Increase In Km Value Research Articles

Immobilization of Urease onto Nanochitosan Enhanced the Enzyme Efficiency: Biophysical Studies and in Vitro Clinical Application on Nephropathy Diabetic Iraqi Patients

Immobilization of enzymes is an effective method for improving the properties and applications of modern enzymes. There are several supports for enzyme immobilization. Because of its unique features, such as inertness and high surface area, chitosan was widely used to immobilize enzymes. Immobilization of urease onto chitosan is a promising approach to treating high urea levels in the blood, however, the immobilization conditions for the best kinetics and enzyme efficiency are still challenging. Herein, we tried to immobilize urease onto nanochitosan (chitosan NPs) through a cross-linker and study the kinetics (km and v max values) and thermodynamics (Ea, ∆H, ∆S, and ∆G) parameters of the enzyme reaction before and after immobilization at different substrate concentration (50, 100, 150, 200, and 250 mg/dl) and incubation temperature (15, 20, 25, 30, 35, and 40°C) under selected optimum conditions. The immobilized urease chitosan NPs was characterized in our previous work using Fourier transform infrared infrared (FTIR), Atomic force force microscopy (AFM), and and imaged here by scanning electron microscopy microscopy (SEM). Results revealed that the highest efficiency % of immobilization (70.38%) was observed at 750 mg/ml chitosan NPs and phosphate buffer pH 7 at 40°C. With an increase of Km value for the immobilized enzyme, however, the efficiency of the enzyme was significantly higher than the free enzyme, p < 0.001 . In addition, the activation energy of the reaction catalyzed by the immobilized enzyme was lower than that of the free enzyme, which suggests that the active site geometry of the immobilized enzyme was more favorable to accommodate the substrate and thus required less energy than that of the free enzyme. The reaction was endothermic by means of positive ∆H. The immobilized urease enzyme was in vitro applied to blood samples of Iraq nephropathy diabetic patients (n = 35) to investigate the effect on serum urease activity and urea level compared to healthy volunteers. Interestingly, the activity of serum urease significantly increased after adding the immobilized enzyme and the level of urea significantly decreased ( p < 0.0001 ) by ∼1.5 folds. Thus, applying an immobilized urease urease to remove urea from blood could be effective in the blood detoxification or dialysis regeneration system of artificial kidney machines.

Effects of 27 CYP3A4 protein variants on saxagliptin metabolism in vitro.

Saxagliptin is a dipeptidyl peptidase 4 (DPP-4) inhibitor widely used in patients with type 2 diabetes. It can increase the amount of insulin after meals and lower blood sugar. CYP450 3A4 (CYP3A4) can metabolize about 30%-40% of therapeutic drugs. Individual differences caused by CYP3A4 genetic polymorphisms can lead to treatment failure, unpredictable side effects, or severe drug toxicity. The aim of this study was to evaluate the catalytic activities of 27 CYP3A4 variants on saxagliptin metabolism in vitro, which were identified in human CYP alleles. We successfully constructed27 kinds of wild-type and variant vectors of pFast-dual-OR-3A4 by overlap extension PCR and prepared 27 kinds of CYP3A4 highly expressed cell microsomes by baculovirus insect cell expression system. The ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) was used to detect the concentrations of the metabolite of saxagliptin (5-hydroxysaxagliptin) and the internal standard. Compared with the wild-type CYP3A4.1, the intrinsic clearance values of most varieties decreased to 1.91%-77.08%. Most of these varieties showed a decrease in Vmax and an increase in Km values compared with wild type. We are the first to report the vitro metabolic data of 27 CYP3A4 variants of the metabolism of saxagliptin which candeepen our understanding of individualized drug use by combining previous studies about the effects of CYP3A4 variants of drug metabolism. With further in vivo studies, we hope it can guide individualized drug use in the clinic when the variants with low metabolic activity to saxagliptin were sequenced in the human body.

