Low Enzyme Concentrations Research Articles

Preparation of cross-linked enzyme aggregates of trehalose synthase via co-aggregation with polyethyleneimine.

Trehalose synthase (TreS) from Meiothermus ruber was co-aggregated with polyethyleneimine (PEI) and precipitated with polyethylene glycol (PEG), followed by cross-linking with glutaraldehyde to obtain TreS-polyethyleneimine cross-linked enzyme aggregates (termed as CLEAs-PEI-PEG). The TreS solution at 0.5 mg mL(-1) protein concentration, with PEI at a mass ratio of 1:0.8 (enzyme/PEI, w/w) and 25 % (w/v) PEG concentration were found to be most adequate for the co-aggregation of TreS. CLEAs-PEI-PEG was most active with glutaraldehyde at a mass ratio of 1:0.5 (enzyme/glutaraldehyde, w/w) to cross-link the co-aggregates. The CLEAs-PEI-PEG prepared in this work had an optimum pH of 6.5 and optimum temperature of 60 °C. For lower concentrations of enzyme, using PEI could enhance the cross-linking efficiency of TreS. The thermal stability and pH tolerance of CLEAs-PEI-PEG were significantly improved. Scanning electron microscopy revealed that the main structure of CLEAs-PEI-PEG showed scaffolding morphology which was constituted by structured ball-like particles with a size of 1-2.5 μm in diameter.

Kinetic studies of a ferredoxin-dependent cyanobacterial nitrate reductase.

A flash photolysis study of electron transfer (ET) kinetics from reduced ferredoxin (photoreduced by Photosystem I) to the ferredoxin-dependent nitrate reductase from the cyanobacterium Synechococcus sp. PCC 7942 has been carried out. In the presence of nitrate, under conditions where only a single electron is transferred to nitrate reductase, the rate of enzyme reduction shows a biphasic concentration dependence: At low enzyme concentrations the dependence is approximately linear, with an estimated second-order rate constant of 7.4 ± 0.8 × 10(7) M(-1) s(-1); at concentrations above 2 μM, the rate increases nonlinearly to an asymptotic value of approximately 300 s(-1), indicating the presence of a rate-limiting step in the process. The spectrum of the one-electron reduced enzyme suggests that Mo centers are largely reduced with a minor contribution of iron-sulfur cluster reduction. Under conditions favoring two-electron reduction of the enzyme, the redox difference spectrum can be accounted for by the oxidation of two reduced ferredoxins, suggesting that the enzyme has completed one full catalytic cycle. The spectral changes observed in the absence of nitrate are significantly different from those seen in the presence of nitrate. Experiments in the absence of nitrate revealed that the singly reduced enzyme exhibits different absorption characteristics and reoxidation kinetics, compared to those observed with nitrate present, and exhibits a much faster binding by reduced ferredoxin than the oxidized enzyme. The implications of these observations for understanding the enzyme mechanism are discussed.

Application of home-made lipase in the production of geranyl propionate by esterification of geraniol and propionic acid in solvent-free system

Abstract This work relates the maximization of geranyl propionate production by esterification in solvent-free system using a lipase extract from Penicillium crustosum. A sequencial strategy of experimental design was carried out followed by a kinetic study. The study of reuse of the non-commercial immobilized enzymatic extract was also carried out. The operating conditions that maximized geranyl propionate production were determined to be 60 °C, geraniol to propionic acid mole ratio of 5:1, 150 rpm and 50 wt% of lipase extract, resulting in a conversion of about 53%. A kinetic study was performed and the results allow to conclude that an excess of alcohol (alcohol to acid mole ratio of 5:1), relatively low enzyme concentration (50 wt%) and 40 °C afforded good conversions after 50 min. The relative activity decreased along the reuse, retaining around 50% of the same after four cycles. The decrease in the activity of the immobilized extract was accompanied by a drop in geranyl propionate conversions. New experimental data on enzymatic esterification of geraniol and propionic acid for geranyl propionate production are reported in this work, showing a promising perspective of using low cost biocatalysts to overcome the well-known drawbacks of the chemical-catalyzed route.

