Analytical solutions for distributions of chemotactic bacteria

This paper reports general and specialized results on analytical solutions to the governing phenomenological equations for chemotactic redistribution and population growth of motile bacteria. It is shown that the number of bacteria cells per unit volume,b, is proportional to a certain prescribed function ofs, the concentration of the critical substrate chemotactic agent, for steady-state solutions through an arbitrary spatial region with a boundary that is impermeable to bacteria cell transport. Moreover, it is demonstrated that the steady-state solution forb ands is unique for a prescribed total number of bacteria cells in the spatial region and a generic Robin boundary condition ons. The latter solution can be approximated to desired accuracy in terms of the Poisson-Green's function associated with the spatial region. Also, as shown by example, closed-form exact steady-state solutions are obtainable for certain consumption rate functions and geometrically symmetric spatial regions. A solutional procedure is formulated for the initialvalue problem in cases for which significant population growth is present and bacteria cell redistribution due to motility and chemotactic flow proceeds slowly relative to the diffusion of the chemoattractant substrate. Finally, a remarkably simple exact analytical solution is reported for a stradily propagating plane-wave which features motility, chemotactic motion and bacteria population growth regulated by substrate diffusion.     

The function and activity of certain membrane enzymes when localized on – and off – the membrane

A group of enzymes known to be involved in group translocation-type transport mechanisms for the uptake of a variety of nucleotide precursors are enzymatically active both in their natural membrane milieu and in aqueous solution. The activity in aqueous solution markedly differ, however, from the enzymatic activity when the enzyme is membrane localized. The adenine phosphoribosyltransferase (PRT) of E. coli (Hochstadt-Ozer and Stadtman, 1971 a) is capable of carrying out an exchange reaction between the base moieties of adenine and AMP without requiring P-ribose-PP as an intermediate; the enzyme in aqueous solution requires P-ribose-PP, indicating a different reaction mechanism in the two environments. Like the adenine PRT of E. coli, the hypo-xanthine PRT of Salmonella typhimurium (Jackman and Hochstadt, 1976) also carried out an exchange reaction on the membrane only and also is more sensitive to a number of inhibitors in aqueous solution relative to the sensitivity when embedded in the membrane. In addition, however, the hypoxanthine PRT, while restricted to hypoxanthine as a substrate in the membrane, also accepts guanine as substrate in its soluble form. The membrane capacities reflect the in situ capacities of the enzyme and the gain of guanine specificity was determined in a guanine PRT deletion strain (Jackman and Hochstadt, 1976). Finally, in mammalian cell lines purine nucleoside phosphorylase, which translocates the ribose moiety of inosine across the plasma membrane of mouse fibroblasts undergoes a 30-fold increase in substrate turnover number upon liberation from the membrane. These data raise two important caveats with respect to study of membrane enzymes and transport. Firstly, an enzyme once solubilized and found to differ kinetically from substrate transport in situ cannot be excluded from participating in translocations in the membrane on the basis of its activity in aqueous solution. Secondly, an enzyme which “appears” largely soluble upon cell rupture cannot be assumed to be a cycloplasmic enzyme because the majority of the solubilized activity may represent only a small fraction of the enzyme molecules highly activated concomitant to their solubilization. In this latter case the ability to activate enzyme still residing on the membrane (e.g., with detergents) would be necessary in order to estimate total membrane associated activity after cell rupture.     

Feeding behaviour of the Japanese loach,Misgurnus anguillicaudatus (Cobitididae)

The feeding behaviour of a Japanese loach,Misgurnus anguillicaudatus was experimentally analyzed. From observations the behaviour patterns were described. The adult fish responded to a piece of thread soaked with filteredTubifex homogenate (Tubifex solution) and showed feeding behaviour. But they ignored an unsoaked piece of thread. LivingTubifex kept in a glass cell failed to elicit feeding behaviour in the fish. A gauze-covered vessel withTubifex solution buried in the sand substrate released feeding behaviour in fish in its vicinity. It follows that the feeding behaviour of this fish is induced by some chemical stimuli. WhenTubifex solution was introduced at the central area of the plastic vessel with a fish, the fish suddenly started the Feeding-crawl and showed Gulp which was composed of Dig and Twist. Using sand particles, it was shown that intake of materials into the digestive tract occurs only at the moment of Twist and not any other time. The standard feeding patterns ofMisgurnus anguillicaudatus are thus quick inhalation of organic matter together with substrate, occurring at the moment of Twist, which is released at the place where the fish received the appropriate chemical stimuli.Misgurnus anguillicaudatus is regarded as a typical “detritus feeder”.     

