Parameters Kcat Research Articles

A New Activity Assay Method for Diamine Oxidase Based on Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry

Copper-containing diamine oxidases are ubiquitous enzymes that participate in many important biological processes. These processes include the regulation of cell growth and division, programmed cell death, and responses to environmental stressors. Natural substrates include, for example, putrescine, spermidine, and histamine. Enzymatic activity is typically assayed using spectrophotometric, electrochemical, or fluorometric methods. The aim of this study was to develop a method for measuring activity using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry based on the intensity ratio of product to product-plus-substrate signals in the reaction mixtures. For this purpose, an enzyme purified to homogeneity from pea (Pisum sativum) seedlings was used. The method employed α-cyano-4-hydroxycinnamic acid as a matrix with the addition of cetrimonium bromide. Product signal intensities with pure compounds were evaluated in the presence of equal substrate amounts to determine intensity correction factors for data processing calculations. The kinetic parameters kcat and Km for the oxidative deamination of selected substrates were determined. These results were compared to parallel measurements using an established spectrophotometric method, which involved a coupled reaction of horseradish peroxidase and guaiacol, and were discussed in the context of data from the literature and the BRENDA database. It was found that the method provides accurate results that are well comparable with parallel spectrophotometry. This method offers advantages such as low sample consumption, rapid serial measurements, and potential applicability in assays where colored substances interfere with spectrophotometry.

Novel dopamine-derived carbon modified fe-based metal–organic framework enhanced aptasensor rendering ultrasensitive electrochemical detection of oxytetracycline

In the present work, an aptasensor with dual binding sites based on Au NPs modified with self-polymerized polydopamine (PDA) and Fe-MOF is successfully constructed. In terms of standard electron transfer, the cyclic voltammetry (CV) analysis showcases a rate constant (ks) of 96.0 s−1, signifying an exceptional electron transfer rate of oxytetracycline at the sensor interface of Apt/Au@PDA@NH2-MIL-101(Fe)/GCE. Further deepening the investigation into electrochemical kinetics using chronoamperometry (CA), we investigated the electrocatalytic parameters (Kcat) of Au@PDA@NH2-MIL-101(Fe) in relation to oxytetracycline. The results divulged an impressive average Kcat value of 3.06 × 105 M−1 S-1. This evidence robustly suggests the superior electrocatalytic activity of Au@PDA@NH2-MIL-101(Fe) when it comes to oxytetracycline oxidation. A custom-designed portable rapid detection platform was developed, achieving detection limits of 6.88 nM under static water flow conditions and 5.56 nM under dynamic conditions. Remarkably, the self-polymerization of PDA on Au NPs not only refines their size but also boosts their interaction with biomolecules, thereby promising excellent biocompatibility. Moreover, the abundance of –COOH groups and unsaturated Fe3+ sites on the NH2-MIL-101(Fe) surface furnishes a considerable number of grafting sites for the aptamer. More importantly, the redox interplay between Au@PDA and NH2-MIL-101(Fe) accelerates electron movement, thus amplifying the electrochemical signal and the detection sensitivity for oxytetracycline.

Computer-aided mining of a psychrophilic cellobiose 2-epimerase from the Qinghai-Tibet Plateau gene catalogue

Cellobiose 2-epimerase (CE) catalyzes the conversion of the lactose into its high-value derivatives, epilactose and lactulose, which has great prospects in food applications. In this study, CE sequences from the Qinghai-Tibet Plateau gene catalogue, we screened these for structural flexibility through molecular dynamics simulation to identify potential psychrophilic CE candidates. One such psychrophilic CE we termed psyCE demonstrated exceptional epimerization activity, achieving an optimum activity of 122.2 ± 1.6 U/mg. Its kinetic parameters (Kcat and Km) for epimerization activity were 219.9 ± 5.6 s−1 and 261.9 ± 18.1 mM, respectively, representing the highest Kcat recorded among known cold-active CEs. Notably, this is the first report of a psychrophilic CE. The psyCE can effectively produce epilactose at 8 °C, converting 20.3 % of 200 mM lactose into epilactose within four hours. These findings suggest that psyCE is highly suitable for cryogenic food processing, and glaciers may serve as a valuable repository of psychrophilic enzymes.