Amino Acid Replacement at Position 228 Induces Fluctuation in the Ω-Loop of KPC-3 and Reduces the Affinity against Oxyimino Cephalosporins: Kinetic and Molecular Dynamics Studies

KPC enzymes are the most common class A carbapenemases globally diffused. The peculiarity of this family of β-lactamases is represented by their ability to hydrolyse all classes of β-lactams, including carbapenems, posing a serious problem to public health. In the present study, seven laboratory mutants of KPC-3 (D228S, D228W, D228M, D228K, D228L, D228I and D228G) were generated by site-saturation mutagenesis to explore the role of residue 228, a non-active site residue. Compared to KPC-3, the seven mutants showed evident differences in kcat and Km values calculated for some penicillins, cephalosporins and carbapenems. In particular, D228S and D228M showed a significant increase of Km values for cefotaxime and ceftazidime. Circular dichroism (CD) experiments have demonstrated that substitution at position 228 does not affect the secondary structure of the mutants. Molecular dynamics (MD) simulations were performed on KPC-3, D228S and D228M uncomplexed and complexed with cefotaxime (substrate). Although the residue 228 is located far from the active site, between α11 helix and β7 sheet in the opposite site of the Ω-loop, amino acid substitution at this position generates mechanical effects in the active site resulting in enzyme activity changes.

Mutational Analysis of Ocriplasmin to Reduce Proteolytic and Autolytic Activity in Pichia pastoris

BackgroundOcriplasmin (Jetrea) is using for the treatment of symptomatic vitreomacular adhesion. This enzyme undergoes rapid inactivation and limited activity duration as a result of its autolytic nature after injection within the eye. Moreover, the proteolytic activity can cause photoreceptor damage, which may result in visual impairment in more serious cases.ResultsThe present research aimed to reduce the disadvantages of ocriplasmin using site-directed mutagenesis. To reduce the autolytic activity of ocriplasmin in the first variant, lysine 156 changed to glutamic acid and, in the second variant for the proteolytic activity reduction, alanine 59 mutated to threonine. The third variant contained both mutations. Expression of wild type and three mutant variants of ocriplasmin constructs were done in the Pichia pastoris expression system. The mutant variants were analyzed in silico and in vitro and compared to the wild type. The kinetic parameters of ocriplasmin variants showed both variants with K156E substitution were more resistant to autolytic degradation than wild-type. These variants also exhibited reduced Kcat and Vmax values. An increase in their Km values, leading to a decreased catalytic efficiency (the Kcat/Km ratio) of autolytic and mixed variants. Moreover, in the variant with A59T mutation, Kcat and Vmax values have reduced compared to wild type. The mix variants showed the most increase in Km value (almost 2-fold) as well as reduced enzymatic affinity to the substrate. Thus, the results indicated that combined mutations at the ocriplasmin sequence were more effective compared with single mutations.ConclusionsThe results indicated such variants represent valuable tools for the investigation of therapeutic strategies aiming at the non-surgical resolution of vitreomacular adhesion.

Armillaria mellea (Vahl) P. Kumm’nın karaciğer glutatyon S-transferaz aktivitesi üzerine baskılayıcı etkileri

Armillaria mellea (Vahl) P. Kumm. commonly-known as the honey fungus is an edible mushroom and its antioxidant, antitumor, neuroprotective, and insulin resistance reductive effects have been well-characterized. Glutathione S-transferases (GST) are the group of detoxification enzymes has a function to conjugate glutathione to the variety of electrophile, making them more water-soluble for excretion. Their inhibition or activation could have profound toxicological or clinical implications. This study is conducted to investigate the inhibitory effects of A. mellea on GST enzymes. Total GST activities were measured using glutathione and 1 chloro-2,4-dinitrobenzene as substrates spectrophotometrically, and enzyme kinetic studies were conducted to determine Vmax and Km values. Additionally, aqueous and methanolic extracts of A. mellea were tested to see how they modulate the kinetic parameters. Vmax for liver GST enzyme was calculated as 443.90 ±11.52 U/mg, Km value for GSH, and CDNB were determined as 4.88± 0.53 mM and 10.43±1.07 mM, respectively. The decrease in Vmax and the increase in Km values with A. mellea extracts demonstrated a mixed-type inhibitory mechanism. Methanolic extract inhibits the GSH-dependent GST activity much more than the CDNB-dependent activity, but aqueous extract mainly affects CDNB-dependent GST activity. In conclusion, A. mellea could suppress the hepatic GST enzymes that might have toxicological consequences such as reduced cellular detoxification of electrophilic xenobiotics as well as alleviated drug resistance in the treatment of several diseases.

