Effect of fluidic transport on the reaction kinetics in lectin microarrays

Lectins are the proteins which can distinguish glycosylation patterns. They are frequently used as biomarkers for progressions of several diseases including cancer. As the lectin microarray based prognosis devices miniaturize the process of glycoprofiling, it is anticipated that their performance can be augmented by integration with microfluidic framework. This is analogous to microfluidics based DNA arrays. However, unlike small oligonucleotide microarrays, it remains uncertain whether the binding reaction-kinetic parameters can be considered invariant of imposed hydrodynamics, for relatively larger and structure sensitive molecules such as lectins. Here we show, using two standard lectins namely Concanavalin A and Abrus Agglutinin, that the steady state binding efficiency unexpectedly declines beyond a critical shear rate magnitude. This observation can be explained only if the associated reaction constants are presumed to be functions of hydrodynamic parameters. We methodically deduce the shear rate dependence of association and dissociation constants from the comparison of experimental and model-simulation trends. The aforementioned phenomena are perceived to be the consequences of strong hydrodynamic perturbations, culminating into molecular structural distortion. The exploration, therefore, reveals a unique coupling between reaction kinetics and hydrodynamics for biomacromolecules and provides a generic scheme towards futuristic microfluidics-coupled biomedical assays.     

Macrophage-stimulating activity of exo-biopolymer from cultured rice bran with <Emphasis Type="Italic">Monascus pilosus</Emphasis>

To find a new use of rice bran, five fungi were examined for the production of exo-biopolymer with macrophage-stimulating activity from rice bran. Among the exo-biopolymers produced from the cultures, Monascus pilosus had the most potent macrophage stimulating activity in a liquid culture rather than in a solid culture. In order to improve the yield of exo-biopolymer with macrophage-stimulating activity, a suitable medium for exo-biopolymer was tested in submerged culture of M. pilosus. The highest amount of exo-biopolymer (13.9 mg/mL) was obtained in a medium containing rice bran as an only carbon source followed by media with additional maltose and sucrose (13.8 and 13.7 mg/mL, respectively). The addition of peptone resulted in the production of high amount of exo-biopolymer (15.1 mg/mL), meanwhile the addition of ammonium chloride resulted in 264.0 μg/mL of glucosamine content. Among eight different kinds of inorganic salts tested, potassium phosphate (0.1%) was the most effective inorganic salt for the mycelial growth and exo-biopolymer production. Therefore the optimal medium composition was as follows (g/L): 20 g of rice bran, 5 g of peptone, and 1 g of KH₂PO₄. The optimal culture pH and time for mycelial growth and exo-biopolymer production was pH 5.0 and 25°C, respectively. The maximum exo-biopolymer (20.1 mg/mL) was observed at the fourth day of cultivation. Exo-biopolymer, a crude polysaccharide fraction, mainly contained neutral sugar (81.8%) with considerable amounts of uronic acid (18.2%). Component sugar analysis showed that the active fraction consisted mainly of arabinose, galactose, glucose, which was digested from starch of rice bran during cultivation, and uronic acid (molar ratio; 0.8:1.0:0.7:0.8).     

Substrate specificity of yeast invertase in transglycosylation reactions

The substrate specificity of purified yeast invertase isolated fromSaccharomyces cerevisiae in transglycosylation reactions was determined. The enzyme is specific for primary alcohols. The yeast activity is a function of the alkyl length and substrate hydrophobicity (n-butyl, isobutyl, isoamyl alcohols).Translated from Khimiya Prirodnykh Soedinenii, No. 1, pp. 69–70, January–February, 2000.     

Studies on the overproduction of indole-containing metabolites by a methanol-utilizing yeast

Production of indole-containing metabolites (“indoles”) from methanol has been studied using a mutant ofHansenula polymorpha resistant to 5-fluorotryptophan. Whereas the wild-type culture produces only a small amount of indoles, the mutant is partially deregulated and overproduces indoles. Indoles production was studied in batch and continuous culture and in a washed-cell system. When the pH was above 4.0, indoles production was growth-associated, in both minimal and complex media, and batch or continuous culture. When the pH was below or equal to 4.0, a low phosphate concentration was found to improve production. In a phosphate-deficient washed-cell suspension system, the addition of an amino acid such as methionine at 5 mM increased specific productivity by more than 60%. Addition of cycloheximide at 50 mg/L decreased residual growth and increased maximum productivity of indoles by more than 60%. When the antibiotic was added at 1000 mg/L, growth was completely inhibited and indoles production continued for about 35 h.     

