Phase diagrams for dextran-PEG aqueous two-phase systems at 22°C

Summary We have determined phase diagrams at 22°C for the aqueous two-phase systems composed of dextran, polyethylene glycol, and water. The effects of polyethylene glycol and dextran molecular weight on phase separation are reported. These phase diagrams provide more complete data for dextran/PEG/water system, and will be needed for the correlation of biomolecule partitioning.     

Molecular level investigations of the inter- and intramolecular interactions of pH-responsive artificial triblock proteins

Intelligent materials that can undergo physical gelation in response to environmental stimuli have potential impacts in the bioengineering and biomedical fields where the entrapment of cellular or molecular species is desired. Here, we utilize atomic force microscopy (AFM) to perform molecular level investigations of designer artificial proteins that undergo physical gelation. These are engineered as triblock copolymers with independent interchain binding and solvent retention functions, namely, two terminal leucine zipper-like peptide sequences and a central alanylglycine rich sequence, respectively. AFM force measurements between probes and surfaces functionalized with molecules of this triblock protein revealed adhesive interactions that increased in average force and frequency as the pH was lowered from pH 11.2 to 7.4 to 4.5, reflecting an increase in the numbers of interacting molecular strands. In bulk solution, lowering the pH results in a viscous liquid to gel transition. The modular design of the triblock protein was also exploited for single molecule force spectroscopy investigations, which revealed altered intramolecular interactions in response to changes in pH. An increased understanding of the inter- and intramolecular forces involved in biomolecule driven gelation processes is not only of great fundamental interest in the study of the biomolecular systems involved but may also prove key in enabling the rational design of new generations of intelligent hydrogel systems.     

Magnetoresistive-based biosensors and biochips

Over the past five years, magnetoelectronics has emerged as a promising new platform technology for biosensor and biochip development. The techniques are based on the detection of the magnetic fringe field of a magnetically labeled biomolecule interacting with a complementary biomolecule bound to a magnetic-field sensor. Magnetoresistive-based sensors, conventionally used as read heads in hard disk drives, have been used in combination with biologically functionalized magnetic labels to demonstrate the detection of molecular recognition. Real-world bio-applications are now being investigated, enabling tailored device design, based on sensor and label characteristics. This detection platform provides a robust, inexpensive sensing technique with high sensitivity and considerable scope for quantitative signal data, enabling magnetoresistive biochips to meet specific diagnostic needs that are not met by existing technologies.     

Comparative Poliovirus Permeability of Silver, Polycarbonate, and Cellulose Membrane Filters

Three types of filters, silver, polycarbonate, and cellulose, were evaluated for permeability toward poliovirus suspended in water, salt-containing, and proteinaceous solvents. The ability of virus to pass through cellulose filters depended on the suspending medium; virus did not pass through cellulose filters in either water or salt solution, whereas the use of protein solutions increased filterability. The virus permeability of both the silver and polycarbonate filters was independent of the suspending medium. Apparently, pore size alone determined their permeability toward poliovirus, and electrostatic forces between filters and the particles being filtered did not appear to play a significant role. Both the silver and polycarbonate filters appear to be promising tools for the separation of viruses from contaminating bacteria and fungi.     

Biospecific fractionation matrices for sequence specific endonucleases

Fractionation of several type II specific restriction endonucleases was achieved by separation on two novel biospecific matrices. The matrices are pyran, a copolymer of divinyl ether of maleic anhydride, and Cibacron Blue F3GA, a blue dye commonly used for the calibration of molecular sieves. Both compounds are insolubilized by coupling to sepharose through a cyanogen bromide linkage and in their soluble form inhibit the restriction endonucleases which we have tested. These affinity matrices can be used to obtain restriction endonucleases from crude extracts after removal of nucleic acids. They have also proven to have a high capacity when used as subsequent steps in enzyme purification. Their additional advantage is the rapid development time and reusability of columns packed with the two matrices.     