Some properties of a papain–agarose reactor provided with a pH monitor

A small reactor of immobilized papain was used to gain some knowledge about the effect of immobilization upon the reactivity of the enzyme towards one substrate and various types of inhibitors. A buffer solution containing benzoyl–arginine ethyl ester as substrate was run through a small column of papain immobilized by attachment to agarose beads. The pH of the effluent was measured continuously and provided the data used to calculate the substrate conversion during passage through the reactor. The operation of the system was checked by determining the substrate conversion as a function of flow rate. It proved to operate as theory demanded. The rate and extent of inhibition were measured after addition of various inhibitors to the buffer–substrate solution. The following quantities of immobilized papain were found to be equal within ±20% to those of the free enzyme in solution: the overall activity, the K_m of benzoyl–arginine ethyl ester, the K_i of the competitive inhibitor benzoylamino-acetonitrile, the rate of inactivation by chloroacetic acid and by chloroacetamide, the rate of activation by cysteine of the mixed disulfide of papain and cysteine, and the rate of spontaneous reactivation of the KCNO–papain adduct. The inactivation by KCNO proved to be strongly pH dependent. This may explain why the rate of the latter reaction is only 66% of the rate with free enzyme. It is concluded that the rates and equilibrium constants measured in the present reactor system are within ±20% of the values of the dissolved enzyme, provided that the reactions are not strongly pH dependent. Calculation showed there was no diffusion limitation.     

Accurate kinetic parameter estimation during progress curve analysis of systems with endogenous substrate production

We have identified an error in the published integral form of the modified Michaelis–Menten equation that accounts for endogenous substrate production. The correct solution is presented and the error in both the substrate concentration, S, and the kinetic parameters V_m, K_m, and R resulting from the incorrect solution was characterized. The incorrect integral form resulted in substrate concentration errors as high as 50% resulting in 7–50% error in kinetic parameter estimates. To better reflect experimental scenarios, noise containing substrate depletion data were analyzed by both the incorrect and correct integral equations. While both equations resulted in identical fits to substrate depletion data, the final estimates of V_m, K_m, and R were different and K_m and R estimates from the incorrect integral equation deviated substantially from the actual values. Another observation was that at R = 0, the incorrect integral equation reduced to the correct form of the Michaelis–Menten equation. We believe this combination of excellent fits to experimental data, albeit with incorrect kinetic parameter estimates, and the reduction to the Michaelis–Menten equation at R = 0 is primarily responsible for the incorrectness to go unnoticed. However, the resulting error in kinetic parameter estimates will lead to incorrect biological interpretation and we urge the use of the correct integral form presented in this study. Biotechnol. Bioeng. 2011;108: 2499–2503. © 2011 Wiley Periodicals, Inc.     

Inactivation kinetics of mushroom tyrosinase in the dimethyl sulfoxide solution

Mushroom tyrosinase (EC 1.14.18.1) is a kind of copper-containing oxidase that catalyzes both the hydroxylation of tyrosine into o-diphenols and the oxidation of o-diphenols into o-quinones and then forms brown or black pigments. In the present paper, the effects of dimethyl sulfoxide on the enzyme activity for the oxidation of L-3,4-dihydroxyphenylalanine (L-DOPA) have been studied. The results show that low concentrations of dimethyl sulfoxide (DMSO) can lead to reversible inactivation of the enzyme, and the IC ₅₀ is estimated to be 2.45 M. Inactivation of the enzyme by DMSO is classified as mixed type. The kinetics of inactivation of mushroom tyrosinase at low concentrations of DMSO solution has been studied using the kinetic method of the substrate reaction. The rate constants of inactivation have been determined. The results show the free enzyme molecule is more fragile than the enzyme–substrate complex in the DMSO solution. It is suggested that the presence of the substrate offers marked protection of this enzyme against inactivation by DMSO.     