Effect of temperature on growth of psychrophilic and psychrotrophic members of Rhodotorula aurantiaca

The thermodependence of growth kinetic parameters was investigated for the Antarctic psychrophilic strain Rhodotorula aurantiaca and a psychrotrophic strain of the same species isolated in Belgium (Ardennes area). Cell production, maximum growth rate (μ_max), and half-saturation constant for glucose uptake (Ks) of both yeasts were temperature dependent. For the two yeasts, a maximum cell production was observed at about 0°C, and cell production decreased when temperature increased. The μ_max values for both strains increased with temperature up to a maximum of 10°C for the psychrophilic strain and 17°C for the psychrotrophic strain. For both yeasts, Ks for glucose was relatively constant at low temperatures. It increased at temperatures above 10°C for the psychrophilic strain and 17°C for the psychrotrophic strain. Although its glucose affinity was lower, the psychrotrophic strain grew more rapidly than the psychrophilicone. The difference in growth rate and substrate affinity was related to the origin of the strain and the adaptation strategy of R. aurantiaca to environmental conditions.     

Calorimetric detection of the toxic effect of androgens on fission yeast

Calorimetry is evaluated for study of the toxic effect of environmental androgens on Schizosaccharomyces pombe cells. The results indicate that androstendione, androstandiendione and dehydrotestosterone inhibited S. pombe heat production rate. Although, the turbidimetric method showed that testosterone (TS) had no influence on growth of S. pombe, calorimetry revealed that there was a shift in growth period in samples with TS.     

Effect of solvent on the kinetics of arylsulfonylation of N-alkylated anilines in a water-propan-2-ol system

The effect of the composition of a water-propan-2-ol solvent on the kinetics of arylsulfonylation of N-ethyl-, N-isopropyl-, and N-butylanilines with 3-nitrobenzenesulfonyl chloride at 298 K was studied. The reaction rate constant increases monotonically with an increase in the water fraction in the solvent from 5 to 30 wt.%. The apparent activation parameters of the reaction of N-butylaniline with 3-nitrobenzenesulfonyl chloride were calculated. No considerable changes in the activation parameters of the reaction were observed on going from pure propan-2-ol to a 10% aqueous solution, which indicates that the mechanism remains unchanged upon solvent change. Propan-2-ol with a water content of 5–30 wt.% can be used in the synthesis of arylsulfonylation products of the amines under study in 98–99% yield. Published in Russian in Izvestiya Akademii Nauk. Seriya Khimicheskaya, No. 6, pp. 937–939, June, 2006.     

Mpp7 controls regioselective Knoevenagel condensation during the biosynthesis of Monascus azaphilone pigments

Targeted inactivation of the mpp7 gene in the Monascus azaphilone pigment (MAzP) biosynthetic gene cluster resulted in the accumulation of monasfluol A (7) and B (8), of which the latter was a novel compound, and the abolition of the main MAzPs. It is thus proposed that 7 and 8 are the products of non-enzymatic Knoevenagel condensation followed by a reduction and that Mpp7 assists in regioselective Knoevenagel aldol condensation during MAzP biosynthesis.     

