The reliability of Michaelis constants and maximum velocities estimated by using the integrated Michaelis–Menten equation

1. Experimental progress curves were simulated for a reaction obeying Michaelis-Menten kinetics. 2. K(m) and V were estimated (a) by fitting the integrated Michaelis-Menten equation to the progress curves, and (b) from the initial slopes of the curves (i.e. from initial velocities). 3. The integrated equation could not be fitted successfully by a non-linear method, so it was transformed and fitted by a linear method. 4. Provided that the initial substrate concentration was greater than K(m) and the data were precise enough, the integrated equation gave parameter estimates which were unbiased and as reliable as those derived from initial velocities although based on fewer experiments. 5. The integrated equation could be used for progress curves of unknown origin.     

An explicit solution for progress curve analysis in systems characterized by endogenous substrate production

The Lambert W function was used to explicitly relate substrate concentration S, to time t, and the kinetic parameters V (m), K (m), and R in the modified Michaelis-Menten equation that accounts for endogenous substrate production. The applicability of this explicit formulation for kinetic parameter estimation by progress curve analysis was demonstrated using a combination of synthetic and experimental substrate depletion data. Synthetic substrate depletion data were generated using S (0) values of 1, 2, and 3 μM and V (m), K (m), and R values of 1.0 μM h(-1), 1.0 μM, and 0.1 μM h(-1), respectively, and contained 5% normally distributed error. Experimental data were obtained from two previously published studies on hydrogen depletion in four experimental systems. In all instances, experimental data were well described by the explicit solution presented in this study. Differential equation solution and iterative S estimation are eliminated with the explicit solution approach, thereby simplifying progress curve analysis in systems characterized by endogenous substrate production.     

Rapid simultaneous determination of metabolic clearance of multiple compounds catalyzed in vitro by recombinant human UDP-glucuronosyltransferases

The purpose of this study was to test the applicability of n-in-one (cocktail) incubations in the determination of intrinsic clearance (Cl(int)) as the slope of the linear portion of the Michaelis-Menten curve (velocity V vs. substrate concentration [S]) where substrate concentrations were low. A rapid, sensitive, and selective liquid chromatography tandem mass spectrometry (LC/MS/MS) method was developed for the analysis of samples produced by single-substrate and n-in-one (seven substrates: entacapone, 17beta-estriol, umbelliferone, 4-methylumbelliferone, tolcapone, hydroxyquinoline, and paracetamol) incubations conducted in 96-well plates with different recombinant UDP-glucuronosyltransferases (UGTs). The Cl(int) values obtained with n-in-one incubations were compared with those obtained in single-compound incubations and with V(max)/K(m) values determined by estimating the enzyme kinetic parameters V(max) and K(m) from the Michaelis-Menten curve. When substrate concentrations were well below their K(m) values, Cl(int) values determined as the slope of the linear part of the Michaelis-Menten fitting correlated well with the values determined as V(max)/K(m) ratios from the Michaelis-Menten curve. The correlation between Cl(int) values determined in single-substrate and n-in-one incubations was high as well. Together, the n-in-one incubations, the determination of Cl(int) values as the slope of the linear part of the Michaelis-Menten fitting, and LC/MS/MS as an analytical method proved to be effective approaches for increasing throughput in the first-phase screening of metabolic properties.     

Rapid determination of enzyme kinetics from fluorescence: overcoming the inner filter effect

Fluorescence change is convenient for monitoring enzyme kinetics. Unfortunately, it loses linearity as the absorbance of the fluorescent substrate increases with concentration. When the sum of absorbance at excitation and emission wavelengths exceeds 0.08, this inner filtering effect (IFE) alters apparent initial velocities, K(m), and k(cat). The IFE distortion of apparent initial velocities can be corrected without doing fluorophore dilution assays. Using the substrate's extinction coefficients at excitation and emission wavelengths, the inner filter effect can be modeled during curve fitting for more accurate Michaelis-Menten parameters. A faster and simpler approach is to derive k(cat) and K(m) from progress curves. Strategies to obtain reliable and reproducible estimates of k(cat) and K(m) from only two or three progress curves are illustrated using matrix metalloproteinase 12 and alkaline phosphatase. Accurate estimates of concentration of enzyme-active sites and specificity constant k(cat)/K(m) (from one progress curve with [S]相似文献   