N‐methylation of histidine to tune tautomeric preferences in histidine‐heme coordination and enzyme‐mimetic catalysis

AbstractEnzymes with active sites involving histidine selectively utilize either the δ‐ or ε‐nitrogen atom (Nδ or Nε) of the histidine imidazole for catalysis. However, evaluating the impact of Nδ and Nε is difficult, and directly integrating noncanonical N‐methylated histidine within enzymes poses risks due to laborious procedures. In this study, we present the self‐assembly of Fmoc‐Histidine (Fmoc‐His) with hemin to create a peroxidase‐mimetic catalyst, in which either the Nε or Nδ of histidine is methylated to modify the tautomeric preferences, thereby tuning hemin catalysis. UV‐vis spectra, 1H‐NMR, and fluorescence experiments elucidate that the N‐methylation of histidine alters the self‐assembly propensity of Fmoc‐His, and affects the binding affinity of histidine to hemin iron, with Fmoc‐δmHis/hemin exhibiting stronger binding than Fmoc‐εmHis/hemin. Theoretical simulation results suggest that εmHis and δmHis ligation produce a saddled structure and planar structure of hemin, respectively, stemming from the disparity of steric hindrance at the Nε and Nδ positions. The significant inhibition of hemin's oxidative activity by Fmoc‐δmHis is observed, likely due to the strong binding of Fmoc‐δmHis, potentially hindering access of the substrate, H2O2, to the hemin iron. Conversely, Fmoc‐εmHis enhances hemin catalysis, surpassing even Fmoc‐His alone. This differential impact of Fmoc‐εmHis and Fmoc‐δmHis on hemin activity is further corroborated by apparent activation energy and kinetic parameters (kcat, kcat/Km). This study sheds light on the heterogeneous biological effects at the nitrogen positions of histidine imidazole and offers insights into designing supramolecular metalloenzymes.

Investigating functional and folding stability of an engineered E. coli L-asparaginase harboring Y176F/S241C mutations

Purpose: L-asparaginase has been widely recognized as a critical component in the treatment of various types of lymphoproliferative disorders, since its introduction in 1960s. However, its use in some cases leads to allergic reactions rendering the continuation of treatment unfeasible. Thus, the development of L-asparaginase from alternative sources or the production of engineered enzymes have always been considered. This study aimed to produce and evaluate a novel enzyme designed based on the sequence of L-asparaginase from E. coli bacteria with Y176F/S241C mutations. Methods: The Y176F/S241C mutant L-asparaginase was successfully expressed as the GST-fusion protein in E. coli, and then was subjected to affinity and size exclusion chromatography. The activity of the purified enzyme was determined based on the released ammonia as the result of substrate hydrolysis using Nessler’s reagent. Chemical denaturation experiment in the presence of increasing concentration of guanidinium chloride was applied to determine the folding stability of the purified enzyme. Results: The mutant enzyme was purified with an efficiency of 77-fold but at a low recovery of 0.7%. The determined kinetic parameters Km, Vmax, kcat, specific activity and catalytic efficiency were 13.96 (mM), 2.218(mM/min), 273.9 (min-1), 237.8 (IU/mg) and 19.62 (mM-1 min-1), respectively. Moreover, unfolding free energy determined by guanidinium chloride induced denaturation for mutated and commercial L-asparaginase enzymes were 8421 J/mol and 5274 J/mol, respectively. Conclusion: The mutant enzyme showed improved stability over the wild-type. Although the expression level and recovery were low, the mutant L-asparaginase demonstrated promising activity and stability, with potential clinical and industrial applications.

Substrate Affinity Is Not Crucial for Therapeutic L-Asparaginases: Antileukemic Activity of Novel Bacterial Enzymes.

L-asparaginases are used in the treatment of acute lymphoblastic leukemia. The aim of this work was to compare the antiproliferative potential and proapoptotic properties of novel L-asparaginases from different structural classes, viz. EcAIII and KpAIII (class 2), as well as ReAIV and ReAV (class 3). The EcAII (class 1) enzyme served as a reference. The proapoptotic and antiproliferative effects were tested using four human leukemia cell models: MOLT-4, RAJI, THP-1, and HL-60. The antiproliferative assay with the MOLT-4 cell line indicated the inhibitory properties of all tested L-asparaginases. The results from the THP-1 cell models showed a similar antiproliferative effect in the presence of EcAII, EcAIII, and KpAIII. In the case of HL-60 cells, the inhibition of proliferation was observed in the presence of EcAII and KpAIII, whereas the proliferation of RAJI cells was inhibited only by EcAII. The results of the proapoptotic assays showed individual effects of the enzymes toward specific cell lines, suggesting a selective (time-dependent and dose-dependent) action of the tested L-asparaginases. We have, thus, demonstrated that novel L-asparaginases, with a lower substrate affinity than EcAII, also exhibit significant antileukemic properties in vitro, which makes them interesting new drug candidates for the treatment of hematological malignancies. For all enzymes, the kinetic parameters (Km and kcat) and thermal stability (Tm) were determined. Structural and catalytic properties of L-asparaginases from different classes are also summarized.