Electrochemical evaluation of glutathione S-transferase kinetic parameters

Glutathione S-transferases (GSTs), are a family of enzymes belonging to the phase II metabolism that catalyse the formation of thioether conjugates between the endogenous tripeptide glutathione and xenobiotic compounds. The voltammetric behaviour of glutathione (GSH), 1-chloro-2,4-dinitrobenzene (CDNB) and glutathione S-transferase (GST), as well as the catalytic conjugation reaction of GSH to CDNB by GST was investigated at room temperature, T=298.15K (25°C), at pH6.5, for low concentration of substrates and enzyme, using differential pulse (DP) voltammetry at a glassy carbon electrode. Only GSH can be oxidized; a sensitivity of 0.14nA/μM and a LOD of 6.4μM were obtained. The GST kinetic parameter electrochemical evaluation, in relation to its substrates, GSH and CDNB, using reciprocal Michaelis–Menten and Lineweaver–Burk double reciprocal plots, was determined. A value of KM~100μM was obtained for either GSH or CDNB, and Vmax varied between 40 and 60μmol/min per mg of GST.

Glycosynthases from Thermotoga neapolitana β-glucosidase 1A: A comparison of α-glucosyl fluoride and in situ-generated α-glycosyl formate donors

TnBgl1A from the thermophile Thermotoga neapolitana is a dimeric β-glucosidase that belongs to glycoside hydrolase family 1 (GH1), with hydrolytic activity through the retaining mechanism, and a broad substrate specificity acting on β-1,4-, β-1,3- and β-1,6-linkages over a range of glyco-oligosaccharides. Three variants of the enzyme (TnBgl1A_E349G, TnBgl1A_E349A and TnBgl1A_E349S), mutated at the catalytic nucleophile, were constructed to evaluate their glycosynthase activity towards oligosaccharide synthesis. Two approaches were used for the synthesis reactions, both of which utilized 4-nitrophenyl β-d-glucopyranoside (4NPGlc) as an acceptor molecule: the first using an α-glucosyl fluoride donor at low temperature (35 °C) in a classical glycosynthase reaction, and the second by in situ generation of the glycosyl donor with (4NPGlc), where formate served as the exogenous nucleophile under higher temperature (70 °C). Using the first approach, TnBgl1A_E349G and TnBgl1A_E349A synthesized disaccharides with β-1,3-linkages in good yields (up to 61%) after long incubations (15 h). However, the GH1 glycosynthase Bgl3_E383A from a mesophilic Streptomyces sp., used as reference enzyme, generated a higher yield at the same temperature with both a shorter reaction time and a lower enzyme concentration. The second approach yielded disaccharides for all three mutants with predominantly β-1,3-linkages (up to 45%) but also β-1,4-linkages (up to 12.5%), after 7 h reaction time. The TnBgl1A glycosynthases were also used for glycosylation of flavonoids, using the two described approaches. Quercetin-3-glycoside was tested as an acceptor molecule and the resultant product was quercetin-3,4′-diglycosides in significantly lower yields, indicating that TnBgl1A preferentially selects 4NPGlc as the acceptor.

Selective oxidation of D-amino acids catalyzed by oligolamellar liposomes intercalated with D-amino acid oxidase.

D-Amino acid oxidase (DAO) is structurally unstable and exhibits broad specificity to D-amino acids. In this work, we fabricated a stable liposomal DAO system with high apparent substrate specificity. Permeability of the membrane composed of POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine) was highly selective between the d-forms of alanine (Ala) and serine (Ser). The permeability coefficient of d-Ala and d-Ser at 25 °C was 3.59 and 0.27 pm/s, respectively, as determined with the dialysis method. On the other hand, the chiral environment of POPC membrane showed no clear selectivity between the enantiomers of Ala or Ser. POPC liposomes encapsulating DAO from porcine kidney selectively catalyzed the oxidation of hydrophobic D-phenylalanine (D-Phe) over D-Ala and D-Ser because of their intrinsic membrane permeability. As a different type of liposomal DAO, the enzyme molecules were conjugated to the surface of activated lipids-bearing liposomes. The activity of liposome-conjugated DAO showed significantly higher stability at 50 °C than free DAO at low enzyme concentrations ranging from 2.5 to 10 mg/L. Then, the DAO-conjugated liposomes were coated with POPC bilayers to give the oligolamellar structure intercalated with the DAO molecules. The additional bilayers allowed to induce the permeability resistance-based substrate specificity and strengthened the stabilizing effect on the DAO activity. The oligolamellar liposomes fabricated can be a colloidal platform for integrating the functions of lipid membrane to stabilize DAO and to modulate its substrate specificity.