Molecular characterization of recombinant arginase of Leishmania donovani

Arginase catalyzes the first committed step in the biosynthesis of polyamines that enable cell growth and hence potential drug target for the treatment of leishmaniasis. The arginase from Leishmania donovani (LdARG) was cloned, overexpressed and characterized. Analysis of the deduced amino acid sequence of LdARG with homologous enzyme from other trypanosomatids arginases identified a non-conserved 12 residues long segment VWGLIERTFLSA from position 161–172. This counter segment in L. mexicana arginase exhibits a different conformation compared with human arginase I. The pH and temperature optima of LdARG were 9.0 and 37 °C, respectively. Biochemical studies revealed that the KM for the substrate L-arginine was 24.76 ± 0.06 mM. Molecular modeling of LdARG studies revealed that the glutamic acid residue at position 288 plays a role in substrate binding. The importance of this glutamic acid residue was validated by constructing a mutant variant of LdARG (E288Q-LdARG) by replacing glutamic acid with glutamine through site-directed mutagenesis. The KM value of mutant variant for L-arginine was found to be 107 ± 0.18 mM. The increase in KM value of E288Q-LdARG as compared to LdARG suggested that substrate binding was significantly affected which could be exploited further. Studies on biochemical and structural characterization of recombinant LdARG will help in evaluating this enzyme as a potential drug target for visceral leishmaniasis.

Biochemical characterization and mutational analysis of alanine racemase from Clostridium perfringens

Clostridium perfringens is a gram-positive, anaerobic, pathogenic bacterium that can cause a wide range of diseases in humans, poultry and agriculturally important livestock. A pyridoxal-5-phosphate-dependent alanine racemase with a function in the racemization of d- and l-alanine is an attractive drug target for C.perfringens and other pathogens due to its absence in animals and humans. In this study alanine racemase from C.perfringens (CPAlr) was successfully expressed and purified in Escherichia coli and biochemically characterized. The purified CPAlr protein was a dimeric PLP-dependent enzyme with high substrate specificity. The optimal racemization temperature and pH were 40°C and 8.0, respectively. The kinetic parameters Km and kcat of CPAlr, determined by HPLC at 40°C were 19.1mM and 17.2s-1 for l-alanine, and 10.5mM and 8.7s-1 for d-alanine, respectively. Gel filtration chromatographic analysis showed that the molecular weight of mutant Y359A was close to monomeric form, suggesting that the inner layer residue Tyr359 might play an essential role in dimer-formation. Furthermore, the mutation at residues Asp171 and Tyr359 resulted in a dramatic increase in Km value and/or decreased in kcat value, indicating that the middle and inner layer residues Asp171 and Tyr359 of CPAlr might have the key role in substrate binding, catalytic activity or oligomerization state through the hydrogen-bonding interaction with the pentagonal ring waters and/or PLP cofactor.

Enhanced Purification Efficiency and Thermal Tolerance of Thermoanaerobacterium aotearoense β-Xylosidase through Aggregation Triggered by Short Peptides.