Colonization and mineralization of palmitic acid byPseudomonas pseudoflava

Pseudomonas pseudoflava and palmitic acid were used to investigate the role of bacterial colonization in the degradation of waterinsoluble organic compounds. Mineralization was measured by trapping the¹⁴CO₂ produced from the labeled substrate, and colonization of the surface of the solid organic chemical was determined by epifluorescence microscopy. In a medium containing solid palmitic acid,P. pseudoflava mineralized the organic substrate at a logarithmic rate. Mineralization was evident before colonization of the surface of the chemical was detected. The rate of appearance of single cells and/or aggregations of cells on the surface of the palmitic acid was essentially the same as the doubling time of free cells in solution. At about 50 hours, mineralization and colonization of the surface stopped. In a salts solution containing solid palmitic acid,P. pseudoflava grew logarithmically in the solution and biphasically on the surface of the palmitic acid. We suggest that the bacterium first metabolizes soluble palmitic acid and later colonizes the solid when the substrate in solution has been depleted.     

Hot‐Substrate Deposition of Hole‐ and Electron‐Transport Layers for Enhanced Performance in Perovskite Solar Cells

Charge transport layers play an important role in determining the power conversion efficiencies (PCEs) of perovskite solar cells (PSCs). However, it has proven challenging to produce thin and compact charge transport layers via solution processing techniques. Herein, a hot substrate deposition method capable of improving the morphology of high‐coverage hole‐transport layers (HTLs) and electron‐transport layers (ETLs) is reported. PSC devices using HTLs deposited on a hot substrate show improvement in the open‐circuit voltage (V_oc) from 1.041 to 1.070 V and the PCE from 17.00% to 18.01%. The overall device performance is then further enhanced with the hot substrate deposition of ETLs as the V_oc and PCE reach 1.105 V and 19.16%, respectively. The improved performance can be explained by the decreased current leakage and series resistance, which are present in PSCs with rough and discontinuous HTLs and ETLs.     

Kinetcs and decay of fumarase activity of immobilized Brevibacterium ammoniagenes cells for continuous production of L-malic acid

The kinetics of the reversible fumarase reaction of immobilized Brevibacterium ammoniagenes cells and the decay behavior of enzyme activity were investigated in a plug flow system. The time course of the reaction in the immobilized cell column was well explained by the time-conversion equation including the apparent kinetic constants of the immobilized cell enzyme. The decay rate of fumarase activity was faster in the upper sections of the column (inlet side of the substrate solution) compared with the lower sections when 1M sodium fumarate (pH 7.0) was continuously passed through the column at 37°C. It was shown that the decay rate of the fumarase activity in the immobilized cell column depends on the flow rate of the substrate solution. The effect of flow rate on the decay rate of enzyme activity was considered to be related to the rate of contamination of enzyme with poisonous substances derived from the substrate solution or to the rate of leakage of enzyme stabilizers and/or enzyme itself from the immobilized cells.     

Asymmetric synthesis of (S)-3-chloro-1-phenyl-1-propanol using Saccharomyces cerevisiae reductase with high enantioselectivity

3-Chloro-1-phenyl-1-propanol is used as a chiral intermediate in the synthesis of antidepressant drugs. Various microbial reductases were expressed in Escherichia coli, and their activities toward 3-chloro-1-phenyl-1-propanone were evaluated. The yeast reductase YOL151W (GenBank locus tag) exhibited the highest level of activity and exclusively generated the (S)-alcohol. Recombinant YOL151W was purified by Ni-nitrilotriacetic acid (Ni-NTA) and desalting column chromatography. It displayed an optimal temperature and pH of 40°C and 7.5–8.0, respectively. The glucose dehydrogenase coupling reaction was introduced as an NADPH regeneration system. NaOH solution was occasionally added to maintain the reaction solution pH within the range of 7.0–7.5. By using this reaction system, the substrate (30 mM) could be completely converted to the (S)-alcohol product with an enantiomeric excess value of 100%. A homology model of YOL151W was constructed based on the structure of Sporobolomyces salmonicolor carbonyl reductase (Protein Data Bank ID: 1Y1P). A docking model of YOL151W with NADPH and 3-chloro-1-phenyl-1-propanone was then constructed, which showed that the cofactor and substrate bound tightly to the active site of the enzyme in the lowest free energy state and explained how the (S)-alcohol was produced exclusively in the reduction process.     

