Aerosol disinfection of bacterial spores by peracetic acid on antibacterial surfaces and other technical materials.

BACKGROUND: The effectiveness of aerosol disinfection processes based on peracetic acid (PAA) might differ depending on the surface targeted. Especially antibacterial, oligodynamic materials have to be regarded as they could cause elevated decomposition of PAA. AIM: This study aimed on the determination of differences in disinfection effectiveness using PAA caused by the treated material. METHODS: Aerosol disinfection of Geobacillus stearothermophilus spores was performed on the antibacterial, oligodynamic materials copper and brass in comparison to stainless steel and aluminium as well as polyvinylchloride, wood, and ceramics. Additionally, the influence of the materials on the decomposition reaction of PAA was evaluated. RESULTS: For aluminium and stainless steel as well as ceramics and polyvinylchloride, a disinfection of 106 spores of Geobacillus stearothermophilus on 40 cm² was obtained by the employment of 60 mL aerosolized disinfectant/m³ on laboratory scale (0.5 m³). For the application on the oligodynamic materials copper and brass an over 30% higher amount of disinfectant was necessary to achieve significant disinfection results, than for the other material surfaces. In contrast to aluminium and stainless steel, copper and brass caused elevated decomposition of PAA what seems to be the reason for the lowered disinfection effectiveness. CONCLUSIONS: Applying aerosol disinfection processes, in addition to parameters such as room size and geometry, the treated materials have to be considered when determining the necessary amount of disinfectant.

Functional characteristics, nutritional value and industrial applications of Madhuca longifolia seeds: an overview.

New sustainable edible oil sources are desired to achieve supply chain flexibility and cost saving opportunities. Non-traditional fruit seeds are being considered because their constituents have unique chemical properties and may augment the supply of nutritional and functional products. Madhuca longifolia Syn. M. indica (Sapotaceae) is an important economic tree growing throughout the subtropical region of the Indo-Pak subcontinent. Information concerning the exact composition of mahua butter (known also as mowrah butter) from fruit-seeds of buttercup or Madhuca tree is scare. Few studies investigated mahua butter for its composition, nutritional value, biological activities and antioxidative properties. In consideration of potential utilization, detailed knowledge on the chemical composition, nutritional value and industrial applications of mahua butter is of major importance. The diversity of applications to which mahua butter can be put gives this substance great industrial importance. This review summarizes recent knowledge on bioactive compounds, functional properties as well as food and non-food industrial applications of mahua butter. Graphical abstractᅟ.

Enzyme catalysis in organic solvents: influence of water content, solvent composition and temperature on Candida rugosa lipase catalyzed transesterification.

In the present study the influence of water content, solvent composition and reaction temperature on the transesterification of 1-phenylpropan-2-ol catalyzed by Candida rugosa lipase was examined. Reactions were carried out in different mixtures of hexane and tetrahydrofurane. The studies showed that an increasing water content of the organic solvent results in an increasing enzyme activity and a decreasing enantiomeric excess. Furthermore, a significant influence of the solvent hydrophilicity both on the enzyme activity and on the enantiomeric excess was found. An increase in solvent hydrophilicity leads to a decrease of enzyme activity and an increase of the enantiomeric excess. This indicates that the enzyme becomes more selective with decreasing flexibility. Similar effects were found by variation of the reaction temperature. Taken together, the decrease in conversion and the increase in selectivity with increasing solvent hydrophilicity are induced by the different water contents on the enzyme surface and not by the solvent itself.

Preparation and characterization of PEGyated Concanavalin A for affinity chromatography with improved stability.