Insights into Chitin-Degradation Potential of Shewanella khirikhana JW44 with Emphasis on Characterization and Function of a Chitinase Gene SkChi65.

Chitin, a polymer of β-1,4-linked N-acetylglucosamine (GlcNAc), can be degraded into valuable oligosaccharides by various chitinases. In this study, the genome of Shewanella khirikhana JW44, displaying remarkable chitinolytic activity, was investigated to understand its chitin-degradation potential. A chitinase gene SkChi65 from this strain was then cloned, expressed, and purified to characterize its enzymatic properties and substrate hydrolysis. Genome analysis showed that, of the 14 genes related to chitin utilization in JW44, six belonged to glycoside hydrolase (GH) families because of their functional domains for chitin binding and catalysis. The recombinant chitinase SkChi65, consisting of 1129 amino acids, was identified as a member of the GH18 family and possessed two chitin-binding domains with a typical motif of [A/N]KWWT[N/S/Q] and one catalytic domain with motifs of DxxDxDxE, SxGG, YxR, and [E/D]xx[V/I]. SkChi65 was heterologously expressed as an active protein of 139.95 kDa best at 37 °C with 1.0 mM isopropyl-β-d-thiogalactopyranoside induction for 6 h. Purified SkChi65 displayed high stability over the ranges of 30-50 °C and pH 5.5-8.0 with optima at 40 °C and pH 7.0. The kinetic parameters Km, Vmax, and kcat of SkChi65 towards colloidal chitin were 27.2 μM, 299.2 μMs-1, and 10,203 s-1, respectively. In addition to colloidal chitin, SkChi65 showed high activity towards glycol chitosan and crystalline chitin. After analysis by thin-layer chromatography, the main products were N,N'-diacetylchitobiose, and GlcNAc with (GlcNAc)2-6 used as substrates. Collectively, SkChi65 could exhibit both exo- and endochitinase activities towards diverse substrates, and strain JW44 has a high potential for industrial application with an excellent capacity for chitin bioconversion.

Structure motif guided mining of MHET hydrolase and development of a two-enzyme cascade for plastics depolymerization at mild temperature

The utilization of polyethylene terephthalate (PET) has caused significant and prolonged ecological repercussions. Enzymatic degradation is an environmentally friendly approach to addressing PET contamination. Hydrolysis of mono(2-hydroxyethyl) terephthalate (MHET), a competitively inhibited intermediate in PET degradation, is catalyzed by MHET degrading enzymes. Herein, we employed bioinformatic methods that combined with sequence and structural information to discover an MHET hydrolase, BurkMHETase. Enzymatic characterization showed that the enzyme was relatively stable at pH 7.5-10.0 and 30-45 ℃. The kinetic parameters kcat and Km on MHET were (24.2±0.5)/s and (1.8±0.2) μmol/L, respectively, which were similar to that of the well-known IsMHETase with higher substrate affinity. BurkMHETase coupled with PET degradation enzymes improved the degradation of PET films. Structural analysis and mutation experiments indicated that BurkMHETase may have evolved specific structural features to hydrolyze MHET. For MHET degrading enzymes, aromatic amino acids at position 495 and the synergistic interactions between active sites or distal amino acids appear to be required for MHET hydrolytic activity. Therefore, BurkMHETase may have substantial potential in a dual-enzyme PET degradation system while the bioinformatic methods can be used to broaden the scope of applicable MHETase enzymes.

Biochemical characterization of L-asparaginase isoforms from Rhizobium etli-the boosting effect of zinc.