To simplify purification and improve heat tolerance of a thermostable β-xylosidase (ThXylC), a short ELK16 peptide was attached to its C-terminus, which is designated as ThXylC-ELK. Wild-type ThXylC was normally expressed in soluble form. However, ThXylC-ELK assembled into aggregates with 98.6% of total β-xylosidase activity. After simple centrifugation and buffer washing, the ThXylC-ELK particles were collected with 92.57% activity recovery and 95% purity, respectively. Meanwhile, the wild-type ThXylC recovery yield was less than 55% after heat inactivation, affinity and desalting chromatography followed by HRV 3C protease cleavage purification. Catalytic efficiency ( Kcat/ Km) was increased from 21.31 mM-1 s-1 for ThXylC to 32.19 mM-1 s-1 for ThXylC-ELK accompanied by a small increase in Km value. Heat tolerance of ThXylC-ELK at high temperatures was also increased. The ELK16 peptide attachment resulted in 6.2-fold increase of half-life at 65 °C. Released reducing sugars were raised 1.3-fold during sugar cane bagasse hydrolysis when ThXylC-ELK was supplemented into the combination of XynAΔSLH and Cellic CTec2.

The development and evaluation of β-glucosidase immobilized magnetic nanoparticles as recoverable biocatalysts

Magnetic nanoparticle (MNP) solid core supports bound with β-glucosidase (βG) were evaluated as recyclable biocatalysts. To determine whether the relative positioning of immobilized βG to MNP surfaces impacts enzyme activity, MNPs were conjugated with βG using a glutaraldehyde cross-linker for proximal binding with different polyethylene glycol spacer-linkers (MW: 200, 400, 1000). MNP surface modifications were validated by X-ray photoelectron spectroscopy and the measurement of saturation magnetization for the bioconjugates revealed minor decreases in magnetic moments relative to core MNPs. All four bioconjugates showed similar binding efficiencies for βG, but measured increases in Km values and decreases in Vmax values, regardless of the spacer length, indicated that the specific activity of βG was lowered upon enzyme immobilization and spacer lengths were negligible. Despite a reduction in βG activity, immobilization conferred thermal stability under conditions in which the native enzyme is inactivated. To evaluate the recycling properties of the bioconjugates, enzyme activity measurements were conducted for ten consecutive rounds at 45 °C. The bioconjugates hydrolyzed total amounts of p-nitrophenyl β-d-glucoside comparable to a single application of unmodified βG following the third recycling round along with significant activity after ten rounds. Based on these results, the enhanced stability of immobilized βG on recoverable MNPs provides a means for reducing the amount of βG required for cellulose hydrolysis, thereby reducing costs associated with ethanol production.

KINETIC AND ENZYME RECYCLING STUDIES OF IMMOBILIZED b-GLUCOSIDASE FOR LIGNOCELLULOSIC BIOMASS HYDROLYSIS

b-glucosidases are very important in the improvement of cellulose degradation rates by diminishing the inhibition of end products. Although, cellulase cost has reduced significantly in last decade but still the high costs of biomass saccharification is one of the main challenges for the commercialization of ethanol from LC biomass. In this work, -glucosidase immobilization in calcium alginate was optimized using response surface methodology. 89% immobilization efficiency was achieved under optimum conditions (i.e., calcium alginate 4.19% (w/v), calcium chloride 0.14 M and -glucosidase 4.42% (v/v) or 57.46 U/mL of reaction mixture). pH and temperature optima of both free and immobilized -glucosidase was found to be 5 and 50 C, respectively. Enzyme kinetics of free and immobilized -glucosidase was done to evaluate Michaelis constant (Km) and Vmax values, using pnitrophenyl- -D-glucopyranoside (pNPG) as the substrate and glucose as inhibitor. Increase in Km value (0.995 mM) for immobilized -glucosidase as compared to the free -glucosidase (0.617 mM) was observed. A decrease in Vmax from (1191.74 mol min-1 for free -glucosidase) to (736.65 mol min-1 immobilized -glucosidase) was observed after immobilization. Glucose was found to inhibit -glucosidase by competitive mode of inhibition (Ki= 3.01 mM). Studies were carried out in a batch reactor using 2% cellobiose, 0.1 M sodium acetate buffer, 5 pH and 50 C. 84% cellobiose to glucose conversion was found in first cycle which was about 67% in fifth cycle. This study suggests that enzyme immobilization improved enzyme stability, substrate inhibition and permits enzyme reusability.