In order to improve its stability, immobilized Concanavalin A (Con A) on Toyopearl adsorbents was conjugated with monomethoxy poly(ethylene glycol) succinimidyl propionate (mPEG-SPA) with different molecular weight. A colorimetric method using ninhydrin is proposed to determine the degree of PEGylation; this method has proved to be easy applicable and reproducible. The PEGylation reaction was studied in detail to elucidate how parameters such as molar ratio of mPEG-SPA to Con A and molecular weight of mPEG-SPA affect the degree of PEGylation. The adsorption isotherms of glucose oxidase (GOD) onto native and PEGylated Con A adsorbents showed that the modification did not alter substantially the specificity of the carbohydrate binding ability of Con A. However, the binding capacity for GOD was slightly reduced probably due to the steric hindrance caused by mPEG chains. Adsorption kinetic studies revealed a lower adsorption rate after PEGylation which was attributed to the steric effect. The dynamic adsorption capacity for modified Con A depended very much on the degree of PEGylation and the molecular weight of mPEG derivatives. The adsorption capacity could be highly preserved for Toyopearl Con A modified by mPEG2k (90% of the original adsorption capacity) even with a degree of PEGylation up to 20% (the ratio of primary amino groups of PEGylated immobilized Con A to that of native immobilized Con A). Studies show that the binding capacity of PEGylated Con A was highly preserved under mild process conditions. PEGylated Con A also exhibited obviously higher stability against more stressful conditions such as the exposure to organic solvents and high temperatures. Conjugation of Con A with mPEG2k provided better adsorption performance thus has greater potential for application in affinity separation processes compared with mPEG5k. The fact that PEGylation stabilizes the properties of Con A may greatly expand the range of applications of unstable proteins to bioprocessing (e.g. biocatalysis and downstream separation) as well as other protein applications (e.g. medication, industrial use, etc.).

Influence of reaction conditions on the enantioselectivity of biocatalyzed C-C bond formations under high pressure conditions.

Benzoylformate decarboxylase (BFD, EC 4.1.1.7) is a homotetrameric thiamine diphosphate (ThDP)-dependent enzyme which catalyzes the synthesis of chiral 2-hydroxyketones accepting a broad range of aldehydes as substrates. In this study the synthesis of 2-hydroxypropiophenone (2-HPP) from benzaldehyde and acetaldehyde was catalyzed by three BFD variants namely BFD F464I, BFD A460I and BFD A460I-F464I. This paper reports the effect of hydrostatic pressure up to 290 MPa when the reactions were carried out at different benzaldehyde concentrations (5-40 mM) as well as at different pH values (7.0-8.5). Acetaldehyde concentration was fixed at 400 mM in all biotransformations. Reactions performed at high benzaldehyde concentrations and at high hydrostatic pressures showed an increase in (R)-2-HPP formation catalyzed by all BFD variants. For BFD A460I-F464I we observed an increase in the ee of (R)-2-HPP up to 80%, whereas at atmospheric conditions this variant synthesizes (R)-2-HPP with an ee of only 50%. Alkaline conditions (up to pH 8.5) and high hydrostatic pressures resulted in an increase of (R)-2-HPP synthesis, especially in the case of BFD A460I and BFD F464I.

Immobilization and characterization of benzoylformate decarboxylase from Pseudomonas putida on spherical silica carrier.

If an adequate biocatalyst is identified for a specific reaction, immobilization is one possibility to further improve its properties. The immobilization allows easy recycling, improves the enzyme performance, and it often enhances the stability of the enzyme. In this work, the immobilization of the benzoylformate decarboxylase (BFD) variant, BFD A460I-F464I, from Pseudomonas putida was accomplished on spherical silica. Silicagel is characterized by its high mechanical stability, which allows its application in different reactor types without restrictions. The covalently bound enzyme was characterized in terms of its activity, stability, and kinetics for the formation of chiral 2-hydroxypropiophenone (2-HPP) from benzaldehyde and acetaldehyde. Moreover, temperature as well as pressure dependency of immobilized BFD A460I-F464I activity and enantioselectivity were analyzed. The used wide-pore silicagel shows a good accessibility of the immobilized enzyme. The activity of the immobilized BFD A460I-F464I variant was determined to be 70% related to the activity of the free enzyme. Thereby, the enantioselectivity of the enzyme was not influenced by the immobilization. In addition, a pressure-induced change in stereoselectivity was found both for the free and for the immobilized enzyme. With increasing pressure, the enantiomeric excess (ee) of (R)-2-HPP can be increased from 44% (0.1 MPa) to 76% (200 MPa) for the free enzyme and from 43% (0.1 MPa) to 66% (200 MPa) for the immobilized enzyme.