Effect of ion and surfactant choice on the recovery of a histidine-tagged protein from tobacco extract using foam fractionation

Tobacco plants can be used for the production of proteins for pharmaceutical applications. One of the most difficult and expensive tasks associated with this technology is isolating the product of interest from the hundreds of other chemicals found in tobacco. We describe a new recovery strategy in which the protein of interest is “tagged” with a histidine structure, which forms a complex with metal ions and a surfactant that will accumulate in the foamate of a foam fractionation step. His-gus, a histidine-tagged enzyme, was selectively recovered in the presence of two different surfactants and two different metal ions. The foam fractionation with N-∈-dodecylamido-N-α, N-α,-bis(carboxymethyl)-l-lysine surfactant and Ni²⁺ ions resulted in an average His-gus activity recovery value of 88% and an activity enrichment of 2.27. The performance of the recovery strategy without tobacco extract resulted in an average activity recovery value of 63.32% and an average activity enrichment value of 5.16, utilizing lauroylethylenediaminetriacetate surfactant and Ni²⁺ ions. It was shown that even though a majority of the native tobacco proteins are removed during the prefoaming step, the presence of tobacco extract does affect the recovery of His-gus.     

growth kinetics ofbacillus stearothermophilus BR219

Bacillus stearothermophilus BR219, a phenol-resistant thermophile, can convert phenol to the specialty chemical catechol. The growth kinetics of this organism were studied in batch, continuous, and im-mobilized-cell culture. Batch growth was insensitive to pH between 6.0 and 8.0, but little growth occurred at 5.5. In continuous culture on a dilute medium supplemented with 10 mM phenol, several steady states were achieved between dilution rates of 0.25 and 1.3 h^-1. Phenol degradation was found to be uncoupled from growth. Immobilized cells grew rapidly in a rich medium, but cell viability plummeted following a switch to a dilute medium supplemented with 5 mM phenol.     

Nutritional profile of food yeast kluyveromyces fragilis biomass grown on whey

Biomass of food yeast Kluyveromyces fragilis (MTCC 188) grown on deproteinized whey supplemented with 0.8% diammonium hydrogen phosphate and 10 ppm indole-3-acetic acid, had a crude protein content of 37%. The true protein content based on nitrogen fractionation procedure was 28.1%. Total nucleic acid content was 4.82%. This amount does not appear to be toxicologically offensive. Crude fiber, ash, and lipid content of K. fragilis dry cells were found to be 4.9%, 16%, and 7.8%, respectively. Essential fatty acids of both ω-3 and ω-6 series were found present in the fat of the yeast and represented 21.5% of the total fatty acids. All the essential amino acids were present in the proteins of K. fragilis; however, sulfur containing amino acids were found in lower amounts. Calculated protein scores indicate moderate biological value. Bvitamins in the biomass were present as expected, but folic acid and pyridoxine were present in high concentration.     

Growth ofschizosaccharomyces pombe on glucose-malte mixtures in continuous cell-recycle cultures kinetics of substrate utilization

The aerobic growth ofSchizosaccharomyces pombe on mixtures of glucose and malate was investigated during continuous high cell density cultures with partial cell-recycle using a membrane bioreactor. Determination of the specific metabolic rates relative to substrates and products allowed the capacity of the yeast to metabolize malic acid under both oxidative metabolism (carbon limited cultures) and oxidofermentative metabolism (carbon sufficient cultures) situations to be characterized. Under carbon limiting conditions, the specific rate of malate utilization was dependent on the residual concentration and a limit for a purely oxidative breakdown without ethanol formation was observed for a characteristic ratio between the rates of substrate consumption qm/qg of 1.63 g.g^-1. In addition, the mass balance analysis revealed the incorporation of malic acid into biomass. In carbon excess environments, the specific rate of malate utilization was dependent on both the residual malate and the specific rate of glucose consumption indicating that in addition to its conversion into ethanol malate can be respiratively metabolized for qm/qg ratios higher than 0.4 g.g^-1.     

A novel biological strategy for morphology control of PbWO4 crystals in tomato extract

PbWO(4) crystals were readily synthesized by reacting lead oxide (PbO(2)) with sodium tungstate (Na(2)WO(4)·2H(2)O) at room temperature in the presence of tomato extract. Biomolecules such as vitamins, proteins in the extract played both the roles of reductant and template. The size and morphology of the PbWO(4) crystals could be controlled by adjusting the concentration of the reactants. When the concentration ratio of PbO(2) and Na(2)WO(4) was increased, the morphologies of the products varied from spherical to fusiform. Room-temperature fluorescence spectra indicated the products had a slight blue shift compared to the solid structure, which may be due to the structure defects in the crystals. The possible mechanism of PbWO(4) crystal growth in tomato extract was discussed.     