Single-molecule fluorescence characterization in native environment

Single-molecule detection (SMD) with fluorescence is a widely used microscopic technique for biomolecule structure and function characterization. The modern light microscope with high numerical aperture objective and sensitive CCD camera can image the brightly emitting organic and fluorescent protein tags with reasonable time resolution. Single-molecule imaging gives an unambiguous bottom-up biomolecule characterization that avoids the "missing information" problem characteristic of ensemble measurements. It has circumvented the diffraction limit by facilitating single-particle localization to ~1 nm. Probes developed specifically for SMD applications extend the advantages of single-molecule imaging to high probe density regions of cells and tissues. These applications perform under conditions resembling the native biomolecule environment and have been used to detect both probe position and orientation. Native, high density SMD may have added significance if molecular crowding impacts native biomolecule behavior as expected inside the cell.     

Aqueous two-phase extraction for downstream processing of amyloglucosidase

A polymer/salt aqueous two-phase system has been successfully employed for separation and purification of amyloglucosidase. The effects of system pH, molecular weight of polymer and composition of the two-phase system on amyloglucosidase partition behaviour in polyethylene glycol (PEG 4000, 6000)/disodium hydrogen phosphate were investigated. Experimental data are explained based on Kim's theoretical model for the prediction of biomolecule partitioning in a PEG/salt system.     

Effect of artificial periodicity in simulations of biomolecules under Ewald boundary conditions: a continuum electrostatics study.

Ewald and related methods are nowadays routinely used in explicit-solvent simulations of biomolecules, although they impose an artificial periodicity in systems which are inherently non-periodic. The consequences of this approximation should be assessed, since they may crucially affect the reliability of computer simulations under Ewald boundary conditions. In the present study we use a method based on continuum electrostatics to investigate the nature and magnitude of possible periodicity-induced artifacts on the potentials of mean force for conformational equilibria in biomolecules. Three model systems and pathways are considered: polyalanine oligopeptides (unfolding), a DNA tetranucleotide (separation of the strands), and the protein Sac7d (conformations from a molecular dynamics simulation). Artificial periodicity may significantly affect these conformational equilibria, in each case stabilizing the most compact conformation of the biomolecule. Three factors enhance periodicity-induced artifacts: (i) a solvent of low dielectric permittivity; (ii) a solute size which is non-negligible compared to the size of the unit cell; and (iii) a non-neutral solute. Neither the neutrality of the solute nor the absence of charge pairs at distances exceeding half the edge of the unit cell do guarantee the absence of artifacts.     

Molecular sieves provoke multiple substitutions in the enzymatic synthesis of fructose oligosaccharide–lauryl esters

The cause of discrepancies in the literature regarding the specificity of immobilized Candida antarctica lipase B in the acylation of oligosaccharides was examined. Molecular sieves, generally used to control the water content during acylation reactions, turned out to have an important role in this. It was proven that molecular sieves alone can catalyze the acylation of fructose oligomers using vinyl laurate, leading to multiple substitution of the oligomers. This effect was the most profound at conditions unfavorable for the enzyme, because this resulted in a relatively high concentration of the chemically produced adducts. The enzyme alone catalyzed the formation of monosubstituted oligomers. It was proven that even solvent pre-drying by molecular sieves already causes the release of catalyzing compounds to the liquid, leading to subsequent catalysis. These findings should be taken into account when applying molecular sieves in this type of reactions in the future. Molecular sieves could, moreover, be used as a catalyst when multiple substitution is desired.     

Synthesis of biological molecules on molecular sieves.

Catalytic properties of aluminosilicates may play a role in the synthesis of biological molecules from simple gaseous molecules commonly found in planetary atmospheres. Urea, amino acids and UV absorbing substances have been obtained by heating CO and NH3 with Linde molecular sieves saturated with Ca+2, NH4+ or Fe+3. The yields of amino acids produced have been determined by an amino acid analyzer. The quantity of urea produced largely depends on the nature of the saturating cation. Experiments using 14CO confirm that the amino acids are not due to contaminants adsorbed on the surface of the molecular sieves.     