β-Galactosidase Immobilization on Chitosan-Hydroxyapatite Complex: Effects of Immobilization Conditions

In this study, ?-galactosidase from Kluyvermoyces lactis was immobilized onto chiosan-hydroxyapatite. Hydroxyapatite (HAP) is a nontoxic ceramic biomaterial generally that has been used for dental applications, drug delivery, immobilization of metals, enzymes and recently some yeast cells. Effects of immobilization parameters including medium temperature and pH, enzyme concentration and coupling time was evaluated to obtain the best reaction conditions. The optimal immobilization pH was determined as pH 7.5 with the highest enzyme activity of 1337.2 U g-1 chitosan-HAP beads and highest immobilization efficiency of 81.4%. Furthermore, the immobilized protein amount showed increase with increasing enzyme concentration as expected. However, highest specific activity (365.2 mg-1 protein) was achieved at low enzyme concentrations. Characterization of immobilized enzyme was tested using o-nitrophenol-?-D-galactopyranoside (ONPG) as a substrate. Immobilized enzyme exhibited better thermostability and higher stability at lower (pH 5.0) and higher (pH 9.0) pH values. Immobilization also resulted in a shift of 0.5 units in pH optimum for maximum enzyme activity. In contrast to optimum pH value, after immobilization no shift occurred in optimum temperature. Furthermore enzyme retained 81.8% of its initial activity during consecutive eight cycles. After 15 days of storage, immobilized enzyme obtained 82.6% of its original activity, whereas free enzyme showed a significant decrease.

Kinetic analysis and enzyme concentration effect relevant to dependence of amylolysis of starch granules on specific surface area concentration

The kinetics and enzyme concentration effects on the amylolysis of rice, maize, and potato starch granules by porcine pancreatic α-amylase (PPA) were analyzed. The K m values of the native starches, when expressed as a function of surface area concentration, were similar to each other. Inconsistencies in V max values can be attributed to the deviations from real values of the surface area concentration. The deviations were confirmed by that the initial rate differences among the native starches with the same apparent surface area concentration were observed at low enzyme concentrations. Kinetic parameters of dimethyl sulfoxide (DMSO)-solubilized native starches showed no significant difference, demonstrating that structural and compositional differences of starches do not affect amylolysis. The resistance of starch granules against amylolysis predominantly affects the V max, and as a result, the k cat is reduced compared with DMSO-solubilized starch. In conclusion, the kinetic analysis and enzyme concentration effect confirmed that surface area concentration mainly affects the initial hydrolysis rate of native starch granules.

A concentration-dependent effect of methanol on Candida antarctica lipase B in aqueous phase

Abstract The influence of methanol on enzyme is of great importance in enzymatic synthesis. The influence of methanol on the activity of Candida antarctica lipase B (CALB) upon hydrolysis of tributyrin was investigated by enzyme kinetics analysis. Dynamic light scattering (DLS) and circular dichroism (CD) was used to measure the variation of hydrodynamic diameter and secondary structure change of CALB at different methanol concentration, respectively. A concentration-dependent influence of methanol on CALB was observed. The activity of CALB slightly increased with less than 1.2 mol l −1 of methanol, followed by sustained decrease at higher methanol concentrations. DLS result indicated that the negative effect of methanol on CALB was supposed to follow different mechanisms due to the significant variation of particle size of CALB at different methanol concentrations. Both reversible competitive inhibition based on enzymatic kinetics analysis and inactivation based on CD spectroscopy study were supposed to contribute to the negative effect of methanol on CALB with methanol concentration in the range of 1.2 mol l −1 to 6 mol l −1 . And after arriving at percolation threshold concentration of 6 mol l −1 , methanol was mainly performed as aggregate and precipitate agent for CALB, which resulted in low effective enzyme concentration in the solution.