In vitro multimodal-effect of Trichilia catigua A. Juss. (Meliaceae) bark aqueous extract in CNS targets

Ethnopharmacological relevanceThe bark of Trichilia catigua A. Juss. (Meliaceae), popularly known as “big catuaba”, is traditionally used in Brazilian folk medicine for its neuroactive potential as memory stimulant, and antinociceptive and antidepressant effects. Aim of the studyTo study the aqueous extract of T. catigua bark as dual inhibitor of monoamine oxidase A (MAO-A) and acetylcholinesterase (AChE). To explore its antioxidant potential through interaction with xanthine/xanthine oxidase (X/XO) pathway, and to attempt a relationship between its phenolic profile and effects displayed. Materials and methodsPhenolic profiling was achieved by HPLC-DAD-ESI/MSn and UPLC-ESI-QTOF-MS analyses. The capacity to inhibit hMAO-A was assessed in vitro, as was that for AChE, evaluated in rat brain homogenates. The direct inhibition of the X/XO pathway and the scavenging of superoxide anion radical were the selected in vitro models to explore the antioxidant potential. The cytotoxic effects were assayed in the human neuronal SH-SY5Y cells by MTT reduction, after direct exposure (24h). ResultsTwenty-six compounds were identified and quantified (551.02 ± 37.61mg/g of lyophilized extract). The phenylpropanoid substituted flavan-3-ols were the most representative compounds (~81% of quantified mass). The extract inhibited hMAO activity in a concentration-dependent manner (IC50 = 121.06 ± 2.13μg/mL). A mixed model of inhibition of AChE activity was observed, reflected by the pronounced increase of Km values and a more discreet effect over the Vmax parameters, calculated from Michaelis-Menten fitted equations. In addition, it was demonstrated that the extract directly inhibits the X/XO pathway (IC50 = 121.06 ± 2.13μg/mL) and also imbalances the oxidative stress acting as superoxide anion radical scavenger (EC50 = 104.42 ± 10.67μg/mL), an oxidative by-product of this reaction. All these neuroprotective and neurotrophic effects were displayed within the non-toxic range of concentrations (0.063–0.500μg/mL) in SH-SY5Y cells. ConclusionsOur results validate the traditional use of T. catigua bark for its neuroactive and neuroprotective potential. A novel approach upon its application towards the management of neurodegenerative and related symptomatology was likewise demonstrated.

Maltase entrapment approach as an efficient alternative to increase the stability and recycling efficiency of free enzyme within agarose matrix

Maltase catalyses the hydrolysis of maltose and is widely used in the synthesis of various food and pharmaceutical products. In the current study, matrix entrapment technique was applied to immobilize maltase within agarose beads. Maximum immobilization yield (76.11%) was achieved at 3% agarose concentration and 5.0mm beads size was found to be optimum combination for maximum catalytic activity of entrapped maltase. It was noticed that entrapment increased the optimum reaction temperature and pH of maltase from 45°C to 60°C and 6.5 to 7.0, respectively with reference to its free enzyme whereas no effect was observed on reaction time. Entrapped maltase showed increase in Km value from 1.717 to 1.912mM ml−1 and decrease in Vmax value from 8411.0 to 6214.0U ml−1 min−1 as compared to free enzyme. Entrapped maltase displayed broad thermal stability up to 80°C whereas; free enzyme completely lost it activity when temperature reached up to 60°C. Scanning electron microscopy of agarose beads before and after maltase entrapment revealed significant morphological change on the matrix surface. Considering the economic feasibility, the entrapped maltase indicated imperative recycling efficiency up to ten reaction cycles.