Simultaneous estimation ofV max,Km, and the rate of endogenous substrate production (R) from substrate depletion data

The nonlinear and 3 linearized forms of the integrated Michaelis-Menten equation were evaluated for their ability to provide reliable estimates of uptake kinetic parameters, when the initial substrate concentration (S₀) is not error-free. Of the 3 linearized forms, the one where t/(S₀–S) is regressed against ln(S₀/S)/(S₀–S) gave estimates ofV _max and K_m closest to the true population means of these parameters. Further, this linearization was the least sensitive of the 3 to errors (±1%) in S₀. Our results illustrate the danger of relying on r² values for choosing among the 3 linearized forms of the integrated Michaelis-Menten equation. Nonlinear regression analysis of progress curve data, when S₀ is not free of error, was superior to even the best of the 3 linearized forms. The integrated Michaelis-Menten equation should not be used to estimateV _max and K_m when substrate production occurs concomitant with consumption of added substrate. We propose the use of a new equation for estimation of these parameters along with a parameter describing endogenous substrate production (R) for kinetic studies done with samples from natural habitats, in which the substrate of interest is an intermediate. The application of this new equation was illustrated for both simulated data and previously obtained H₂ depletion data. The only means by whichV _max, K_m, and R may be evaluated from progress curve data using this new equation is via nonlinear regression, since a linearized form of this equation could not be derived. Mathematical components of computer programs written for fitting data to either of the above nonlinear models using nonlinear least squares analysis are presented.     

A new linear plot for standard curves in kinetic substrate assays extended above the Michaelis-Menten constant: application to a luminometric assay of glycerol

In conventional kinetic substrate assays the standard curve is plotted as observed reaction rate, upsilon obs, versus added substrate concentration, Sadd, and has a linearity limited to Sadd much less than Km. From this plot the blank reaction rate, upsilon bl, is easily estimated but not the contaminating substrate concentration, Scon, present in reagents (unless it is the only blank source). Thus the actual substrate concentration, S = Scon + Sadd, cannot be estimated as required for the various linear plots based on the Michaelis-Menten equation. We have derived an expression, (upsilon obs - upsilon bl)/Vapp = Sadd/(Kmapp + Sadd), containing only those parameters measured for a conventional standard curve (Vapp and Kmapp are obtained from a plot of (upsilon obs - upsilon bl) versus (upsilon obs - upsilon bl)/Sadd). A plot of (upsilon obs - upsilon bl)/Vapp versus Sadd/(Kmapp + Sadd) can be used as a standard curve with the following advantages over the conventional standard curve: (a) For all kinetic substrate assays it is identical and connects the points (0, 0) and (1, 1). Thus deviations from true Michaelis-Menten kinetics or erroneous kinetic constants are easily detected. (b) Since it is linear even above Km, the analytically useful range is considerably extended. (c) For assays with a wide dynamic range it can be used in lin-lin or log-log form. The procedure is illustrated for a kinetic assay of glycerol (Kmapp = 40 mumol/liter). The plot was found to be entirely linear in the range 0.07-100 mumol/liter (glycerol concentration in cuvette).     

Kinetic analysis of urea-inactivation of beta-galactosidase in the presence of galactose

The effect of galactose on the inactivation of purified beta-galactosidase from the black bean, Kestingiella geocarpa, in 5 M urea at 50 degrees C and at pH 4.5, was determined. Lineweaver-Burk plots of initial velocity data in the presence and absence of urea and galactose were used to determine the relevant K(m) and V(max) values, with p-nitrophenyl beta-D-galactopyranoside (PNPG) as substrate, S. The inactivation data were analysed using the Tsou equation and plots. Plots of ln([P](infinity) - [P](t) ) against time in the presence of urea yielded the inactivation rate constant, A. Plots of A vs [S] at different galactose concentrations were zero order showing that A was independent of [S]. Plots of [P](infinity) vs [S] were used to determine the mode of inhibition of the enzyme by galactose, and slopes and intercepts of the 1/[P](infinity) vs. 1/[S] yielded k(+0) and k '(+0), the microscopic rate constants for the free enzyme and the enzyme-substrate complex, respectively. Plots of k(+0) and k '(+0) vs. galactose concentrations showed that galactose protected the free enzyme and not the enzyme-substrate complex against urea inactivation via a noncompetitive mechanism at low galactose concentrations and a competitive pattern of inhibition at high galactose concentrations. The implication of the different modes of inhibition in protecting the free enzyme was discussed.     