L-Asparaginases, divided into three structural Classes, catalyze the hydrolysis of L-asparagine to L-aspartic acid and ammonia. The members of Class 3, ReAIV and ReAV, encoded in the genome of the nitrogen fixing Rhizobium etli, have the same fold, active site, and quaternary structure, despite low sequence identity. In the present work we examined the biochemical consequences of this difference. ReAIV is almost twice as efficient as ReAV in asparagine hydrolysis at 37°C, with the kinetic KM, kcat parameters (measured in optimal buffering agent) of 1.5mM, 770s-1 and 2.1mM, 603s-1, respectively. The activity of ReAIV has a temperature optimum at 45°C-55°C, whereas the activity of ReAV, after reaching its optimum at 37°C, decreases dramatically at 45°C. The activity of both isoforms is boosted by 32 or 56%, by low and optimal concentration of zinc, which is bound three times more strongly by ReAIV then by ReAV, as reflected by the KD values of 1.2 and 3.3μM, respectively. We also demonstrate that perturbation of zinc binding by Lys→Ala point mutagenesis drastically decreases the enzyme activity but also changes the mode of response to zinc. We also examined the impact of different divalent cations on the activity, kinetics, and stability of both isoforms. It appeared that Ni2+, Cu2+, Hg2+, and Cd2+ have the potential to inhibit both isoforms in the following order (from the strongest to weakest inhibitors) Hg2+ > Cu2+ > Cd2+ > Ni2+. ReAIV is more sensitive to Cu2+ and Cd2+, while ReAV is more sensitive to Hg2+ and Ni2+, as revealed by IC50 values, melting scans, and influence on substrate specificity. Low concentration of Cd2+ improves substrate specificity of both isoforms, suggesting its role in substrate recognition. The same observation was made for Hg2+ in the case of ReAIV. The activity of the ReAV isoform is less sensitive to Cl- anions, as reflected by the IC50 value for NaCl, which is eightfold higher for ReAV relative to ReAIV. The uncovered complementary properties of the two isoforms help us better understand the inducibility of the ReAV enzyme.

Characterization of a novel ribose-5-phosphate isomerase B from Curtobacterium flaccumfaciens ZXL1 for D-allose production.

Enzymatic preparation of rare sugars as an alternative to traditional sweeteners is an effective strategy to achieve a low-calorie healthy diet. Ribose-5-phosphate isomerase B (RpiB) is a key enzyme in the non-oxidative branch of the catalytic pentose phosphate pathway. Here, we investigated the potential of Curtobacterium flaccumfaciens ZXL1 (C. flaccumfaciens ZXL1) derived RpiB (CfRpiB) in D-allose preparation. The optimal reaction conditions for recombinant CfRpiB were found experimentally to be pH 7.0, 55°C, and no metal ions. The kinetic parameters Km, kcat, and catalytic efficiency kcat/Km were 320mM, 4769s-1, and 14.9mM-1s-1 respectively. The conversion of D-allulose by purified enzyme (1g L-1 ) to D-allose was 13% within 1h. In addition, homology modeling and molecular docking were used to predict the active site residues: Asp13, Asp14, Cys72, Gly73, Thr74, Gly77, Asn106, and Lys144.

Characterization of the enzyme kinetics of EMP and HMP pathway in Corynebacterium glutamicum: reference for modeling metabolic networks

The model of intracellular metabolic network based on enzyme kinetics parameters plays an important role in understanding the intracellular metabolic process of Corynebacterium glutamicum, and constructing such a model requires a large number of enzymological parameters. In this work, the genes encoding the relevant enzymes of the EMP and HMP metabolic pathways from Corynebacterium glutamicum ATCC 13032 were cloned, and engineered strains for protein expression with E.coli BL21 and P.pastoris X33 as hosts were constructed. The twelve enzymes (GLK, GPI, TPI, GAPDH, PGK, PMGA, ENO, ZWF, RPI, RPE, TKT, and TAL) were successfully expressed and purified by Ni2+ chelate affinity chromatography in their active forms. In addition, the kinetic parameters (Vmax, Km, and Kcat) of these enzymes were measured and calculated at the same pH and temperature. The kinetic parameters of enzymes associated with EMP and the HMP pathway were determined systematically and completely for the first time in C.glutamicum. These kinetic parameters enable the prediction of key enzymes and rate-limiting steps within the metabolic pathway, and support the construction of a metabolic network model for important metabolic pathways in C.glutamicum. Such analyses and models aid in understanding the metabolic behavior of the organism and can guide the efficient production of high-value chemicals using C.glutamicum as a host.