A systematic methodology for defining coarse-grained sites in large biomolecules

Coarse-grained (CG) models of biomolecules have recently attracted considerable interest because they enable the simulation of complex biological systems on length-scales and timescales that are inaccessible for atomistic molecular dynamics simulation. A CG model is defined by a map that transforms an atomically detailed configuration into a CG configuration. For CG models of relatively small biomolecules or in cases that the CG and all-atom models have similar resolution, the construction of this map is relatively straightforward and can be guided by chemical intuition. However, it is more challenging to construct a CG map when large and complex domains of biomolecules have to be represented by relatively few CG sites. This work introduces a new and systematic methodology called essential dynamics coarse-graining (ED-CG). This approach constructs a CG map of the primary sequence at a chosen resolution for an arbitrarily complex biomolecule. In particular, the resulting ED-CG method variationally determines the CG sites that reflect the essential dynamics characterized by principal component analysis of an atomistic molecular dynamics trajectory. Numerical calculations illustrate this approach for the HIV-1 CA protein dimer and ATP-bound G-actin. Importantly, since the CG sites are constructed from the primary sequence of the biomolecule, the resulting ED-CG model may be better suited to appropriately explore protein conformational space than those from other CG methods at the same degree of resolution.     

Influence of different magnetites on properties of magnetic Pseudomonas aeruginosa immobilizates used for biosurfactant production

During the last decades, whole‐cell immobilization has been used successfully in many bioprocesses. In particular, it is aimed at implementing continuous production processes, reaching higher production rates, and reusing the biocatalyst. In some cases, effective retention of immobilizates in the bioprocess is not feasible by membranes or sieves due to pore plugging or undesired losses of immobilizates. In the present publication, it is reported about the investigation of magnetic immobilizates of Pseudomonas aeruginosa for application in continuous biosurfactant production of rhamnolipids by foam fractionation and retention of entrained immobilizates by high‐gradient magnetic separation from foam. Different materials and methods were tested with respect to important parameters, such as stability, diffusion properties or magnetic separation. Good magnetic separation of immobilizates was achieved at 5% (w/w) magnetite loading. Best results in terms of homogeneous embedding, good diffusion properties, and stability enhancement vis‐à‐vis pure alginate beads was achieved with alginate beads with embedded Bayoxide® magnetite or MagPrep® silica particles. Although polyurethane immobilizates showed higher stabilities compared with alginate beads, rhamnolipid diffusion in immobilizates was superior in magnetic alginate beads. Regarding bead production, smaller immobilizates were achieved with suspension polymerization compared to droplet extrusion by the JetCutting® technology. In total, magnetic immobilizates are a promising tool for an easier handling of biocatalysts in a continuous biological production process, but they have to be adapted to the current production task.© 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Prefractionation of protein samples for proteome analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis

To isolate high molecular weight (HMW) or low-abundance proteins we exploited the high resolving power provided by the molecular sieves of polyacrylamide gel matrices. Rice-leaf protein extracts were applied to a single well of an SDS-polyacrylamide gel with prestained molecular size markers at both ends. After electrophoresis, the gel was cut into 4 segments according to size, and each segment was ground in extraction buffer. The eluted proteins were separated from the gel matrix by centrifugation followed by acetone precipitation, and the precipitated proteins were subjected to SDS-PAGE and 2-DE. The SDS-PAGE-based prefractionation method provided non-overlapping discrete sample pools. About 27% more protein spots were detected in the fractionated samples than in the unfractionated samples, and 17% were enhanced. The improvement was especially prominent in the case of HMW proteins. Well-separated HMW proteins were analyzed by MALDI-TOF mass spectrometry. The molecular masses of the identified proteins in the > 48 kDa gel segment were distributed between 50 and 112 kDa, thus validating this prefractionation method. Identified HMW proteins with similar mass but different pI were mostly isoforms. Thus SDS-PAGE-based size prefractionation provides improved separation and detection of HMW proteins.     