A test for measuring the effects of enzyme inactivation

In the single-enzyme, single-substrate reaction with non-mechanism-based enzyme inactivation, the formation of the product and inactivation of the enzyme occur independently. For this reaction, we show that the steady-state hypothesis is applicable even when degradation of the enzyme occurs. An equation for the rate of product formation has been derived and it shows Michaelis-Menten kinetics with an apparent Michaelis-Menten constant K(M)(app)=K(M)+K(delta) where K(delta) is the enzyme inactivation constant. Use of a Lineweaver-Burk plot yields values for K(M)(app), which can be used to estimate K(delta) and, consequently, the degree of enzyme inactivation in a particular experiment. We employ this methodology to estimate the inactivation constant for the arsenate reductase catalyzed production of arsenite with appreciable enzyme inactivation.     

A new multi-wavelength model-based method for determination of enzyme kinetic parameters

Lineweaver-Burk plot analysis is the most widely used method to determine enzyme kinetic parameters. In the spectrophotometric determination of enzyme activity using the Lineweaver-Burk plot, it is necessary to find a wavelength at which only the substrate or the product has absorbance without any spectroscopic interference of the other reaction components. Moreover, in this method, different initial concentrations of the substrate should be used to obtain the initial velocities required for Lineweaver-Burk plot analysis. In the present work, a multi-wavelength model-based method has been developed and validated to determine Michaelis-Menten constants for some enzyme reactions. In this method, a selective wavelength region and several experiments with different initial concentrations of the substrate are not required. The absorbance data of the kinetic assays are fitted by non-linear regression coupled to the numeric integration of the related differential equation. To indicate the applicability of the proposed method, the Michaelis-Menten constants for the oxidation of phenanthridine, 6-deoxypenciclovir and xanthine by molybdenum hydroxylases were determined using only a single initial concentration of the substrate, regardless of any spectral overlap.     

The original Michaelis constant: translation of the 1913 Michaelis-Menten paper

Nearly 100 years ago Michaelis and Menten published their now classic paper [Michaelis, L., and Menten, M. L. (1913) Die Kinetik der Invertinwirkung. Biochem. Z. 49, 333-369] in which they showed that the rate of an enzyme-catalyzed reaction is proportional to the concentration of the enzyme-substrate complex predicted by the Michaelis-Menten equation. Because the original text was written in German yet is often quoted by English-speaking authors, we undertook a complete translation of the 1913 publication, which we provide as Supporting Information . Here we introduce the translation, describe the historical context of the work, and show a new analysis of the original data. In doing so, we uncovered several surprises that reveal an interesting glimpse into the early history of enzymology. In particular, our reanalysis of Michaelis and Menten's data using modern computational methods revealed an unanticipated rigor and precision in the original publication and uncovered a sophisticated, comprehensive analysis that has been overlooked in the century since their work was published. Michaelis and Menten not only analyzed initial velocity measurements but also fit their full time course data to the integrated form of the rate equations, including product inhibition, and derived a single global constant to represent all of their data. That constant was not the Michaelis constant, but rather V(max)/K(m), the specificity constant times the enzyme concentration (k(cat)/K(m) × E(0)).     

Purification and characterization of carbonic anhydrase from bovine stomach and effects of some known inhibitors on enzyme activity

Carbonic anhydrase (CA) was purified from four different cell localisation (outer peripheral, cytosolic, inner peripheral and integral) in bovine stomach using affinity chromatography with Sepharose-4B-L-tyrosine sulphanilamide. During the purification steps, the activity of the enzyme was measured using p-nitrophenyl acetate at pH 7.4. Optimum pH and optimum temperature values for all CA samples were determined, and their K(m) and V(max) values for the same substrate by Lineweaver-Burk graphics. The extent of purification for all CA localizations was controlled by SDS-PAGE. The K(m) values at optimum pH and 20 degrees C were 0.625 mM, 0.541 mM, 0.785 mM and 0.862 mM with p-nitro phenyl acetate, for all CA localizations. The respective V(max) values at optimum pH and 20 degrees C were 0.875 micromol/L min, 0.186 micromol/L min, 0.214 micromol/L min and 0.253 micromol/L min with the same substrate. The K(i) and I50 values for the inhibitors sulphanilamide, KSCN, NaN3 and acetazolamide were determined for all the CA localizations.     