Correlation of Waist-to-Hip Ratio and Oxidative Stress in Patients of Acute Myocardial Infarction

Obesity is associated with increased oxidative stress and lipid peroxidation is thought to play a crucial role in the generation of atherosclerotic lesions of Acute Myocardial Infarction (AMI). The case-control study contained 120 subjects divided in two groups, 60 patients with AMI and equal number of age- and sex-matched healthy subjects as controls. Ratio of Waist Circumference (WC) to Hip Circumference (HC) (waist-to-hip ratio) of all the subjects was recorded. Antioxidant status of the individuals was determined by measuring the serum levels of Glutathione Peroxidase (GPx) and Superoxide Dismutase (SOD). Estimation of Malondialdehyde (MDA), a marker of lipid peroxidation was used as a surrogate marker of free radical activity. WHR was found to be significantly higher in patients of AMI as compared to controls. MDA levels were significantly high and antioxidants molecules GPx and SOD were significantly decreased in AMI patients as compared with control (p<0.001). WHR correlated positively to serum level of MDA and inversely to the serum level of antioxidant enzymes. High Waist-to-hip ratio is associated with high concentrations of malondialdehyde level and low concentration of antioxidant's enzyme. This results in increased oxidative stress, a major causative factor of AMI.

Evidence that Pulsed Electric Field Treatment Enhances the Cell Wall Porosity of Yeast Cells

The application of rectangular electric pulses, with 0.1-2 ms duration and field intensity of 2.5-4.5 kV/cm, to yeast suspension mediates liberation of cytoplasmic proteins without cell lysis. The aim of this study was to evaluate the effect of pulsed electric field with similar parameters on cell wall porosity of different yeast species. We found that electrically treated cells become more susceptible to lyticase digestion. In dependence on the strain and the electrical conditions, cell lysis was obtained at 2-8 times lower enzyme concentration in comparison with control untreated cells. The increase of the maximal lysis rate was between two and nine times. Furthermore, when applied at low concentration (1 U/ml), the lyticase enhanced the rate of protein liberation from electropermeabilized cells without provoking cell lysis. Significant differences in the cell surface of control and electrically treated cells were revealed by scanning electron microscopy. Data presented in this study allow us to conclude that electric field pulses provoke not only plasma membrane permeabilization, but also changes in the cell wall structure, leading to increased wall porosity.

Enzymatic Synthesis of Dipalmitin in Supercritical Carbon Dioxide and Mechanism Study

Dipalmitin was enzymatically synthesized from palmitic acid and glycerol in supercritical carbon dioxide (scCO2), and optimum reaction conditions were determined. An organic solvent (tert-butanol) system was also investigated, but the highest conversion of palmitic acid (81%) and highest yield of dipalmitin (69.44%) were obtained in scCO2 with an extremely low enzyme concentration, 0.34 g/L, the volumetric concentration of enzyme in scCO2. The immobilized enzyme showed excellent operational stability in scCO2 under the experimental conditions. A phase behavior study indicated that the difference in solubilities of the products in scCO2 resulted in an improved selectivity for dipalmitin. A phase transfer model was designed to illustrate the reaction process in scCO2. The enzyme kinetic parameters in the scCO2 system and tert-butanol solvent system were also evaluated using a ping-pong Bi–Bi mechanism with inhibition, and palmitic acid in scCO2 was to have the lowest inhibition (KB/KiB = 4.356), which is due to the low viscosity and high diffusivity of scCO2.