Temperature selects for static soil enzyme systems to maintain high catalytic efficiency

Knowledge on enzymatic mechanisms of acclimation to temperature is required to predict the effects of warming on decomposition of soil organic matter – the largest C stock in terrestrial ecosystems. Based on Michaelis–Menten kinetics we tested the hypothesis that enzyme affinity to substrate (Km) is more sensitive to warming at cold than at warm temperatures. We also predicted a gradual increase in Km values with increasing temperature. The kinetic parameters of six enzymes involved in cycles of C, (cellobiohydrolase, β-glucosidase and xylanase), P (phosphatase), and N (leucine-aminopeptidase, tyrosine-aminopeptidase) were determined after one month of soil incubation at a temperature range 0–40 °C (with 5° increment).Contrary to our hypothesis, the increase in Km with temperature was not gradual for most tested enzymes. Within large range of temperatures from 0 to 15 °C (phosphatase), 0–20 °C (enzymes involved in C cycle) and 0–40 °C (proteases) the hydrolytic activity was governed by enzymes with nearly constant substrate affinity. Temperature, therefore, mainly selected for soil enzyme systems maintaining static Km. The catalytic efficiency of the enzymes (Vmax/Km) increased from low to intermediate temperatures (0–20 °C) as a result of linear increase of Vmax at constant Km. Static Km values were explained either by low flexibility (high structural stability) of a single enzyme type, which catalyzed the reaction over a broad temperature range, or by production of multiple isoenzymes each with different temperature optima but with similar affinity to substrate. Thus, maintaining static Km with temperature increase ensured high enzyme efficiency within a low and intermediate soil temperature range.

Contribution to catalysis of ornithine binding residues in ornithine N5-monooxygenase

The SidA ornithine N5-monooxygenase from Aspergillus fumigatus is a flavin monooxygenase that catalyzes the NADPH-dependent hydroxylation of ornithine. Herein we report a mutagenesis study targeting four residues that contact ornithine in crystal structures of SidA: Lys107, Asn293, Asn323, and Ser469. Mutation of Lys107 to Ala abolishes activity as measured in steady-state oxygen consumption and ornithine hydroxylation assays, indicating that the ionic interaction of Lys107 with the carboxylate of ornithine is essential for catalysis. Mutation of Asn293, Asn323, or Ser469 individually to Ala results in >14-fold increases in Km values for ornithine. Asn323 to Ala also increases the rate constant for flavin reduction by NADPH by 18-fold. Asn323 is unique among the four ornithine binding residues in that it also interacts with NADPH by forming a hydrogen bond with the nicotinamide ribose. The crystal structure of N323A complexed with NADP+ and ornithine shows that the nicontinamide riboside group of NADP is disordered. This result suggests that the increase in flavin reduction rate results from an increase in conformational space available to the enzyme-bound NADP(H). Asn323 thus facilitates ornithine binding at the expense of hindering flavin reduction, which demonstrates the delicate balance that exists within protein-ligand interaction networks in enzyme active sites.

Purification and characterization of a phenoloxidase in the hemocytes of Ephestia kuehniella Zeller (Lepidoptera: Pyralidae): effects of insect growth regulators and endogenous inhibitors

A phenoloxidase was extracted and purified from hemocytes of Ephestia kuehniella by using ammonium sulfate, Sepharyl G-100 and DEAE-Cellulose fast flow chromatographies. At the final stage of purification, a protein was purified by molecular mass of 78.5 kDa, specific activity of 1.17 U/mg protein, recovery of 20.48% and purification fold of 16.71. The purified PO showed the highest activity at pH 4–5 and temperatures of 35–40 °C. Na+, K+, Mn+, Zn2+ and Mg2+ decreased activity of the purified PO but Ca2+ and Cu2+ increased the enzymatic activity. EDTA (General chelating agent), DTC (Copper chelating agent) and EGTA (Calcium chelating agent) significantly decreased PO activity but TTHA (Magnesium chelating agent) showed no statistically significant effects. Kinetic parameters of the purified enzyme showed the highest Vmax when L-DOPA was used as substrate but no significant differences were observed in case of Km for used L-DOPA, pyrocatechol and hydroquinone. In vitro inhibition of the purified PO by using two insect growth regulators, Hexaflumuron and Pyriproxyfen, revealed IC50 of 96.41 and 38.59 µg/ml for these compounds, respectively. Kinetic studies using different concentrations of L-DOPA and IC50 concentrations of the two IGRs revealed the increase of Km value versus control and competitive inhibition. Finally, column chromatography of hemolymph revealed peak III showing endogenous inhibitors of phenoloxidase by molecular weight of 27.3 that showed competitive inhibition on the PO.