The measurement of true initial rates is not always absolutely necessary to estimate enzyme kinetic parameters

In the chapters dealing with enzyme reactions, the authors of all Biochemistry textbooks and of even more specialized texts consider that the characteristic parameters (kcat and Km) must be determined under initial or steady-state rate conditions. This implies the transformation of a very limited proportion of substrate (at most 10–20%) or a continuous recording of the product or substrate concentration vs. time. Both options can present practical difficulties. Is it possible to get around these very stringent conditions? Here we show that in the most favourable cases up to 70% of the substrate can be converted resulting in systematic errors on the parameters (that can easily be taken account of) if the simple Henri-Michaelis–Menten equation is utilised. Alternatively, the integrated form of the same equation directly yields excellent estimates of the same parameters. Our observations should greatly facilitate the task of researchers who study systems in which measurements of the reaction progress are painstaking or when substrate concentrations close to the detection limit must be used. The general conclusion is that it is not always absolutely necessary to determine initial or steady-state rates to obtain reliable estimations of the enzyme kinetic parameters..

Chimeric versus isolated proteins: Biochemical characterization of the NADP+-dependent formate dehydrogenase from Pseudomonas sp. 101 fused with the Baeyer-Villiger monooxygenase from Thermobifida fusca

The expression of multifunctional proteins can facilitate the setup of a biotechnology process that requires multiple functions absolved by different proteins. Herein the functional and conformational characterization of a formate dehydrogenase-monooxygenase chimera enzyme is presented. The fused enzyme (FDH-PAMO) was prepared by linking the C-terminus of the mutant NADP+-dependent formate dehydrogenase from Pseudomonas sp. 101 (FDH) to the N-terminus of the NADPH-dependent monooxygenase from Thermobifida fusca (PAMO) through a peptide linker of 9 amino acids (ASGGGGSGT) generating a chimera protein of 107,056 Da. The catalytic properties (e.g., kinetic parameters kcat and Km), stability, fluorescence and circular dichroism spectra showed that the so-obtained chimera enzyme FDH-PAMO retains the same functional and conformational properties of the two parental enzymes. Furthermore, SEC chromatographic analysis indicated that, in solution (pH 7.4), FDH-PAMO assembles to tetramers (up to 4.2 %) due to the propensity of FDH and PAMO to form dimers, up to 96.6 % and 6.2 %, respectively. This study provides valuable insights into the structural stability of a thermostable protein (e.g., PAMO) after increasing its size through fusion with another similarly sized thermostable protein (e.g., FDH).

Cloning, Expression, and Characterization of Family A DNA Polymerase from Massilia aurea

Mau DNA polymerase is a family A DNA polymerase isolated from Massilia aurea. In this study, a recombinant plasmid, His6-tagged Mau-pET28c, was constructed. His-tagged Mau was expressed in Escherichia coli Rosseta 2 (DE3) competent cells and, after optimization of purification conditions, was successfully isolated via a two-step purification system by Ni2+-chelating affinity chromatography followed by heparin affinity chromatography. The biochemical properties of Mau DNA polymerase were investigated next. This polymerase showed maximal polymerase activity at 30 °C, pH 8.4–8.8, 2–10 mM MgCl2, and 10–40 mM KCl. Kinetic parameters of correct and incorrect dNTP incorporation as well as DNA-binding affinity were determined too. KdNTPd,app values were found to be 16 µM for correct dNTP and 200–500 µM for incorrect dNTP. The kinetic parameter kcat turned out to be 0.2 s−1 for correct dNTP incorporation and an order of magnitude less for incorrect dNTP incorporation. It was demonstrated that Mau DNA polymerase has 5′→3′ and 3′→5′ exonuclease activities associated with the main activity.

Real time monitoring and evaluation of the inhibition effect of fucoxanthin against α-amylase activity by using QCM-A.