Importance of product inhibition in the kinetics of the acylase hydrolysis reaction by differential stopped flow microcalorimetry

The hydrolysis of N-acetyl-L-methionine, N-acetylglycine, N-acetyl-L-phenylalanine, and N-acetyl-L-alanine at 298.35K by porcine kidney acylase I (EC 3.5.1.14) was monitored by the heat released upon mixing of the substrate and enzyme in a differential stopped flow microcalorimeter. Values for the Michaelis constant (K(m)) and the catalytic constant (k(cat)) were determined from the progress of the reaction curve employing the integrated form of the Michaelis-Menten equation for each reaction mixture. When neglecting acetate product inhibition of the acylase, values for k(cat) were up to a factor of 2.3 larger than those values determined from reciprocal initial velocity-initial substrate concentration plots for at least four different reaction mixtures. In addition, values for K(m) were observed to increase linearly with an increase in the initial substrate concentration. When an acetate product inhibition constant of 600+/-31M(-1), determined by isothermal titration calorimetry, was used in the progress curve analysis, values for K(m) and k(cat) were in closer agreement with their values determined from the reciprocal initial velocity versus initial substrate concentration plots. The reaction enthalpies, Delta(r)H(cal), which were determined from the integrated heat pulse per amount of substrate in the reaction mixture, ranged from -4.69+/-0.09kJmol(-1) for N-acetyl-L-phenylalanine to -1.87+/-0.23kJmol(-1) for N-acetyl-L-methionine.     

NMR for direct determination of K(m) and V(max) of enzyme reactions based on the Lambert W function-analysis of progress curves

(1)H NMR spectroscopy was used to follow the cleavage of sucrose by invertase. The parameters of the enzyme's kinetics, K(m) and V(max), were directly determined from progress curves at only one concentration of the substrate. For comparison with the classical Michaelis-Menten analysis, the reaction progress was also monitored at various initial concentrations of 3.5 to 41.8mM. Using the Lambert W function the parameters K(m) and V(max) were fitted to obtain the experimental progress curve and resulted in K(m)=28mM and V(max)=13μM/s. The result is almost identical to an initial rate analysis that, however, costs much more time and experimental effort. The effect of product inhibition was also investigated. Furthermore, we analyzed a much more complex reaction, the conversion of farnesyl diphosphate into (+)-germacrene D by the enzyme germacrene D synthase, yielding K(m)=379μM and k(cat)=0.04s(-1). The reaction involves an amphiphilic substrate forming micelles and a water insoluble product; using proper controls, the conversion can well be analyzed by the progress curve approach using the Lambert W function.     

Reduced lung endothelial angiotensin-converting enzyme activity in Watanabe hyperlipidemic rabbits in vivo

We investigated pulmonary endothelial function in vivo in 12- to 18-mo-old male Watanabe heritable hyperlipidemic (WHHL; n = 7) and age- and sex-matched New Zealand White (n = 8) rabbits. The animals were anesthetized and artificially ventilated, and the chest was opened and put in total heart bypass. The single-pass transpulmonary utilizations of the angiotensin-converting enzyme (ACE) substrate [(3)H]benzoyl-Phe-Ala-Pro (BPAP) and the 5'-nucleotidase (NCT) substrate [(14)C]AMP were estimated, and the first-order reaction parameter A(max)/K(m), where A(max) is the product of enzyme mass and the catalytic rate constant and K(m) is the Michaelis-Menten constant, was calculated. BPAP transpulmonary utilization and A(max)/K(m) were reduced in WHHL (1.69 +/- 0.16 vs. 2.9 +/- 0.44 and 599 +/- 69 vs. 987 +/- 153 ml/min in WHHL and control rabbits, respectively; P < 0.05 for both). No differences were observed in the AMP parameters. BPAP K(m) and A(max) values were estimated separately under mixed-order reaction conditions. No differences in K(m) values were found (9.79 +/- 1 vs. 9.9 +/- 1.31microM), whereas WHHL rabbit A(max) was significantly decreased (5.29 +/- 0.88 vs. 7. 93 +/- 0.8 micromol/min in WHHL and control rabbits, respectively; P < 0.05). We conclude that the observed pulmonary endothelial ACE activity reduction in WHHL rabbits appears related to a decrease in enzyme mass rather than to alterations in enzyme affinity.     