Steady-state, Pre-steady-state, and Single-turnover Kinetic Measurement for DNA Glycosylase Activity

Human 8-oxoguanine DNA glycosylase (OGG1) excises the mutagenic oxidative DNA lesion 8-oxo-7,8-dihydroguanine (8-oxoG) from DNA. Kinetic characterization of OGG1 is undertaken to measure the rates of 8-oxoG excision and product release. When the OGG1 concentration is lower than substrate DNA, time courses of product formation are biphasic; a rapid exponential phase (i.e. burst) of product formation is followed by a linear steady-state phase. The initial burst of product formation corresponds to the concentration of enzyme properly engaged on the substrate, and the burst amplitude depends on the concentration of enzyme. The first-order rate constant of the burst corresponds to the intrinsic rate of 8-oxoG excision and the slower steady-state rate measures the rate of product release (product DNA dissociation rate constant, k(off)). Here, we describe steady-state, pre-steady-state, and single-turnover approaches to isolate and measure specific steps during OGG1 catalytic cycling. A fluorescent labeled lesion-containing oligonucleotide and purified OGG1 are used to facilitate precise kinetic measurements. Since low enzyme concentrations are used to make steady-state measurements, manual mixing of reagents and quenching of the reaction can be performed to ascertain the steady-state rate (k(off)). Additionally, extrapolation of the steady-state rate to a point on the ordinate at zero time indicates that a burst of product formation occurred during the first turnover (i.e. y-intercept is positive). The first-order rate constant of the exponential burst phase can be measured using a rapid mixing and quenching technique that examines the amount of product formed at short time intervals (<1 sec) before the steady-state phase and corresponds to the rate of 8-oxoG excision (i.e. chemistry). The chemical step can also be measured using a single-turnover approach where catalytic cycling is prevented by saturating substrate DNA with enzyme (E>S). These approaches can measure elementary rate constants that influence the efficiency of removal of a DNA lesion.

Pivotal role of P450–P450 interactions in CYP3A4 allostery: the case of α-naphthoflavone

We investigated the relationship between oligomerization of CYP3A4 (cytochrome P450 3A4) and its response to ANF (α-naphthoflavone), a prototypical heterotropic activator. The addition of ANF resulted in over a 2-fold increase in the rate of CYP3A4-dependent debenzylation of 7-BFC [7-benzyloxy-4-(trifluoromethyl)coumarin] in HLM (human liver microsomes), but failed to produce activation in BD Supersomes or Baculosomes containing recombinant CYP3A4 and NADPH-CPR (cytochrome P450 reductase). However, incorporation of purified CYP3A4 into Supersomes containing only recombinant CPR reproduced the behaviour observed with HLM. The activation in this system was dependent on the surface density of the enzyme. Although no activation was detectable at an L/P (lipid/P450) ratio ≥750, it reached 225% at an L/P ratio of 140. To explore the relationship between this effect and CYP3A4 oligomerization, we probed P450-P450 interactions with a new technique that employs LRET (luminescence resonance energy transfer). The amplitude of LRET in mixed oligomers of the haem protein labelled with donor and acceptor fluorophores exhibited a sigmoidal dependence on the surface density of CYP3A4 in Supersomes™. The addition of ANF eliminated this sigmoidal character and increased the degree of oligomerization at low enzyme concentrations. Therefore the mechanisms of CYP3A4 allostery with ANF involve effector-dependent modulation of P450-P450 interactions.

The development and use of an ELISA-based method to follow the distribution of cellulase monocomponents during the hydrolysis of pretreated corn stover

