Michaelis-Menten Kinetic Model Research Articles

Respiration rate model for mature green capsicum (Capsicum annum L.) under closed aerobic atmospheric conditions

The respiration rate of green capsicum cv. ‘Swarna’ fruits harvested at mature green stage were determined under closed system at 10, 15 and 20 oC temperatures. A simple Michaelis-Menten kinetic model coupled with Arrhenius-type equation, which describes temperature as a function of respiration rate, was used to model the respiration rate of capsicum. The respiration rate model parameters at defined temperature were estimated by fitting the model to the experimental set of data by non-linear regression analysis method. The respiration rate of green mature capsicum was influenced by the temperature. The Arrhenius equation well described the relationship between enzyme kinematics model parameters and temperature. The values of Michaelis-Menten constant for oxygen (Kmo2) and carbon dioxide (Kmco2) were found to vary from 2.14 to 3.92 and 1.33 to 3.24, respectively at different temperature. Experimental and predicted RRO2 values for mature green capsicum was found to be ranged from 9.54 to 14.54 and 11.81 to 17.52 mg/kg-h, respectively. Whereas, the experimental and predicted RRCO2 were 20.1 to 38.51 and 22.15 to 39.83 mg/ kg-h, respectively.

Treatment of Chlorobenzene-Contaminated Gas Stream in a Biotrickling Filter Inoculated with <i>Ralstonnia pickettii</i> L2

Biotrickling filter (BTF) inoculated with Ralstonniapickettii L2 was established to treat waste gas containing chlorobenzene (CB). Results revealed BTF could achieve more than 80% removal efficiency of CB under the conditions of <0.6 g·m-3inlet concentration and >30 s empty bed residence time (EBRT). The mass ratio of carbon dioxide produced to the mixture of benzene, toluene, and oxylene (BTo-X) removed was approximately 2.10, indicating that 89.5% mineralization of the incoming CB vapor. The degradation of CB in the BTF followed Michaelis-Menten kinetic model, and the maximum specific degradation rate (rmax) was 76.3g·m-3·h-1. AWCD values indicated that the microganisms in the BTF showed the high microbial metabolic activities. Real-time PCR indicated that Ralstoniapickettii L2 could still maintain its stability andactivity in the BTF under different conditions.

Chemiluminescence lateral flow immunoassay based on Pt nanoparticle with peroxidase activity

A lateral flow immunoassay (LF-immunoassay) with an enhanced sensitivity and thermostability was developed by using Pt nanoparticles with a peroxidase activity. The Pt nanoparticles were synthesized by citrate reduction method, and the peroxidase activity of Pt nanoparticles was optimized by adjusting reaction conditions. The peroxidase activity was estimated by using Michaelis–Menten kinetics model with TMB as a chromogenic substrate. The kinetics parameters of KM and Vmax were calculated and compared with horseradish peroxidase (HRP). The thermal stability of the Pt nanoparticles was compared with horseradish peroxidase (HRP) according to the storage temperature and long-term storage period. The feasibility of lateral flow immunoassay with a chemiluminescent signal band was demonstrated by the detection of human chorionic gonadotropin (hCG) as a model analyte, and the sensitivity was determined to be improved by as much as 1000-fold compared to the conventional rapid test based on colored gold-colloids.

In vitro glucuronidation of Armillarisin A: UDP-glucuronosyltransferase 1A9 acts as a major contributor and significant species differences

1. This study is performed to investigate liver microsomal glucuronidation of Armillarisin A (A.A), an effective cholagogue drug, aiming at characterizing the involved UDP-glucuronosyltranferases (UGT) and revealing potential species differences.2. A.A glucuronidation in human liver microsomes (HLM) generates one metabolite (M2) glucuronidated at the phenol hydroxyl group, obeying Michaelis–Menten kinetic model. Multiple isoforms including UGT1A1, 1A7, 1A9 and 2B15 can catalyze A.A glucuronidation. Kinetic assays and chemical inhibition studies both demonstrate that UGT1A9 is responsible for A.A glucuronidation in HLM. A.A glucuronidation in Cynomolgus monkey microsomes (CyLM) also follows Michaelis–Menten model, but can additionally catalyze the traced glucuronosyl substitution at the alcohol hydroxyl group (M1). The reactions in liver microsomes from Sprague-Dawley rats (RLM), ICR/CD-1 mouse (MLM), Beagle dog (DLM) all display biphasic kinetics and only M2 is detected. HLM, RLM and CyLM exhibit very similar catalytic activities towards A.A glucuronidation, with the intrinsic clearance values of respective 38, 37 and 37 μL/min/mg, which are much higher than MLM and DLM.3. This in vitro study indicates that UGT1A9 acts as a major contributor to A.A glucuronidation in human liver, and the reaction displays large species differences.