Inhibition by N'-nitrosonornicotine of the catalytic activity of glutamate dehydrogenase in alpha-ketoglutarate amination

The effect of N'-nitrosonornicotine (NNN), one of the tobacco-specific nitrosamines, on the catalytic activity of glutamate dehydrogenase (GLDH) in the alpha-ketoglutarate amination, using reduced nicotinamide adenine dinucleotide as coenzyme, was studied by a chronoamperometric method. The maximum reaction rate of the enzyme-catalyzed reaction and the Michaelis-Menten constant, or the apparent Michaelis-Menten constant, were determined in the absence and presence of NNN. NNN remarkably inhibited the bio-catalysis activity of GLDH, and was a reversible competitive inhibitior with K(i), estimated as 199 micromol l(-1) at 25 degrees C and pH 8.0.     

Analysis of the Escherichia coli ribosome-ribosomal subunit equilibrium using pressure-induced dissociation

Hydrostatic pressure can be used to perturb the ribosome-ribosomal subunit equilibrium. We have used glutaraldehyde fixation and subsequent sucrose gradient analysis to determine the equilibrium concentrations of Escherichia coli 70 S, 50 S, and 30 S particles at pressures from 1 to 1400 atm. This method is shown to be sufficiently rapid and free of interfering ribosomal aggregation artifacts when performed at Mg2+ concentrations below 8 mM. We show directly that the E. coli ribosome is in equilibrium with its subunits and that the pressure-sensitive reaction is appropriately described by the expression: In Kp = ln K0 + (P delta V/RT), where Kp and K0 are the equilibrium constants at pressure P and 1 atm, respectively, and delta V is the change in molecular volume that occurs during the reaction. The method provides values for K0 under different conditions, and the effects of Mg2+ ion can be readily ascertained. K0 and delta V were also estimated by a method of fitting computer-generated sucrose gradient profiles to experimental profiles. Determination of delta H0, delta S0, and delta V0 at 5 mM Mg2+ are presented. The results are discussed in the context of previous thermodynamic studies of the E. coli ribosome.     

Immobilization of tyrosinase and alcohol oxidase in conducting copolymers of thiophene functionalized poly(vinyl alcohol) with pyrrole

Immobilization of tyrosinase and alcohol oxidase is achieved in the copolymer of pyrrole with vinyl alcohol with thiophene side groups (PVATh-co-PPy) which is a newly synthesized conducting polymer. PVATh-co-PPy/alcohol oxidase and PVATh-co-PPy/tyrosinase electrodes are constructed by the entrapment of enzyme in conducting copolymer matrix during electrochemical copolymerization. For tyrosinase and alcohol oxidase enzymes, catechol and ethanol are used as the substrates, respectively. Kinetic parameters: maximum reaction rates (V(max)) and Michaelis-Menten constants (K(m)) are obtained. V(max) and K(m) are found as 2.75 micromol/(minelectrode) and 18 mM, respectively, for PVATh-co-PPy/alcohol oxidase electrode and as 0.0091micromol/(minelectrode) and 40 mM, respectively, for PVATh-co-PPy/tyrosinase electrode. Maximum temperature and pH values are investigated and found that both electrodes have a wide working range with respect to both temperature and pH. Operational and storage stabilities show that although they have limited storage stabilities, the enzyme electrodes are useful with respect to operational stabilities.     

Inhibition of beta-lactamase of Bacillus licheniformis 749/C by compound PS-5, a new beta-lactam antibiotic.