BackgroundIt is widely recognised that fast, effective hydrolysis of pretreated lignocellulosic substrates requires the synergistic action of multiple types of hydrolytic and some non-hydrolytic proteins. However, due to the complexity of the enzyme mixture, the enzymes interaction with and interference from the substrate and a lack of specific methods to follow the distribution of individual enzymes during hydrolysis, most of enzyme-substrate interaction studies have used purified enzymes and pure cellulose model substrates. As the enzymes present in a typical “cellulase mixture” need to work cooperatively to achieve effective hydrolysis, the action of one enzyme is likely to influence the behaviour of others. The action of the enzymes will be further influenced by the nature of the lignocellulosic substrate. Therefore, it would be beneficial if a method could be developed that allowed us to follow some of the individual enzymes present in a cellulase mixture during hydrolysis of more commercially realistic biomass substrates.ResultsA high throughput immunoassay that could quantitatively and specifically follow individual cellulase enzymes during hydrolysis was developed. Using monoclonal and polyclonal antibodies (MAb and PAb, respectively), a double-antibody sandwich enzyme-linked immunosorbent assay (ELISA) was developed to specifically quantify cellulase enzymes from Trichoderma reesei: cellobiohydrolase I (Cel7A), cellobiohydrolase II (Cel6A), and endoglucanase I (Cel7B). The interference from substrate materials present in lignocellulosic supernatants could be minimized by dilution.ConclusionA double-antibody sandwich ELISA was able to detect and quantify individual enzymes when present in cellulase mixtures. The assay was sensitive over a range of relatively low enzyme concentration (0 – 1 μg/ml), provided the enzymes were first pH adjusted and heat treated to increase their antigenicity. The immunoassay was employed to quantitatively monitor the adsorption of cellulase monocomponents, Cel7A, Cel6A, and Cel7B, that were present in both Celluclast and Accellerase 1000, during the hydrolysis of steam-pretreated corn stover (SPCS). All three enzymes exhibited different individual adsorption profiles. The specific and quantitative adsorption profiles observed with the ELISA method were in agreement with earlier work where more labour intensive enzyme assay techniques were used.

High concentrations of cellulosic ethanol achieved by fed batch semi simultaneous saccharification and fermentation of waste-paper

A fundamental goal of second generation ethanol production is to increase the ethanol concentration to 10% (v/v) or more to optimise distillation costs. Semi simultaneous saccharification and fermentations (SSSF) were conducted at small pilot scale (5L) utilising fed-batch additions of solid shredded copier paper substrate. Early addition of Accellerase® 1500 at 16FPU/g substrate and 30U/g β-glucosidase followed by substrate only batch addition allowed low final equivalent enzyme concentrations to be achieved (3.7FPU/g substrate) whilst maintaining digestion. Batch addition resulted in a cumulative substrate concentration equivalent to 65% (w/v). This in turn resulted in the production of high concentrations of ethanol (11.6% v/v). The success of this strategy relied on the capacity of the bioreactor to perform high shear mixing as required. Further research into the timing and number of substrate additions could lead to further improvement in overall yields from the 65.5% attained.

Micropipet Tip-Based Miniaturized Electrochemical Device Combined with Ultramicroelectrode and Its Application in Immobilization-Free Enzyme Biosensor

In this study, a simple miniaturized microliter electrochemical device was constructed using a disposable micropipet tip and a reproducible carbon fiber ultramicroelectrode. The novel electrochemical device set the electrochemical reaction in a micropipet tip containing an ultramicroelectrode. We investigated the feasibility of the designed electrochemical device by cyclic voltammetric measurements of redox probe. Its application in an immobilization-free enzyme electrochemical biosensor was also evaluated. Horseradish peroxidase and glucose oxidase were selected to test sensor feasibility. Our results showed that the micropipet tip-based electrochemical device could detect low substrate or enzyme concentration or enzymatic reaction rate. The electrochemical device was applied to analyze the glucose content in human blood samples. With the advantages of low cost, easy operation, rapid detection and high reproducibility, this design provides a new approach in immobilization-free enzyme biosensor construction. Integrated with an ultramicroelectrode, our micropipet tip-based electrochemical device could replace most normal electrodes and electrochemical cells in common laboratories for electroanalysis.