Directed-Evolution Analysis of Human Cytochrome P450 2A6 for Enhanced Enzymatic Catalysis

Cytochrome P450 2A6 (P450 2A6) is the major enzyme responsible for the oxidation of coumarin, nicotine, and tobacco-specific nitrosamines in human liver. In this study, the catalytic turnover of coumarin oxidation was improved by directed-evolution analysis of P450 2A6 enzyme. A random mutant library was constructed using error-prone polymerase chain reaction (PCR) of the open reading frame of the P450 2A6 gene and individual mutant clones were screened for improved catalytic activity in analysis of fluorescent coumarin 7-hydroxylation. Four consecutive rounds of random mutagenesis and screening were performed and catalytically enhanced mutants were selected in each round of screening. The selected mutants showed the sequentially accumulated mutations of amino acid residues of P450 2A6: B1 (F209S), C1 (F209S, S369G), D1 (F209S, S369G, E277K), and E1 (F209S, S369G, E277K, A10V). E1 mutants displayed approximately 13-fold increased activity based on fluorescent coumarin hydroxylation assays at bacterial whole cell level. Steady-state kinetic parameters for coumarin 7-hydroxylation and nicotine oxidation were measured in purified mutant enzymes and indicated catalytic turnover numbers (kcat) of selected mutants were enhanced up to sevenfold greater than wild-type P450 2A6. However, all mutants displayed elevated Km values and therefore catalytic efficiencies (kcat/Km) were not improved. The increase in Km values was partially attributed to reduction in substrate binding affinities measured in the analysis of substrate binding titration. The structural analysis of P450 2A6 indicates that F209S mutation is sufficient to affect direct interaction of substrate at the active site.

Quick and low cost immobilization of proteinases on polyesters: Comparison of lactobacilli cell-envelope proteinase and trypsin for protein degradation

Cell-envelope proteinases (CEPs) are a class of proteolytic enzymes produced by lactic acid bacteria and have several industrially relevant applications. However, soluble CEPs are economically unfavorable for such applications due to their poor stability and lack of reusability. In a quest to prepare stable biocatalysts with improved performance, CEP from Lactobacillus delbrueckii subsp. lactis 313 and trypsin (as a model enzyme) were immobilized onto nonwoven polyester fabrics in a three-step protocol including ethylenediamine activation and glutaraldehyde crosslinking. Immobilization gave protein loading yields of 21.9% (CEP) and 67.7% (trypsin) while residual activity yields were 85.6% (CEP) and 4.1% (trypsin). The activity of the immobilized enzymes was dependent on pH, but was retained at elevated temperatures (40–70°C). An increase in Km values was observed for both enzymes after immobilization. After 70 days of storage, the immobilized CEP retained ca. 62% and 96% of initial activity when the samples were stored in a lyophilized form at -20°C or in a buffer at 4°C, respectively. Both immobilized CEP and trypsin were able to hydrolyze proteins such as casein, skimmed milk proteins and bovine serum albumin. This immobilization protocol can be used to prepare immobilized biocatalyst for various protein degradation processes.

Understanding carbamoyl phosphate synthetase (CPS1) deficiency by using the recombinantly purified human enzyme: Effects of CPS1 mutations that concentrate in a central domain of unknown function