Interaction of cellulose-based cationic polyelectrolytes with mucin.

Mucoadhesivity of water-soluble polymers is an important factor, when testing their suitability for controlled drug delivery systems. For this purpose, the interaction of new cationic cellulose polyelectrolytes with lyophilized mucin was investigated by means of turbidimetric titration, microscopy and measurement of zeta potential and particle size changes in the system. Results show that the cellulose derivatives interact with mucin. This interaction became stronger if cellulose macromolecules contained positively charged groups and an electrostatic interaction with the negatively charged mucin particles occurred. Under certain conditions flocculation of mucin particles by the cellulose polyelectrolyte was observed.

Influence of the hydrostatic pressure and pH on the asymmetric 2-hydroxyketone formation catalyzed by Pseudomonas putida benzoylformate decarboxylase and variants thereof.

Benzoylformate decarboxylase (BFD) from Pseudomonas putida is a thiamine diphosphate-dependent (ThDP) enzyme that catalyzes the asymmetric C--C bond formation to (S)-2-hydroxypropiophenone [(S)-HPP] starting from benzaldehyde and acetaldehyde. The enantioselectivity of BFD was shown to be a function of temperature and substrate concentration. It can additionally be changed by site-directed mutagenesis on hot spot positions in the active site. In this article, we present the effect of hydrostatic pressure up to 250 MPa on the enantioselectivity for the recombinant wtBFD as well as for the variants BFD F464I, BFD A460I, and BFD A460I-F464I. A general tendency toward lower amounts of (S)-HPP could be observed at increasing pressures. For two of these variants an increase in pressure even caused an inversion in the enantioselectivity and thus increasing enantiomeric excesses, respectively. A pressure-induced increase in enantioselectivity could therefore be observed for the first time in biocatalysis to the best of our knowledge. Furthermore, the pH is shown to be a parameter that also significantly influences the enantioselectivity of the reaction mentioned above.

Small angle neutron scattering as sensitive tool to detect ligand-dependent shape changes in a plant lectin with beta-trefoil folding and their dependence on the nature of the solvent.

The galactoside-specific Viscum album L. agglutinin (VAA) is a potent biohazard akin to ricin and a mitogen for immune and tumor cells. These activities depend on cell surface binding to glycans. It is an open question whether the process of ligand binding alters the lectin's shape. Small angle neutron scattering (SANS) experiments revealed that the carbohydrate ligand lactose induced a decrease of the radius of gyration of dimeric VAA from 54.5 +/- 1 to 49.5 +/- 1 A in water. Apparently, VAA in aqueous solution and at the concentrations tested at 3.6 mg/ml and above adopts a compacted structure as response to ligand binding. In contrast to the behavior in aqueous solution, lactose binding in DMSO resulted in an increase of the lectin's radius of gyration from 49 +/- 1 to 55.5 +/- 1 A. Because shape changes may be reflected in the thermostability of the protein, this parameter was examined by activity assays of protein exposed to 60 degrees C and 70 degrees C and by differential scanning calorimetry (DSC). In line with the lactose-induced conformational alterations revealed by the SANS experiments, lactose presence enhanced the thermostability of VAA in water. Thus, binding of the carbohydrate ligand in solution can entail changes in shape and thermostability in the case of the tested plant lectin.

Evaluation of two different Concanavalin A affinity adsorbents for the adsorption of glucose oxidase.