A gradually slowing travelling band of chemotactic bacteria

A model for describing the motion of chemotactic bacteria in a capillary tube containing substrate is treated. Chemotactic substrate threshold effects are included in the chemotactic response coefficient. The ratio of the substrate threshold, s _T, to the substrate level far ahead of the travelling band, s , is used as a small parameter in developing an asymptotic solution of near travelling wave form.Also at: Membrane Filtration Technology, Kiryat Weizmann, Rehovot, Israel.Also at: Department of Mathematical Sciences, Rensselaer Polytechnic Institute, Troy, NY 12181, USA.     

Oxidation of Various Substrates by Mitochondria as Related to the Concentration of Osmotics in the Medium

Wheat (Triticum aestivumL.) root mitochondria were used to investigate the effect of solute concentration in the medium on the rates of succinate, malate, -ketoglutarate, and glutamate oxidation; the corresponding values of the apparent Michaelis constant (K _M); and the changes in the organelle volumes. The oxidation rates and the K _M values for all substrates were lower in the 0.5 M sucrose solution than in the 0.3 M solution. Under high osmotic concentration, mitochondria did not shrink. On the contrary, in the absence of the substrate, mitochondria swelled more in the 0.5 M sucrose solution than in the 0.3 M solution. This effect was absent when the substrate was added. The authors conclude that the decrease in the oxidation rate imposed by sucrose was not related to the hindered influx of substrates caused by matrix contraction. Rather, the osmotic effect of sucrose is related to the changes in the structure of mitochondrial enzyme complexes.     

Optimization of Medium Components for Production of Cellulases by Melanocarpus sp. MTCC 3922 under Solid-state Fermentation

Summary The medium components for the production of extracellular cellulases by Melanocarpus sp. MTCC 3922 were optimized using solid-state fermentation. Melanocarpus sp. cultured in optimized medium containing 1.5% urea, and 0.12% KH₂PO₄ along with a trace element solution and surfactant (Tween 20), produced endoglucanase (142.4 U/g of substrate), Avicel-adsorbable endoglucanase (27.0 U/g of substrate), Avicelase (0.65 U/ g of substrate), FPase (39.9 U/g of substrate) and β-glucosidase (109.0 U/g of substrate) activities. The presence of sulphate ions in traces stimulated endoglucanase yields. The IEF fractionation of the crude proteins from Melanocarpus sp. showed the expression of 3, 1 and 11 isoforms of endoglucanase, β-glucosidase and xylanase, respectively.     

Yeast alcohol dehydrogenase immobilized in a glutaraldehyde–albumin matrix: Kinetics and cofactor diffusional effects*

This study was carried out to define how the overall rate of reaction would be influenced by different degrees of diffusional resistance to cofactor transport within an oxidoreductase membrane matrix. To accomplish this, 0.7–6.6μM yeast alcohol dehydrogenase was immobilized in an albumin matrix crosslinked with 2.5 or 5.0% glutaraldehyde to give 102–1685 μM thick membranes. The enzyme half-life was at least doubled at pH 7.5 or 8.8 on immobilization. Values of the kinetic constants for the soluble and immobilized enzyme were determined at 25°C and pH 8.8 over the range of 0.01–1.0M bulk solution concentration of ethanol as substrate and 140–1000μM bulk solution concentration of nicotinamide adenine dinucleotide (NAD⁺) as cofactor, to give essentially single substrate kinetics in NAD⁺. Equilibrium partitioning of ethanol and NAD⁺ between the solution and membrane was measured and used in the data analysis. The four kinetic constants for the soluble enzyme agreed with literature values; and all increased with immobilization of the enzyme. The Michaelis constants for NAD⁺ and for ethanol were greater for the immobilized enzyme. The diffusional resistance to NAD⁺ transport, presented in terms of the Thiele modulus, showed that the overall rate of reaction was decreased by about 50% even at values of the modulus as low as 2.0.     