The main symptoms of diabetes are hyperglycemia and insulin resistance. The inhibition of the starch digestion enzymes could effectively regulate starch digestion and glucose absorption, thereby slowing or treating the symptoms of postprandial hyperglycemia. Herein, we used fucoxanthin isolated from Undaria pinnatifida stems, as α-amylase inhibitor, and monitored the interactions of both biomolecules by using quartz crystal microbalance-admittance (QCM-A) instrument. All the processes of α-amylase hydrolysis of starch were also dynamically tracked by using amylose-immobilized QCM technology. In our work, we found that the kinetic parameter (k off, k on, and k cat) values obtained by the QCM-A analysis were relatively consistent compared to the kinetic parameter values obtained by the conventional Michaelis-Menten analysis. For the inhibitory reactions, the results showed that fucoxanthin significantly reduced the activity of α-amylase in a dose-dependent manner. The QCM-A technology shown to be an excellent approach in obtaining comprehensive and accurate kinetic parameters, thereby providing real and accurate data for kinetic studies. It is helpful to clarify the mechanism of action of fucoxanthin on α-amylase, which further proved the potential of fucoxanthin to improve and treat postprandial hyperglycemia.

Biochemical Insights into the functionality of a novel thermostable β-amylase from Diocleareflexa

β-amylases are exohydrolases that catalyze the hydrolysis of α-1,4 glycosidic linkages in polysaccharides to produce maltose. Despite various applications of plant β-amylases, thermolability of the enzyme has been a major challenge that continuously prompts the search for thermostable β-amylases. In this study, β-amylase was isolated from an underutilized legume, marble vine (Dioclea reflexa) and purified to apparent homogeneity, using a combination of 30–80% ammonium sulfate precipitation, ion exchange and gel filtration chromatography. It was characterized for its physicochemical properties. The enzyme gives a purification fold and specific activity of 12.25 and 9.43 U/mg respectively, it has a molecular weight of 56.23 kDa using 12% SDS-PAGE. The kinetic parameters Km and Kcat of the enzyme for p-nitrophenyl-maltopentaoside were 0.22 mM and 1340 s−1. The enzyme had an optimum pH of 6.0 while the optimum temperature was 60 °C. while the thermal inactivation rate constants (kd) value of the enzyme was 0.002, thermal inactivation (ΔG) was 99.1 kJ/mol, ΔH and ΔS were 27.7 kJ/mol and −305.80 J/mol.K respectively at 333 k respectively. The thermal inactivation of the enzyme followed first-order kinetics at 60 °C. The enzyme was relatively stable for 140 min retaining 70% of its initial activity at 60 °C and 67% of the activity at pH 6.0. The activity of purified D. reflexa β-amylase was enhanced in the presence of Zn2+, Ca2+ and Mg2+ and strongly inhibited by Cu2+ and Hg2+. Remarkably, the enzyme exhibited thermostable properties which increase its prospects as a veritable potential enzyme for various industrial and biotechnological applications.

Interface investigation the protein interaction interface between malate dehydrogenase and citrate synthase

Enzymes complexes (metabolons) support the metabolic reactions through substrate channeling. Several enzymes in the Krebs cycle are found in protein‐protein metabolons including mitochondrial malate dehydrogenase (MDH2) and citric synthase (CS). While the weak transitory interaction between these enzymes has been demonstrated, the key residues of human MDH2 involved in the interface with CS has not been identified. The purpose of this study is to probe possible residues of MDH responsible for binding to CS. Because cytosolic MDH (MDH1) poorly binds to CS, we initially identified unique sequences that were involved in or adjacent to reported or predicted binding sites of MDH2 and CS. Four regions were selected and residues corresponding to the primary sequence of cytosolic MDH1 were substituted in place of mitochondrial MDH2. Computational models of all the monomeric constructs were made using phyre2 homology modeling, subsequent refinement of the monomers using Galaxy Refine and construction of the dimer forms using the appropriate template in PyMol. Dimers were then refined using Galaxy Refine Complex to give replicate structures of the final models. Potential metabolon formation was explored using HawkDock with the dimer versions of MDH and Citrate Synthase either with or without restraints imposed by published metabolon models.Wild‐type human genes of MDH1, MDH2, and CS were codon optimized for bacterial expression and cloned into the C‐terminus of each gene in a pET28a expression vector. Each MDH1‐MDH2 substitution (DS1‐DS4) were constructed by Gibson cloning. Resulting structure and functional impacts were determined by melting points and kinetic parameters (specific activity, Km, Vmax and Kcat) in purified proteins. Interactions between wild‐type MDH1/2 and CS were compared to MDH2 DS mutants. To qualitatively demonstrate interactions, pull‐down assays were performed in the absence and presence of crowding agents, glycerol, PEG or Ficoll 70. Finally wild‐type MDH 1 and 2 vs MDH2 mutants were subjected to competitive pull‐down assays to identify regions responsible for isozyme specific interactions. This work will show four of the potential interacting regions between MDH and CS and lead to a better understanding of dynamics of this metabolic pair.