By use of a new computer-assisted u.v.-spectrophotometric assay method, the kinetic parameters of the reaction catalysed by Bacillus licheniformis 749/C beta-lactamase were re-examined and the mode of inhibition of the enzyme by compound PS-5, a novel beta-lactam antibiotic, was studied with benzylpenicillin as substrate. (1) The fundamental assay conditions for the determination of Km and V were examined in detail with benzylpenicillin as substrate. In 0.1 M-sodium/potassium phosphate buffer, pH 6.8, at 30 degrees C, initial substrate concentrations of benzylpenicillin above 0.7 mM were very likely to lead to substrate inhibition. The Km value of the enzyme for benzylpenicillin at initial concentrations from 1.96 to 0.07 mM was calculated to be 97-108 microM. (2) The Km values of the enzyme for 6-aminopenicillanic acid, ampicillin and cephaloridine were found to be 25, 154-161 and 144-161 microM respectively. (3) Compound PS-5 was virtually unattacked by Bacillus licheniformis 749/C beta-lactamase. (4) The activity of the enzyme was diminished by compound PS-5, to extents depending on the duration of incubation and the concentration of the inhibitor. The rate of inactivation of the enzyme by compound PS-5 followed first-order kinetics. (5) In an Appendix, a new computer-assisted u.v.-spectrophotometric enzyme assay method, in which a single reaction progress curve of time-absorbance was analysed by the integrated Michaelis-Menten equation, was devised for the accurate and precise determination of the kinetic constants of beta-lactamase. For conversion of absorbance readings into molar substrate concentrations, the initial or final absorbance reading that was independent of the reaction time was used as the basis of calculation. In calculation of Km and V three systematic methods of data combination were employed for finer analysis of the reaction progress curve. A list of the computer program named YF6TAIM is obtainable from the author on request or as Supplementary Publication SUP 50100 (12 pages) from the British Library Lending Division, Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., on the terms indicated in Biochem. J. (1978) 169, 5.     

Characterization of the <Emphasis Type="Italic"> ugpG </Emphasis>gene encoding a UDP-glucose pyrophosphorylase from the gellan gum producer <Emphasis Type="Italic"> Sphingomonas paucimobilis </Emphasis>ATCC 31461

The ugpGgene, which codes for a UDP-glucose pyrophosphorylase (UGP) (or glucose-1-phosphate uridylyltransferase; EC 2.7.7.9) in Sphingomonas paucimobilis ATCC 31461, was cloned and sequenced. This industrial strain produces the exopolysaccharide gellan, a new commercial gelling agent, and the ugpG gene may convert glucose-1-phosphate into UDP-glucose in the gellan biosynthetic pathway. The ugpG gene is capable of restoring the capacity of an Escherichia coli galU mutant to grow on galactose by functional complementation of its deficiency for UDP-glucose pyrophosphorylase activity. As expected, the predicted gene product shows strong homology to UDP-glucose pyrophosphorylases from several bacterial species. The N-terminal region of UgpG exhibits the motif GXGTRXLPXTK, which is highly conserved among bacterial XDP-sugar pyrophosphorylases, and a lysine residue (K(192)) is located within a VEKP motif predicted to be essential for substrate binding or catalysis. UgpG was purified to homogeneity as a heterologous fusion protein from crude cell extracts prepared from IPTG-induced cells of E. coli, using affinity chromatography. Under denaturing conditions, the fusion protein S-UgpG-His(6) migrated with an estimated molecular mass of 36 kDa [corresponding to the predicted molecular mass of native UgpG (31.2 kDa) plus 5 kDa for the S and histidine tags). Kinetic analysis of UgpG in the reverse reaction (pyrophosphorolysis) showed a typical Michaelis-Menten substrate saturation pattern. The apparent K(m) and V(max) values estimated for UDP-glucose were 7.5 microM and 1275 micromol/min/g.     

Removal of fiber from vines by solid state fermentation/enzymatic degradation: a comparison of flax and kudzu retting

Kinetic data describing the decomposition of the outer sheath of kudzu vines (undergoing a solid fermentation process in a glass beaker of soil) have been analyzed to determine the two constants, K(m)/S(o) and V/S(o), where S(o) is the initial substrate concentration, K(m) the Michaelis constant, and V the maximum product rate. The kinetic data are expressed by a simple time-varying desheathing index, obtained from the number of spatula scrapings required to reach the desired hard cellulosic fibers (vascular bundles) of the plant. A simple relationship between the desheathing index, D.I. and the dimensionless product concentration, P/S(o), is proposed to relate the D.I. data and enzyme kinetic concentration data. Thus, the Michaelis-Menten enzyme kinetic parameters can be estimated from easily obtained physical (non-chemical data; the D.I.(t) measurements). This low energy process for processing vines into valuable fibers is similar to the traditional microbial method for recovering flax fibers for linen cloth, by retting of the flax plant vines, except there is no unbound liquid water present in the soil medium.