Concanavalin A (Con A) was selected as ligand and thus immobilized onto two different supports, namely the polymeric Toyopearl and the inorganic silica, with the protection of its binding sites provided during the coupling procedure. The prepared Con A affinity adsorbents were then employed to evaluate their adsorption behaviour for the enzyme glucose oxidase (GOD). The immobilization kinetics showed that the immobilization of Con A on silica supports was much faster than that on Toyopearl supports, which could highly reduce the possibility of the denaturation of Con A. The optimal adsorption conditions for binding of GOD onto the ligand were determined in terms of the pH value and the ionic strength of the adsorption medium. The adsorption isotherms for binding GOD onto two Con A affinity adsorbents fitted well with the Langmuir equation. The maximum adsorption capacity q(m) of Toyopearl Con A and silica Con A were 7.9 mg/ml and 4.9 mg/ml, with a dissociation constant K(d) of 4.8 x 10(-7)M and 2.6 x 10(-6)M, respectively. Due to the less diffusive resistance, silica Con A showed both higher adsorption and desorption rates for GOD when compared with Toyopearl Con A. The nonspecific adsorption of GOD was less than 8% for both end-capped Toyopearl and silica supports. The dynamic adsorption of GOD for five times repeated processes showed a high stability for both prepared adsorbents. All the results indicate a good suitability of both Con A adsorbents for affinity adsorption of GOD.

Molecular dynamics simulations of pea (Pisum sativum) lectin structure with octyl glucoside detergents: the ligand interactions and dynamics.

The mitogenic pea (Pisum sativum) lectin is a legume protein of non-immunoglobulin nature capable of specific recognition of glucose derivatives without altering its structure. Molecular dynamics simulations were performed in a realistic environment to investigate the structure and interaction properties of pea lectin with various concentrations of n-octyl-beta-d-glucopyranoside (OG) detergent monomers distributed inside explicit solvent cell. In addition, the diffusion coefficients of the ligands (OG, Ca2+, Mn2+, and Cl-) and the water molecules were also reported. The structural flexibility of the lectin was conserved in all simulations. The self-assembly of OG monomers into a small micelle at the hydrophobic site of the lectin was noticed in the simulation with 20 OG monomers. The interaction energy analysis concludes that the lectin was appropriately termed an adaptive structure. One or rarely two binding sites were observed at an instant in each simulation that were electrostatically favoured for the OG to interact with the surface amino acid residues. Enhanced binding of OG to the pea lectin was quantified in the system containing only Ca2+ divalent ions. Interestingly, no binding was observed in the simulation without divalent ions. Furthermore, the lectin-ligand complex was stabilized by multiple hydrogen bonds and at least one water bridge. Finally, the work was also in accordance with the published work elsewhere that the simulations performed with different initial conditions and using higher nonbonded cutoffs for the van der Waals and electrostatic interactions provide more accurate information and clues than the single large simulation of the biomolecular system of interest.

Chemical indices and methods of multivariate statistics as a tool for odor classification.

Industrial and agricultural off-gas streams are comprised of numerous volatile compounds, many of which have substantially different odorous properties. State-of-the-art waste-gas treatment includes the characterization of these molecules and is directed at, if possible, either the avoidance of such odorants during processing or the use of existing standardized air purification techniques like bioscrubbing or afterburning, which however, often show low efficiency under ecological and economical regards. Selective odor separation from the off-gas streams could ease many of these disadvantages but is not yet widely applicable. Thus, the aim of this paper is to identify possible model substances in selective odor separation research from 155 volatile molecules mainly originating from livestock facilities, fat refineries, and cocoa and coffee production by knowledge-based methods. All compounds are examined with regard to their structure and information-content using topological and information-theoretical indices. Resulting data are fitted in an observation matrix, and similarities between the substances are computed. Principal component analysis and k-means cluster analysis are conducted showing that clustering of indices data can depict odor information correlating well to molecular composition and molecular shape. Quantitative molecule describtion along with the application of such statistical means therefore provide a good classification tool of malodorant structure properties with no thermodynamic data needed. The approximate look-alike shape of odorous compounds within the clusters suggests a fair choice of possible model molecules.

Detection, isolation and partial characterization of an immunostimulating glycoprotein from Rhodococcus fascians.

In a search for novel immunostimulating substances we detected that culture supernatants of the gram-positive phytopathogenic bacterium, Rhodococcus fascians, were able to induce cytokine release (TNF(alpha)) from mouse peritoneal macrophages. Monoclonal antibodies were generated against the active principle, and were employed for its isolation and partial characterization as a high molecular (MW>100 kDa) glycoprotein. In addition, methods practicable for its biotechnological preparation and several ELISA variants for its determination were developed.