Operation of packed-bed immobilized cell reactor featuring active β-galactosidase inclusion body-containing recombinant <Emphasis Type="Italic">Escherichia coli</Emphasis> cells

In this study, we developed a packed-bed immobilized cell reactor containing active β-gal (β-galactosidase) inclusion body (IB)-containing Escherichia coli (E. coli) cells in alginate beads. This packed-bed reactor was operated using a substrate feed solution 0.72 ∼ 38.4 mM ONPG (o-nitrophenyl-β-D-galactoside) prepared in Z buffer supplemented with chloroform and 0.1% SDS (sodium dodecyl sulfate). The production rate of ONP (o-nitrophenol) in the reactor containing cells that were incubated with α-MG (α-methyl D-glucospyranoside) or D-fucose after induction was superior to those prepared with cells that were not incubated with α-MG or D-fucose. The ONP production rate was increased proportionally with ONPG concentration in the substrate feed up to a concentration of 38.4 mM. However, as the ONPG concentration was increased in the substrate feed solution, galactose inhibition inside the alginate beads was increased. This most likely occurred due to problems with diffusion. In addition, partial breakage of alginate beads was observed during the later periods of operation. In this study, we demonstrated that active β-gal IB-containing E. coli cells were sustained in the immobilized cell reactor during operation. Particularly, these findings demonstrate the feasibility of using active IBs in an enzymatic reaction without the need for any purification step. In addition, we showed that these IB-containing cells could be directly used in an immobilized reactor.     

Keratinolysis by Absidia cylindrospora and Rhizomucor pusillus: biochemical proof

Absidia cylindrospora and Rhizomucor pusillus, causal agents of phycomycoses, were cultured on sterile natural keratins in a mineral solution and the keratin degradation products analyzed. The excess of sulphur was removed by oxidation to inorganic sulphate and thiosulphate, which were the main products of sulphitolysis of keratin. The proteolytic activity of the two fungi depended on the nature of the keratin substrate. Human scalp hair was the most favoured keratin substrate by both the fungi.     

Glucoamylase and glucose oxidase preparations and their combined application for conversion of maltose to gluconic acid

The kinetic behavior of a system of multiple enzyme in solution has been studied in a variable volume batch reactor at pH 5, controlled dissolved oxygen concentration, and T = 30°C. The enzymes used were glucoamylase (R. delemar), glucose oxidase (A. niger), and gluconolactonase (A. niger), all of which are important commercial biocatalysts, and a disaccharide was employed as the starting substrate. This study includes the basic kinetic properties of individual enzymes and interactions between components of the reaction mixture. Classical Michaelis–Menten single substrate or two substrate kinetic with parameters based on initial rate data predict correctly the batch time course of the sequential reaction network.     

Enhanced Asymmetric Reduction of Ethyl 3‐Oxobutyrate by Baker's Yeast via Substrate Feeding and Enzyme Inhibition

The moderate enantioselectivity of wild form baker's yeast can be considerably increased either by using continuous feeding to maintain a low substrate concentration throughout the reaction, or by the selective inhibition of competing enzymatic pathways. The reduction of ethyl 3‐oxobutyrate to ethyl (S)‐3‐hydroxybutyrate was used as a model reaction. With the substrate feeding method, the enantioselectivity could be increased from 75 % to as high as 98 %. The increased selectivity originates from the much higher substrate binding constant of the (R)‐specific enzymes, so that these enzymes remain essentially inactive if a low concentration of ethyl 3‐oxobutyrate is maintained in the bioreactor. Alternatively, the enantioselectivity of baker's yeast can be improved by selectively blocking competing enzymatic pathways. It was found that vinyl acetate is a selective inhibitor for the (R)‐specific enzymes. Ethyl (S)‐3‐hydroxybutyrate with an enantiomeric excess of 98 % was obtained by pre‐incubation of baker's yeast in 100 mM of vinyl acetate solution for 1 h. These results suggest that by selecting appropriate process conditions, natural baker's yeast can be a competitive biocatalyst for the large‐scale production of chiral secondary alcohols.     

The Structure of 5-Cyclohexyl-2′-Deoxyuridine as Studied by X-Ray Crystallography and NMR Spectroscopy

Abstract

5-Cyclohexyl-2′-deoxyuridine (I) is an example of a 5-substituted pyrimidine 2′-deoxynucleoside which exhibits no antiviral activity and which is not a substrate for either cellular or viral (herpes) kinases. Despite the fact that a cursory inspection of NMR spectra of the compound, taken in DMSO-d ₆ solution, suggested that the compound had a normal conformation, we here show that in the crystal and in aqueous solution (analysed by 2D NMR techniques), the conformation of this nucleoside has a syn-glycosidic and C4′-exo (₄E) sugar pucker conformation.