied as building block for several compounds of great industrial interest. In this work we demonstrated for the first time the direct transesterification of BoE with different acyl donors by free and immobilized lipases. Influence of acetonitrile (ACN) and cyrene (CYE) in the stability of lipases from Candida rugosa and Candida antarctica B by differential scanning Nano fluorimetry (NanoDSF), at substrate concentrations of 20–300 mM as well as the alkyl chain effect for synthesis of the carbohydrate fatty acid esters (CFAEs) were carried out at 55 °C. Best results were obtained with Novozym® 435 in ACN and ethyl octanoate (C8) with 74% of conversion against 58% in CYE and ethyl palmitate (C16). In general it was observed that increasing on hydrophobicity of acyl donor contributed to greater regioselectivity for the hydroxyl bound to carbon 4 with up to 87% for C16 in ACN. Thermostability profile of CalB and CR suggested higher stabilty in CYE with values of Tm = 61 °C for CalB and Tm = 53 °C for CR. Finally, the kinetic parameters were obtained through the Michaelis-Menten constants generated by the double-reciprocal plot and indicated an inhibition by the LG substrate. Moreover, catalytic efficiency data suggest higher enzyme-substrate affinity in CYE.

Highly sensitive colorimetric detection of Cr (VI) in water through nano-confinement effect of CTAB-MoS2/rGO nanocomposites

Cr (VI) can invade the human body through digestion, respiratory tract, skin, and mucous membrane; therefore, the detection of Cr (VI) in water has extreme environmental significance. In this study, the intercalated CTAB broadened the interspacing of the MoS2, ensured that the interlayer gap between S-Mo-S molecules was unblocked, and exposed more reactive sites. CTAB-MoS2 grows vertically on the fewer layers of reduced graphene oxide (rGO) and forms a nanowall structure through a C-S bond. The unique nanowall structure of CTAB-MoS2/rGO constitutes the nano-confinement space, and CTAB changes the surface electrical property of CTAB-MoS2/rGO nanocomposites. Cr (VI) ions were fixed on the surfaces of the composites by electrostatic attraction. Based on the nano-confinement and electrostatic attraction effect of CTAB-MoS2/rGO, the sensitive colorimetric detection of Cr (VI) was achieved, and the detection limit was 1.25 nM (S/N = 3). The catalytic activity conforms to the Lineweaver-Burk equation; the reaction velocity (Vmax) and Michaelis-Menton constant (Km) of CTAB-MoS2/rGO nanocomposites for Cr (VI) detection are 14.85 × 10−7 Ms−1 and 1.09 mM, respectively. The colorimetric sensing method not only has a high selectivity for the Cr (VI) but also can be utilized for the determination of Cr (VI) in real samples. Consequently, the colorimetric sensing method provides a broad range of environmental monitoring prospects.