Inherent Regulation of EAL Domain-catalyzed Hydrolysis of Second Messenger Cyclic di-GMP

The universal second messenger cyclic di-GMP (cdG) is involved in the regulation of a diverse range of cellular processes in bacteria. The intracellular concentration of the dinucleotide is determined by the opposing actions of diguanylate cyclases and cdG-specific phosphodiesterases (PDEs). Whereas most PDEs have accessory domains that are involved in the regulation of their activity, the regulatory mechanism of this class of enzymes has remained unclear. Here, we use biophysical and functional analyses to show that the isolated EAL domain of a PDE from Escherichia coli (YahA) is in a fast thermodynamic monomer-dimer equilibrium, and that the domain is active only in its dimeric state. Furthermore, our data indicate thermodynamic coupling between substrate binding and EAL dimerization with the dimerization affinity being increased about 100-fold upon substrate binding. Crystal structures of the YahA-EAL domain determined under various conditions (apo, Mg(2+), cdG·Ca(2+) complex) confirm structural coupling between the dimer interface and the catalytic center. The built-in regulatory properties of the EAL domain probably facilitate its modular, functional combination with the diverse repertoire of accessory domains.

The Enzymatic Hydrolysis of Oil Palm Empty Fruit Bunches to Xylose

Empty fruit bunches (EFB) of oil palm are the lignocellulosic waste from crude palm oil production. The current practice to deal with the waste is either to burn EFB for energy production or to spread them back on the field as a fertilizer. Both options offer a limited additional value to the industry compared to the use of EFB as a renewable resource for chemicals production. To be used as the raw materials for chemicals production, EFB needs to be hydrolysed first to its sugar-monomer content. This manuscript presents the study of enzymatic hydrolysis of EFB by xylanolytic enzyme. The study covers the evaluation of commercial xylanolytic enzyme in hydrolysing EFB, the effect of temperature, pH, substrate concentration and potential inhibitors in the EFB hydrolysis process, and the influences of thermal pretreatment for enhancing the yield of hydrolysis. The results of this study lead to an increase in the enzymatic hydrolysis process of EFB. The maximum hydrolysis yield was obtained at temperature of 60 ℃ and pH of 5. Both inhibitors, xylose and glucose, affected the hydrolysis process. The results showed that the thermal pretreatment of EFB enhanced the enzymatic hydrolysis yield. Enzymatic hydrolysis was shown to follow Michaelis Menten kinetic model, and the kinetic parameters were obtained to be Km = 22.16 g/L and Vm = 0.17 g/L/min.

Bioethanol Fermentation from Non-Treated and Pretreated Corn Stover using Aspergillus Oryzae