Molecular dynamics characterization of n-octyl-beta-D-glucopyranoside micelle structure in aqueous solution.

n-Octyl-beta-D-glucopyranoside (OG) is a non-ionic glycolipid, which is used widely in biotechnical and biochemical applications. All-atom molecular dynamics simulations from two different initial coordinates and velocities in explicit solvent have been performed to characterize the structural behaviour of an OG aggregate at equilibrium conditions. Geometric packing properties determined from the simulations and small angle neutron scattering experiment state that OG micelles are more likely to exist in a non-spherical shape, even at the concentration range near to the critical micelle concentration (0.025 M). Despite few large deviations in the principal moment of inertia ratios, the average micelle shape calculated from both simulations is a prolate ellipsoid. The deviations at these time scales are presumably the temporary shape change of a micelle. However, the size of the micelle and the accessible surface areas were constant during the simulations with the micelle surface being rough and partially elongated. Radial distribution functions computed for the hydroxyl oxygen atoms of an OG show sharper peaks at a minimum van der Waals contact distance than the acetal oxygen, ring oxygen, and anomeric carbon atoms. This result indicates that these atoms are pointed outwards at the hydrophilic/hydrophobic interface, form hydrogen bonds with the water molecules, and thus hydrate the micelle surface effectively.

Evaluation of cellulose-based biospecific adsorbents as a stationary phase for lectin affinity chromatography.

Macroporous cellulose Granocel was evaluated as a matrix for the immobilization of two lectins Concanavalin A (ConA) (108 kDa) and Wheat Germ Agglutinin (WGA) (36 kDa). Two different methods were employed for the immobilization of the lectins via their protein moieties by a Schiff's bases reaction. One of them results in covalent coupling of the lectin directly to the support and the other gives the attachment through a long spacer arm which benefits the immobilization of voluminous ConA molecules. The adsorbents were characterized by the glycoproteins sorption recording adsorption kinetic data and isotherms. The adsorbents demonstrated high affinity to glycoproteins with a sorption capacity in the column up to 7.4 mg/ml support and a high recovery (up to 93%). The adsorption isotherms of glucose oxidase (GOD) onto ConA adsorbents reveals an adsorption behavior with high and low affinity binding sites. The dissociation constant K(d) of the ligand-sorbate complex is approximately 1 x 10(-6) and 0.4 x 10(-5)M, respectively. It was supposed that the second step is related to the sorption of solvated GOD onto already adsorbed GOD forming sorbate dimers.

Enhancement of enzymatic digestion of Antarctic krill and successive extraction of selenium organic compounds by ultrasound treatment.

In a previous work we described the isolation of selenium organic species from Antarctic krill after enzymatic hydrolysis. In this paper we present the results of the influence of ultrasonication on the enzymatic treatment and the successive isolation of selenomethionine. We showed that ultrasound-assisted enzymatic digestion leads to quantitative release of selenium in the soluble fraction and recovery of selenomethionine from the krill protein within a time 2 orders of magnitude shorter. The solubilised sample was analysed by size-exclusion chromatography and the selenomethionine content was quantified by high-performance liquid chromatography-inductively coupled plasma mass spectrometry. In total, 99% of the selenomethionine in the krill hydrolysate was recovered from the chromatographic fractions. It corresponds to 35% of the total selenium content in Antarctic krill. Monitoring by microscopy of the changes in the structure of the krill samples during ultrasonication suggested that the enhancement of the ultrasound-assisted enzymatic reaction was mainly due to decrease of mass transfer limitations. A reference experiment for ultrasound-assisted enzymatic digestion of cell-free protein in a homogeneous system does not exclude direct influence of the ultrasound energy on the enzyme-substrate interaction.

Comparison of modified supports on the base of glycoprotein interaction studies and of adsorption investigations.