Effect of alkali additives on the kinetics of WO3+CCl4 reaction

The chlorination kinetics of alkali-added (K and Li) tungsten trioxide were studied by thermogravimetry, using gaseous CCl₄ as chlorinating agent. The reactivity of the modified samples was compared to the results on the chlorination of pure WO₃. Similar apparent activation energies were found for the pure and alkali-added samples. However, potassium additive resulted in a strong decrease of the initial reaction rate, while surface lithium has no influence on it.During the chlorination a continuous decrease of the linear reaction rate was observed for both samples, which was explained by retarding effects of surface by-products and alkali additives. For describing the isothermal TG curves an appropriate kinetic model, based on the monotonously increasing inhibition of these species was assumed. The curve calculated with this model fits well to the experimental results.

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Zusammenfassung Mittels Thermogravimetrie wurde die Chlorierungskinetik von alkaliversetztem (K und Li) Wolframtrioxid untersucht, wobei CCl₄ als Chlorierungsreagens fungierte. Die Reaktivität der modifizierten Proben wurden mit der von reinem WO₃ verglichen. Für die reinen und für die alkaliversetzten Proben wurden ähnliche scheinbare Aktivierungsenergien gefunden. Der Zusatz von Kalium verursacht jedoch eine starke Abnahme der ursprünglichen Reaktionsgeschwindigkeit, während oberflächiges Lithium keinen Effekt zeigt.Während der Chlorierung wurde eine ständige Senkung der linearen Reaktionsgeschwindigkeit für beide Proben festgestellt, was mit Rückhalteeffekten von oberflächigen Nebenprodukten und Alkalizusätzen erklärt wird. Zur Beschreibung der isothermen TG-Kurven wurde ein entsprechendes kinetisches Modell angenommen, welches auf einer monoton steigenden Inhibition dieser Proben basiert. Die auf der Grundlage dieses Modelles berechnete Kurve stimmt recht gut mit den experimentellen Ergebnissen überein.

     

Temperature and pH effects on the kinetics of 2-aminophenol auto-oxidation in aqueous solution

The kinetics of the auto-oxidation of 2-aminophenol (OAP) to 2-amino-phenoxazin-3-one (APX) was followed in air-saturated aqueous solutions and the influence of temperature and pH on the auto-oxidation rate was studied. The kinetic analysis was based on a spectrophotometric method following the increase of the absorbance of APX. The process follows first order kinetics according to the rate law—d[OAP]/dt=k′[OAP]. The experimental data, within the pH range 4–9.85, were analyzed using both differential and incremental methods. The temperature variation of the overall rate constant was studied at pH=9.85 within the range 25–50°C and the corresponding activation energy was evaluated.     

The thermal decomposition of N , N -dimethyl-3-oxaglutaramic acid and the kinetics of its second-stage thermal decomposition reaction

N,N-dimethyl-3-oxa-glutaramic acid was purified and characterized by ¹H-NMR, Fourier transform infrared spectroscopy (FT-IR) and elemental analysis. The thermal decomposition of the title compound was studied by means of thermogravimetry differential thermogravimetry (TG-DTG) and FT-IR. The kinetic parameters of its second-stage decomposition reaction were calculated and the decomposition mechanism was discussed. The kinetic model function in a differential form, apparent activation energy and pre-exponential constant of the reaction are 3/2 [(1−α)^1/3−1]⁻¹, 203.75 kJ·mol⁻¹ and 10^17.95s⁻¹, respectively. The values of ΔS ^≠, ΔH ^≠ and ΔG ^≠ of the reaction are 94.28 J·mol⁻¹·K⁻¹, 203.75 kJ·mol⁻¹ and 155.75 kJ·mol⁻¹, respectively. Supported by the National Natural Science Foundation of China (Grant No. 20106009)     