A comparison was studied for non-treated and pretreated corn stover with dilute alkaline peroxide and dilute acid treatment respectively for bioethanol production by simultaneous saccharification and fermentation (SSF) process in a continuous stirred batch bioreactor using fungi Aspergillus oryzae . The optimum parameters for bioethanol fermentation were: time, 48 h; pH, 6.0; temperature, 50oC; stirring speed, 35 rpm; and corn stover loading, 35 g/L. The maximum concentration of bioethanol at optimum fermentation process parameters were 0.762 g/g, 0.799 g/g and 0.819 g/g for non-treated, dilute acid and dilute alkaline peroxide pretreated corn stover respectively. Maximum yields of bioethanol were 0.399 g/L h, 0.420 g/L.h and 0.431 g/L h of corn stover at optimum parameters for non-treated, dilute acid and dilute alkaline peroxide pretreated corn stover respectively. The sp. growth rate (m) were 5.30 s −1 , 5.54 s −1 , and 5.93 s −1 and maximum sp. growth rate (m max ) were 10.60 s −1 , 11.08 s −1 and 11.86 s −1 using Monod model for non-treated, dilute acid and dilute alkaline peroxide pretreated corn stover respectively. The Monod parameter (K s ) were 33.87 g/L, 34.21 g/L and 34.85 g/L for non-treated, dilute acid and dilute alkaline peroxide pretreated corn stover respectively. The sp. enzyme activity (ν) were 1192 min −1 , 1463 min −1 and 1559 min −1 and maximum sp. growth rate (ν max ) were 2384 min −1 , 2926 min −1 and 3118 min −1 using Michaelis-Menten enzyme kinetic model for non-treated, dilute acid and dilute alkaline peroxide pretreated corn stover respectively. The Michaelis-Menten parameter (K m ) were 34.42 g/L, 34.63 g/L and 34.85 g/L for non-treated, dilute acid and dilute alkaline peroxide pretreated corn stover respectively. The first order rate constants (k) were 0.014 h −1 , 0.016 h −1 and 0.019 h −1 for non-treated, dilute acid and dilute alkaline peroxide pretreated corn stover for continuous stirred batch bioreactor respectively. DOI: http://dx.doi.org/10.3329/cerb.v16i1.6659 Chemical Engineering Research Bulletin 16(2013) 33-44

Lipase production from a novel thermo-tolerant and extreme acidophile Bacillus pumilus using palm oil as the substrate and treatment of palm oil-containing wastewater

The thermo-tolerant and extreme acidophilic microorganism Bacillus pumilus was isolated from the soil collected from a commercial edible-oil extraction industry. Optimisation of conditions for the lipase production was conducted using response surface methodology. The optimum conditions for obtaining the maximum activity (1,100 U/mL) of extremely acidic thermostable lipase were fermentation time, 96 h; pH, 1; temperature, 50 °C; and concentration of palm oil, 50 g/L. After purification, a 7.1-fold purity of lipase with specific activity of 5,173 U/mg protein was obtained. The molecular weight of the thermo-tolerant acidophilic lipase (TAL) was 55 kDa. The predominant amino acid in the TAL was glycine. The functional groups of lipase were determined by Fourier transform infrared spectroscopy. TAL exhibited enhanced activity (114 %) with dimethyl sulphoxide (20 %, v/v), and it showed a moderate activity with methanol, hexane and benzene. The optimum conditions for the treatment of palm oil in wastewater using the TAL were found to be time, 3 h; pH, 1; temperature, 50 °C with pseudo second-order kinetic constant of 1.88 × 10(-3) L mol(-1) min(-1). The Michaelis-Menten enzyme kinetic model and the nonlinear kinetic model were evaluated for the TAL. TAL established hydrolysis efficiency of 96 % for palm oil in wastewater at 50 °C.

Oxidative coupling of 2-aminophenol to 2-amino-phenoxazine-3-one catalyzed by organotin (IV)–copper (I) cyanide coordination polymers as heterogeneous catalysts

The activity of organotin (IV)–copper (I) cyanide coordination polymers [Ph3SnCu (CN)2·L] where L=1,2-bis(4-pyridyl)ethane (bpe) 1, 4,4′-bipyridine (bpy) 2, methylpyrazine (mepyz) 3 or trans-1,2-bis(4-pyridyl)ethene (tbpe) 4 has been investigated as heterogeneous catalysts for the biomimetic oxidative coupling of 2-aminophenol (OAP) to 2-aminophenoxazine-3-one (APX). The rate of OAP consumption was determined by measuring the amount of dioxygen consumed using a gas burette. All the coordination polymeric catalysts showed high activity for auto-oxidation of OAP. The coordination polymer 4 has been found to be the most reactive catalyst. The rate of auto-oxidation reaction of OAP catalyzed by the coordination polymer 4 has been found to increase with increasing the pH from 7 to 9 then decreased at higher pH. The rate of auto-oxidation reaction showed linear dependence on catalyst concentration and pressure of dioxygen. The rate of reaction has been found to fit a Michaelis–Menten kinetic model for saturation of catalyst sites with increasing OAP concentration. The catalyst 4 showed no deactivation after six runs.