Tetrachlorofluorescein TInsP 5 as a Substrate Analog Probe for Measuring Phytase Activity in Surface Water: Proof of Concept

An innovative approach for measuring phytase activity (PA) in surface water is presented. A substrate analog of -inositol hexakis(dihydrogen) phosphate (InsP), commonly referred to as phytic acid, 1--5--(1-oxo-1-(2' ,4,7,7' -tetrachloro-3',6'-dihydroxy-3-oxo-3H-spiro[isobenzofuran-1,9'-xanthen]-6-yl)-5,8,11-trioxa-2-azatridecan-13-yl)-inositol 1,2,3,4,6-pentakis--(dihydrogen) phosphate, referred to as tetrachlorofluorescein (TET) tethered (T)InsP, has been developed that can be used to monitor the (phytase-catalyzed) phosphate ester bond-cleavage reaction. Test phytases, (wheat [4-] and [3-] phytase) sequentially remove phosphate groups from TET TInsP, producing dephosphorylated probe species that were readily separated by reversed-phase high-performance liquid chromatography (RP-HPLC). Because dephosphorylated probe species retain the TET group, highly sensitive quantification could be achieved using fluorescence detection (excitation/emission ' = 245/540 nm). Calibration curves for TET TInsP, which could be used as a standard for quantifying all probe species, were linear ( > 0.999) over the range of concentrations tested. Phytase-generated dephosphorylated probe species were characterized or identified using RP-HPLC with mass spectrometry. Results of mass spectrometry analysis show that the RP-HPLC system was capable of distinguishing between dephosphorylated probe species at the regioisomeric level. The TET TInsP molecular probe was used to successfully measure PA in pond water. We found that the PA associated with the particulate plus water-soluble fraction was greater than that observed for the water-soluble fraction alone. Moreover, it appeared that 4- and 3-phytase were active in pond water based on an analysis of the chromatographic profile (i.e., elution sequence) of dephosphorylated probe species produced. The advent of a fluorescent substrate analog of InsP affords environmental scientists with the means to unambiguously quantify an extremely small amount of phytase-generated dephosphorylated product(s), enabling the measurement of PA over a reasonably short time duration, in an environmental sample containing low concentrations of enzyme.

Enzymatic Degumming of Silk with Microbial Proteases

Degumming of Chinese bivoltine silk with alkaline proteases from various microbial sources was investigated and compared with commercial enzymes. Among the proteases tested, two fungal and two actinomycete proteases were promising, which showed weight loss similar to conventional method (19.58% to 21.78%). Conidiobolus brefeldianus and BOA-2 proteases were best enzymes, which showed weight loss similar to conventional method with low enzyme concentrations and in shorter time. No significant differences were found in tensile strength or elongation at break by enzymatic degumming indicating that there was no damage to the fiber. Scanning electron micrographs showed the sericin deposits were removed and the fibers were separated.

MODELING THE OPTIMAL CENTRAL CARBON METABOLIC PATHWAYS UNDER FEEDBACK INHIBITION USING FLUX BALANCE ANALYSIS

Metabolism is a complex process for energy production for cellular activity. It consists of a cascade of reactions that form a highly branched network in which the product of one reaction is the reactant of the next reaction. Metabolic pathways efficiently produce maximal amount of biomass while maintaining a steady-state behavior. The steady-state activity of such biochemical pathways necessarily incorporates feedback inhibition of the enzymes. This observation motivates us to incorporate feedback inhibition for modeling the optimal activity of metabolic pathways using flux balance analysis (FBA). We demonstrate the effectiveness of the methodology on a synthetic pathway with and without feedback inhibition. Similarly, for the first time, the Central Carbon Metabolic (CCM) pathways of Saccharomyces cerevisiae and Homo sapiens have been modeled and compared based on the above understanding. The optimal pathway, which maximizes the amount of the target product(s), is selected from all those obtained by the proposed method. For this, we have observed the concentration of the product inhibited enzymes of CCM pathway and its influence on its corresponding metabolite/substrate. We have also studied the concentration of the enzymes which are responsible for the synthesis of target products. We further hypothesize that an optimal pathway would opt for higher flux rate reactions. In light of these observations, we can say that an optimal pathway should have lower enzyme concentration and higher flux rates. Finally, we demonstrate the superiority of the proposed method by comparing it with the extreme pathway analysis.