Immobilization and characterization of latex cysteine peptidases on different supports and application for cow’s milk protein hydrolysis

Calotropis procera cysteine peptidases (CpCPs) have been used for reducing cow’s milk allergenicity or as rennet in cheesemaking. Due to their residual presence in food products, the present study evaluated, for the first time, different protocols for their immobilization on different supports. Although the yield of immobilization on sulfopropyl-agarose (99%, at pH 7.0) was better than when using DEAE- and MANAE-agarose (40% and 15%, respectively), the derivatives had low recovered activity (4%). On the other hand, MANAE-agarose at pH 10.0 (200 mM buffer) exhibited the highest recovered enzymatic activity (~23%). Regarding the covalent immobilization, the peptidases immobilized on glyoxyl-agarose (glyoxyl-CpCPs) showed broader pH stability (pH 3.0–10.0), 60-fold more stable at 60 °C, and retained 70% of their initial activities after five reaction cycles, even though the immobilization has induced some structural changes analyzed by Fourier-Transform Infrared (FTIR) spectroscopy as well as altered some of enzyme kinetic parameters (Vmax, Km, Kcat, and catalytic efficiency). In addition, this biocatalyst (glyoxyl-CpCPs) hydrolyzed the major cow’s milk allergens (whey proteins) to a greater extent (65%) than the soluble enzymes (8%) and a commercial hypoallergenic formula (50%).

Impact of enzyme turnover on the dynamics of the Michaelis–Menten model

Enzymatic (metabolic rate) processes are traditionally modelled by means of Michaelis–Menten type reactions. The experimental setup is usually performed in vitro also denoted as a ’closed system’. In this paper we explore the impact of enzyme turnover on the classical Michaelis–Menten model by modifying it to include enzyme turnover, specifically through zeroth-order synthesis and first-order degeneration of the enzyme. It is shown how enzyme turnover significantly alters the dynamics of substrate, free- and bound enzyme, and impacts the rate with which substrate is converted to a metabolite P. Qualitative and quantitative estimates are derived for the effect of the parameters ksyn, kdeg and kcat on the dynamics of substrate, and free- and bound enzyme.The model integrates four distinct processes, each characterised with its own parameter(s): (i) substrate–enzyme binding, characterised by kon and koff; (ii) the catalytic process, characterised by kcat; (iii) simultaneous re-generation of free enzyme; and (iv) turnover of free enzyme, characterised by kdeg. The properties of the open Michaelis–Menten model have a direct bearing on the drug discovery process, the translation of data to the human situation and on explaining deviating clinical metabolic observations.

Thermally Stable and Reusable Ceramic Encapsulated and Cross-Linked CalB Enzyme Particles for Rapid Hydrolysis and Esterification

Candida antarctica lipase B (CalB) enzyme was encapsulated and cross-linked by silica matrix to enhance its thermal stability and reusability, and demonstrated an enzymatic ability for rapid hydrolysis and esterification. Silica encapsulated CalB particles (Si-E-CPs) and silica cross-linked CalB particles (Si-CL-CPs) were prepared as a function of TEOS concentration. The particle size analysis, thermal stability, catalytic activity in different pHs, and reusability of Si-E-CPs and Si-CL-CPs were demonstrated. Furthermore, the determination of the CalB enzyme in Si-E-CPs and Si-CL-CPs was achieved by Bradford assay and TGA analysis. Enzymatic hydrolysis was performed against the p-nitrophenyl butyrate and the catalytic parameters (Km, Vmax, and Kcat) were calculated by the Michaelis–Menten equation and a Lineweaver–Burk plot. Moreover, enzymatic synthesis for benzyl benzoate was demonstrated by esterification with an acyl donor of benzoic acid and two acyl donors of benzoic anhydride. Although the conversion efficiency of Si-CL-CPs was not much higher than that of native CalB, it has an efficiency of 91% compared to native CalB and is expected to be very useful because it has high thermal and pH stability and excellent reusability.