Internal and external mass transfer limitations on the activity of immobilised acid urease derivatives differing in enzyme loading

The specific activity of several Eupergit® C 250 L immobilised acid urease derivatives with enzyme loadings (YP/B) of 48–171mg of bovine serum albumin equivalent (BSAE) per gram of dry support (ds) in a model wine solution was satisfactorily reconstructed using the simultaneous film and intraparticle diffusion and Michaelis-Menten kinetic model by assuming that the immobilisation procedure did not alter the intrinsic kinetic parameters of free acid urease. The behaviour of crunched biocatalysts resulted to be practically coincident with that of the free enzyme, while that of the whole beads was affected by diffusional effects. The model prediction capability was also established in long-time reaction tests using two other biocatalysts at different dry bead concentrations (cBd). The effect of YP/B was finally simulated in the pseudo-1st order regime in the presence of a stagnant or highly-stirred medium. At the limiting Sherwood number of 2.0 and YP/B>48mg BSAE g−1 ds, the calculated specific activity was greatly affected by external mass transfer. To help designing stirred bioreactors, the substrate reduction to be achieved in a given time interval was empirically related to cBd and YP/B.

5,10,15,20-Tetrakis-(4-sulfonatophenyl)porphyrinatocobalt(II) supported on ion exchange resin as reusable and effective catalyst for the oxidative coupling of 2-aminophenol to 2-aminophenoxazine-3-one

Abstract 5,10,15,20-Tetrakis-(4-sulfonatophenyl)porphyrinatocobalt(II) supported on Amberlite ion exchange resin showed good catalytic activity as heterogeneous catalyst for the biomimetic oxidative coupling of 2-aminophenol (OAP) to 2-aminophenoxazine-3-one (APX). The reaction rate was found to fit a Michaelis–Menten kinetic model for saturation of catalyst site with increasing OAP concentration. Recycling of the Co(II)TPPS-resin system showed no loss of activity after six successive runs.

Molecular Hydrogels with Esterase‐like Activity

AbstractThe development of molecular hydrogels that can be applied for mimicking bioactive molecules attracts extensive interests of researchers in fields of self‐assembly. In this study, we reported on several molecular hydrogels based on naphthylacetic acid‐peptides containing L‐histidine formed by the heating‐cooling process. All hydrogels exhibited higher activity to hydrolyze 4‐nitrophenyl acetate (4‐NPA) than the free L‐histidine probably due the high density of L‐histidine residue at the surface of self‐assembled nanofibers. To calculate the 4‐NPA hydrolysis rates, the Michaelis‐Menten enzyme kinetics model was made. Among these gels, the gel of Nap‐GFFYGHY possesses the highest enzyme activity of making the ester bond cleavage, which is approximately 25 times higher than that of the control (free L‐histidine and Nap‐GFFYGYY). These results indicate that molecular hydrogels with self‐assembled nanofibers have great potential for the generation of self‐assembled multivalent materials.

Glucuronidation of the broad-spectrum antiviral drug arbidol by UGT isoforms

The aim of this work was to identify the uridine glucuronosyltransferase (UGT) isoforms involved in the metabolism of the broad-spectrum antiviral drug arbidol. A human liver microsome (HLM) incubation system was employed to catalyse the formation of arbidol glucuronide. The glucuronidation activity of commercially recombinant UGT isoforms towards arbidol was screened. A combination of kinetic analysis and chemical inhibition study was used to determine the UGT isoforms involved in arbidol's glucuronidation. The arbidol glucuronide was detected when arbidol was incubated with HLMs in the presence of UDP-glucuronic acid. The Eadie-Hofstee plot showed that glucuronidation of arbidol was best fit to the Michaelis-Menten kinetic model, and K(m) and apparent V(max) were calculated to be 8.0 ± 0.7 μm and 2.03 ± 0.05 nmol/min/mg protein, respectively. Assessment of a panel of recombinant UGT isoforms revealed that UGT1A1, UGT1A3 and UGT1A9 could catalyse the glucuronidation of arbidol. Kinetic analysis and chemical inhibition study demonstrated that UGT1A9 was the predominant UGT isoform involved in arbidol glucuronidation in HLMs. The major contribution of UGT1A9 towards arbidol glucuronidation was demonstrated in this study.