The features of matrices, suitable for affinity chromatography, have been extensively investigated and got subject for several reviews. But these investigations show, that there is still a lack in adsorbent characterization and a demand of comparative investigations of adsorbents, based on different materials with a range of different surface functionalities. In this work the performance of self-prepared silica and cellulose-based adsorbents were compared with commercially available polymeric supports. A model system was chosen comprising the lectins concanavalin A (ConA) and wheat germ agglutinin (WGA), which were covalently attached to the support matrices, combining the selectivity of the lectin-sugar interaction with the chemical and mechanical properties of the support that influence the efficacy of the prepared adsorbent. The verification of the different supports provides information about tayloring carbohydrate specific lectin adsorbents. The characterization outlines the main features of the different adsorbents and takes into account the properties of the pure supports. It encompasses immobilization kinetics and isotherms as well as the description of the binding capacity of the adsorbents by depicting adsorption isotherms. The separation performances were also investigated in terms of glycoprotein purification factors and recoveries. Further, detailed information about binding of GOD to immobilized ConA are obtained.

Isolation of selenium organic species from antarctic krill after enzymatic hydrolysis.

Total selenium content and its distribution in the soluble and insoluble protein-bound fractions obtained after aqueous extraction of antarctic krill samples were determined. About 26% of the total selenium (2.4 microg g-1 dry weight) was found in the supernatant; the rest was in the pellet. Isolation of low molecular selenium-containing fractions was also performed by enzymatic digestion of the protein, followed by size-exclusion chromatography in conjunction with atomic absorption spectrometry. From the applied various proteinases (pronase E, subtilisin Carlsberg, trypsin, chymotrypsin, proteinase and proteinase N from Bacillus subtilis and Novo 0.6 MPX enzyme), the treatment with pronase E led to best recovery of selenium. About 96% of the total Se was found in the hydrolysate, mainly in low molecular weight fractions. Eighty percent of the Se species were in fractions with molecular weights in the range of amino acids and short peptides. High-performance liquid chromatography/inductively coupled plasma mass spectrometry (HPLC-ICP-MS) allowed the identification of selenomethionine and the assumption that selenocystine or its derivatives were the main species in these fractions.

Decontamination of polyaromatic hydrocarbons from soil by steam stripping: mathematical modeling of the mass transfer and energy requirement.

For cleaning of contaminated soil from polyaromatic hydrocarbons (PAH), a thermal separation process is applied. The process uses superheated steam that is supplied through a nozzle together with a suspension (approximately 40% soil content) of the contaminated soil into a tube reactor. In the reactor, the soil suspension is vaporized, and the PAH are stripped from the soil at temperatures of 140-300 degrees C. In a cyclone, a solid-vapor separation is carried out, and after going through a condenser, a separation of the condensed water and the PAH is obtained. For improvement of the economical performance, a heat recovery is integrated. This is realized by preheating the water/stream supplied to the evaporator by cooling the vapor steam leaving the reactor. For the mathematical description of the process, the removal of the PAH from the soil is considered to take place by a desorption process. Sorption isotherms are measured by batch experiments and can be described by isotherms of Langmuir type. A dispersion model is used to describe the mass transfer of the process. The process is mathematically modeled for instationary and stationary operation. The simulation predicts the lowest energy consumption at a good cleaning performance at a steam-to-suspension ratio of 5.

Process development in affinity separation of glycoconjugates with lectins as ligands.

Process development for affinity separation is a crucial prerequisite for a successful biospecific isolation of biological active substances like glycoconjugates or enzymes. The functionalization of polymer and silica based adsorbents and their influence on the adsorption behaviour of the modified adsorbents are presented. Improvement of the immobilization conditions for different lectins lead to a stable binding of more than 90% within 4 h of ligands applied to the immobilization solution. The prepared adsorbents are characterized according to specificity, stability and capacity. The isolation of the glycoprotein fetuin from fetal calf serum with wheat germ agglutinin adsorbents and the purification of horseradish peroxidase with concanavalin A (Con A) adsorbent are described. The Langmuir model, using glucose oxidase as glycoprotein and Con A adsorbents, expresses the sorption behaviour. The fixed bed separation is represented by the dispersion model. The process simulation supports the process development evaluating design parameters and investigating and optimizing process conditions. The influence of the flow as well as the concentration of contaminants competing with the valuable product for the ligands on the separation performance are demonstrated and discussed.