Modeling in biological chemistry. From biochemical kinetics to systems biology

A brief review on biochemical kinetics in the twentieth century mainly concerned with enzyme kinetics and cooperative processes is presented. Molecular biology and, in particular, structural biology provided the basis for modeling biological phenomena at the molecular level. Structure was recognized as the ultimate and only level at which biological processes find an explanation that is satisfactory for chemists and physicists. A new epoch in biology was initiated by successful extensions of the molecular approach from individual molecules and reactions to the cellular and organismic level. Starting with sequencing of whole genomes in the 1980s more and more techniques became available that are suitable for upscaling from molecules to cells. A series of research programs was initiated: genomics dealing with sequencing the DNA of whole organisms, proteomics considering all proteins of a cell and their interactions, metabolomics studying all metabolic reactions of a cell or an organism, and functional genomics or systems biology aiming at an exploration of the dynamics of complete biological entities. At the same time computational facilities have experienced an unexpected development in speed of calculations and storing devices. At present computer simulations of whole cells at molecular resolution are within reach. The challenge for the theorist in biology is to develop methods for handling the enormously complex networks of gene regulation and metabolism in such a way that biological questions can be addressed. This goal cannot be achieved by dynamical systems theory alone. What is needed is a joint effort from different mathematical disciplines supported by empirical knowledge and tools from discrete mathematics to informatics. Two sections with selected examples from our own laboratory dealing with structural bioinformatics of RNA and with a dynamical systems approach to gene regulation are added.     

The kinetics of poly(N-isopropylacrylamide) microgel latex formation

Conversion versus time curves were measured for poly(N-isopropylacrylamide) microgel latexes prepared by polymerization in water with sodium dodecyl sulfate, SDS. Polymerization rates increased with temperature with methylenebisacrylamide crosslinking monomer consumed faster thanN-isopropylacrylamide. The particle diameter decreased with increasing concentrations of SDS in the polymerization recipe and there was evidence that the rate of polymerization increased somewhat with SDS concentration. Particle formation occurred by homogeneous nucleation as micelles were absent.Comparison of particle size distributions from dynamic light scattering to those from a centrifugal sizer led to the conclusion that larger particles within a specific latex were less swollen with acetonitrile than were the smaller ones. This was interpreted as evidence for the polymer in larger particles having a higher crosslink density. Particle swelling was estimated from swelling ratios defined as the particle volume at 25 °C divided by the volume at 50 °C. In the absence of crosslinking poly(N-isopropylacrylamide) linear chains would disolve at 25 °C. The swelling results indicated that the average crosslink density in the particles decreased with conversion. This was explained by the observation that the methylenebisacrylamide was consumed more quickly and is typical of crosslinking in emulsion polymerization where polymer particles have high polymer concentrations at their birth.     

橙色红曲菌As3.4384及其诱变体发酵产物中的桔霉素和色素的高效液相色谱研究

对橙色红曲菌As3.4384及其诱变菌株大米固态发酵物中桔霉素和色素的HPLC分离进行了优化,采用Symmetry C18(250mm×4.6mmi.d.,5μm)色谱柱,流动相为乙腈-水(磷酸调pH3.0,77∶23,V/V),分别采用紫外检测器(UV=254nm)、荧光检测器(λex=331nm,λem=500nm)及两者串联对桔霉素和色素进行了监测。通过ESI-MS对色素进行了鉴定,并测定了经橙色红曲菌As3.4384及其诱变菌株于30℃发酵14d后的大米、糯米、小黄米、燕麦米和红米中桔霉素含量。结果表明:ESI-MS鉴定的两种色素分别为红曲菌素和红曲黄素,橙色红曲菌As3.4384发酵后的大米、糯米、小黄米、燕麦米和红米中桔霉素含量分别为151.4、100.5、5.06、80.5和150.5mg/kg,而诱变菌株发酵后的大米、糯米、小黄米、燕麦米和红米中桔霉素含量分别为0.218、3.86、0.223、2.45和4.05mg/kg。     

The effect of cell density on the production of xylitol fromd-xylose by yeast

The rate of xylitol production from D-xylose increased with increasing yeast cell density. The optimal temperature for xylitol production is 36‡ C, and the optimal pH range is from 4.0 to 6.0. At high initial yeast cell concentration of 26 mg/mL, 210 g/L of xylitol was produced from 260 g/L of D-xylose after 96 h of incubation with an indicated yield of 81% of the theoretical value.