Direct electrochemistry of Penicillium chrysogenum catalase adsorbed on spectroscopic graphite

The voltammetric studies of Penicillium chrysogenum catalase (PcCAT) adsorbed on spectroscopic graphite, showed direct electron transfer (DET) between its active site and the electrode surface. Analogous tests performed with the commercially available bovine catalase revealed that mammalian enzyme is much less efficient in the DET process. Both catalases were found capable to catalyse the electrooxidation of phenol, but differed in the specifics of catalytic action. At an applied potential of 0.45V the non-linear regression showed the kinetics of the bioelectrochemical oxidation catalysed by the PcCAT obeyed the Hill equation with a binding constant K=0.034±0.002M2 (Hill's coefficient n=2.097±0.083, R2=0.997), whilst the catalytic action of the bovine catalase was described by the Michaelis–Menten kinetic model with the following parameters: Vmax,app=7.780±0.509μA, and KM,app=0.068±0.070molL−1. The performance of the electrode reaction was affected by the electrode potential, the pH, and temperature. Based on the effect of pH and temperature on the electrode response in presence of phenol a tentative reaction pathway of its bioelectrocatalytic oxidation has been hypothesised. The possible application of these findings in biosensing phenol up to concentration 30mM at pHs below 7 and in absence of oxidising agents (oxygen or H2O2) was considered.

Enzymatic hydrolysis of cod liver oil for the fatty acids production

The present paper investigates the comparative enzymatic hydrolysis of cod liver oil with Candida cylindracea lipase (CCL) and Candida rugosa lipase (CRL) in biphasic solvent system for the production of free fatty acids (FFAs). These two lipases were characterized for their physiochemical properties with methods such as BET surface area analyzer, particle size analyzer and Fourier transform infrared (FT-IR) spectroscopy to find the relationship between the characteristic and activity. The yield of FFAs from cod liver oil was studied by varying suitable reaction conditions such as pH, temperature, agitation speed, water and solvent concentrations. The maximum FFAs yields of 36.3 and 26.1wt% FFAs (equivalent to 122.9 and 88.6μmoles FFAs formed/ml) were obtained for CCL and CRL respectively under the most suitable reaction conditions. The Michaelis–Menten kinetic model was found to fit satisfactorily for the hydrolysis of cod liver oil with both CCL and CRL enzymes and the kinetic parameters such as maximum rate of reaction (Vmax) and rate constant (KM) were calculated. The activation energy (E=21.3kJ/mol) demonstrates that the hydrolysis of cod liver oil is kinetically controlled and free from mass transfer limitations under the experimental conditions studied.

Kinetics of Hydrolyzing Isolated Soy Protein by an Endopeptidase and its Conceptual Application in Process Engineering

A response study and the effects of different parameters (pH, temperature and enzyme dose) on kinetics of isolated soy protein hydrolysis by a trypsin-like endopeptidase (TL1) were conducted. Degree of hydrolysis (%DH) data varied at different times under different hydrolysis conditions. Fitting the kinetics data to Michaelis-Menten kinetics model did not result in reasonable kinetic parameters, which implied that Michaelis-Menten kinetics was invalid for such a hydrolysis process. A kinetics model proposed by (Gonzalez-Tello, Camacho, Jurado, Paez, & Guadix, 1994) was found to fit the kinetics curve well and resulted in acceptable model parameters. A simple simulation example was performed to demonstrate the concept of how the kinetics equation could be applied in process engineering.