Carbamoyl phosphate synthetase 1 deficiency (CPS1D) is an inborn error of the urea cycle that is due to mutations in the CPS1 gene. In the first large repertory of mutations found in CPS1D, a small CPS1 domain of unknown function (called the UFSD) was found to host missense changes with high frequency, despite the fact that this domain does not host substrate-binding or catalytic machinery. We investigate here by in vitro expression studies using baculovirus/insect cells the reasons for the prominence of the UFSD in CPS1D, as well as the disease-causing roles and pathogenic mechanisms of the mutations affecting this domain. All but three of the 18 missense changes found thus far mapping in this domain in CPS1D patients drastically decreased the yield of pure CPS1, mainly because of decreased enzyme solubility, strongly suggesting misfolding as a major determinant of the mutations negative effects. In addition, the majority of the mutations also decreased from modestly to very drastically the specific activity of the fraction of the enzyme that remained soluble and that could be purified, apparently because they decreased Vmax. Substantial although not dramatic increases in Km values for the substrates or for N-acetyl-L-glutamate were observed for only five mutations. Similarly, important thermal stability decreases were observed for three mutations. The results indicate a disease-causing role for all the mutations, due in most cases to the combined effects of the low enzyme level and the decreased activity. Our data strongly support the value of the present expression system for ascertaining the disease-causing potential of CPS1 mutations, provided that the CPS1 yield is monitored. The observed effects of the mutations have been rationalized on the basis of an existing structural model of CPS1. This model shows that the UFSD, which is in the middle of the 1462-residue multidomain CPS1 protein, plays a key integrating role for creating the CPS1 multidomain architecture leading us to propose here a denomination of “Integrating Domain” for this CPS1 region. The majority of these 18 mutations distort the interaction of this domain with other CPS1 domains, in many cases by causing improper folding of structural elements of the Integrating Domain that play key roles in these interactions.

Total phenolics, antioxidant and xanthine oxidase inhibitory activity of three colored onions (Allium cepa L.)

Methanolic extracts from bulbs of three onion (Allium cepa L.) varieties: red (Pusa Red), white (Pusa White Round), and yellow (Arka Pitamber) and the CHCl 3, Et 2O, EtOAc, and n-BuOH fractions obtained from those extracts were investigated for their total phenolics and antioxidant activity. The antioxidant activity measurements, expressed as total antioxidant status (TAS), were 5.2–50.18, 2.2–35.6 and 3.5–42.2 mg ml−1 for red, white and yellow onion bulb extracts, respectively, as compared to trolox (IC 50, 40.2 mg ml−1). The total phenolic content of the samples was 62–186, 56–156 and 68–165 mg GAE ml−1 for red, white and yellow onion bulb extracts, respectively, with all solvents. There was a positive correlation between the total phenolic content and antioxidant activity for MeOH extracts and EtOAc fractions (R2=0.96, 0.8 respectively). Of the selected colored onion bulbs the red onion (IC 50=14.2 μg ml−1) revealed more potent xanthine oxidase inhibitory activity to those of yellow (IC 50=15.5 μg ml−1) and white onion (IC 50=17.0 μg ml−1) in vitro and as compared to control allopurinol (IC 50=10.5 μg ml−1). The kinetic study by xanthine oxidase revealed that the white and yellow onion extracts were non-competitive inhibitors, as Km values were constant, while Vmax consequently decreased with increased inhibitor concentration. In the red onion extract there was an increase in Km values while Vmax remained constant at all extract concentrations, thus confirming a competitive type of inhibition by the Lineweaver–Burk equation. These findings suggest that these onion varieties can be considered as potential sources of antioxidants and XO inhibitory agents.

Immobilization of horseradish peroxidase in phospholipid-templated titania and its applications in phenolic compounds and dye removal

In this study, horseradish peroxidase (HRP) was encapsulated in phospholipid-templated titania particles through the biomimetic titanification process and used for the treatment of wastewater polluted with phenolic compounds and dye. The encapsulated HRP exhibited improved thermal stability, a wide range of pH stability and high tolerance against inactivating agents. It was observed an increase in Km value for the encapsulated HRP (8.21mM) when compared with its free counterpart. For practical applications in the removal of phenolic compounds and dye by the encapsulated HRP, the removal efficiency for phenol, 2-chlorophenol, Direct Black-38 were 92.99%, 87.97%, and 79.72%, respectively, in the first treatment cycle. Additionally, the encapsulated HRP showed better removal efficiency than free HRP and a moderately good capability of reutilization.