Kinetics of Hydrolyzing Isolated Soy Protein by an Endopeptidase and its Conceptual Application in Process Engineering

A response study and the effects of different parameters (pH, temperature and enzyme dose) on kinetics of isolated soy protein hydrolysis by a trypsin-like endopeptidase (TL1) were conducted. Degree of hydrolysis (%DH) data varied at different times under different hydrolysis conditions. Fitting the kinetics data to Michaelis-Menten kinetics model did not result in reasonable kinetic parameters, which implied that Michaelis-Menten kinetics was invalid for such a hydrolysis process. A kinetics model proposed by (Gonzalez-Tello, Camacho, Jurado, Paez, & Guadix, 1994) was found to fit the kinetics curve well and resulted in acceptable model parameters. A simple simulation example was performed to demonstrate the concept of how the kinetics equation could be applied in process engineering.

Enzymatic modification of secondary sludge by lipase and laccase to improve the nylon/sludge composite properties

Secondary sludge from pulp and paper mills can be considered as potential filler for composite industry. Enzymatic modifications of the waste secondary sludge from pulp and paper mills to reduce the hydrophobicity and increase the molecular weight have been carried out by lipase and laccase, respectively. The enzymatic modification was performed to enhance the reinforcing capability of the secondary sludge for further composite production. The lipid content of the secondary sludge, which was measured to be 6 ± 0.5%, was hydrolyzed by lipase from Candida rugosa and the structural changes were followed by Fourier transform infrared (FTIR) spectroscopy. Laccase from Trametes versicolor was tested for its activity and reaction rate in the secondary sludge and the alkali-extracted lignin. Characterization of the sludge before and after the laccase treatment was carried out by FTIR spectroscopy. High pressure size exclusion chromatography (HPSEC) was applied to determine the molecular weight distribution of the lignin samples and also as a means for comparing modified and unmodified samples. Biokinetic parameters for the Michaelis-Menten kinetic model as a function of dissolved oxygen concentrations were determined the Km values to be 3.491 and 2.318 g/m3 for sludge and the alkali extract, respectively. The FTIR results on the laccase-treated secondary sludge showed clear changes in the molecular structure, which was mainly attributed to the crosslinking reactions and generation of new bonds. Moreover, the HPSEC results revealed that laccase modifies the sludge by increasing the molecular weight. The manufactured nylon/sludge composites showed lower tensile strength for the lipase treated sludge/nylon composite. However, the laccase-treated sludge/nylon composite showed a statistically significant increase in the mechanical strength, which is attributed to the increase in the components molecular weight.

Cinética de Inactivación de la Enzima Peroxidasa, Color y Textura en Papa Criolla (Solanum tuberosum Grupo phureja) sometida a tres Condiciones de Escaldado

The objective of this work was to determine the inactivation kinetics of peroxidase enzyme and the change in color and texture in golden potato tubers (Solanum tuberosum phureja group) exposed to blanching with water at 80°C, 90°C and to saturated steam at 93°C. The heat transfer coefficients for the blanch water ranged between 214 and 230 W °C -1 m -2 while for saturated steam they showed an average of 84.5 W °C -1 m -2 . At different pre-cooking times the golden potato tuber showed an area that developed enzymatic browning, which was fitted to the Michaelis-Menten kinetic model and another area where the color change decreased due to enzyme inactivation, which corresponded to first order kinetics, in a similar way as the texture behavior. The inactivation of peroxidase enzyme followed the Lumry-Eyring mechanism.

Kinetic Behaviour of Free Lipase and Mica-Based Immobilized Lipase Catalyzing the Synthesis of Sugar Esters

The utilization of natural mica as a biocatalyst support in kinetic investigations is first described in this study. The formation of lactose caprate from lactose sugar and capric acid, using free lipase (free-CRL) and lipase immobilized on nanoporous mica (NER-CRL) as a biocatalyst, was evaluated through a kinetic study. The apparent kinetic parameters, K(m) and V(max), were determined by means of the Michaelis-Menten kinetic model. The Ping-Pong Bi-Bi mechanism with single substrate inhibition was adopted as it best explains the experimental findings. The kinetic results show lower K(m) values with NER-CRL than with free-CRL, indicating the higher affinity of NER-CRL towards both substrates at the maximum reaction velocity (V(max,app)>V(max)). The kinetic parameters deduced from this model were used to simulate reaction rate data which were in close agreement with the experimental values.