The solvent-free thermal dehydration of tetritols on zeolites

A new alditol dehydration method at high temperatures, in the presence of molecular sieves without solvent in an argon atmosphere, is described. Investigations on tetritols have been carried out. Products arising after the intramolecular and intermolecular elimination of water, with retention or inversion of the configuration of asymmetric carbon atoms, were observed. Complete analytical separation of reaction products was achieved by means of GLC. The chemical structures of the compounds obtained were assigned using co-injection with standards, GLC-CIMS and GLC-EIMS analyses. Two intermolecular dehydration products of tetritols were isolated by HPLC and identified by 1H and 13C NMR spectroscopy.     

An expedient one-step synthesis of polysubstituted guanidinoglucosides using HgO-4A molecular sieves as catalyst

A novel one-step method of preparing polysubstituted guanidinoglucosides using peracetylated methyl 6-deoxy-6-thioureidoglucosides as starting materials and employing HgO in combination with molecular sieves as an efficient catalyst is reported. The structures of three methyl 2,3,4-tri-O-acetyl-6-[N(2)-(benzothiazol-2-yl)-N(3)-(oxydi-1,2-ethandiyl)]guanidino-6-deoxy-alpha-d-glucopyranosides were unambiguously confirmed by X-ray crystallography. The methodology affords new compounds in good yields and also provides a promising route for the synthesis of carbamate-protected guanidines.     

Protein refolding assisted by molecular tube based α-cyclodextrin as an artificial chaperone

In this study, we evaluated, for the first time, the application of molecular tube based alpha-cyclodextrin for improving the refolding yields of two different enzymes: carbonic anhydrase and alkaline phosphatase. Our results indicate that under the optimal developed refolding environments, the denatured carbonic anhydrase and alkaline phosphatase were refolded with a yield of 51 and 61% using 15 and 5 mg/ml of the molecular tube, respectively. Regardless of lower refolding yields compared with liquid-phase artificial chaperone assisted approach, the new technique (solid-phase artificial chaperone assisted refolding) benefits from easier and faster separation of the refolded product from the refolding environment, recycling of the stripping agent, and finally, significantly less environmental effect at the industrial levels. However, further improvements in solid-phase artificial chaperone assisted technique are needed either through synthesizing better stripping agents or by optimizing and defining better refolding environments.     

Integral equation theories for predicting water structure around molecules

Water plays a crucial role in the structure and function of proteins and other biological macromolecules; thus, theories of aqueous solvation for these molecules are of great importance. However, water is a complex solvent whose properties are still not completely understood. Statistical mechanical integral equation theories predict the density distribution of water molecules around a solute so that all particles are fully represented and thus potentially both molecular and macroscopic properties are included. Here we discuss how several theoretical tools we have developed have been integrated into an integral equation theory designed for globular macromolecular solutes such as proteins. Our approach predicts the three-dimensional spatial and orientational distribution of water molecules around a solute. Beginning with a three-dimensional Ornstein-Zernike equation, a separation is made between a reference part dependent only on the spatial distribution of solvent and a perturbation part dependent also on the orientational distribution of solvent. The spatial part is treated at a molecular level by a modified hypernetted chain closure whereas the orientational part is treated as a Boltzmann prefactor using a quasi-continuum theory we developed for solvation of simple ions. A potential energy function for water molecules is also needed and the sticky dipole models of water, such as our recently developed soft-sticky dipole (SSD) model, are ideal for the proposed separation. Moreover, SSD water is as good as or better than three point models typically used for simulations of biological macromolecules in structural, dielectric and dynamics properties and yet is seven times faster in Monte Carlo and four times faster in molecular dynamics simulations. Since our integral equation theory accurately predicts results from Monte Carlo simulations for solvation of a variety of test cases from a single water or ion to ice-like clusters and ion pairs, the application of this theory to biological macromolecules is promising.     

Hybrid filtering of feed stream noise from oscillating yeast cultures by combined Kalman and neural network configurations

Large continuous flow bioreactors are often under the influence of noise in the feed stream(s). Prior removal of noise is done by filters based either on specific algorithms or on artificial intelligence. Neither method is perfect. Hybrid filters combine both methods and thereby capitalize on their strengths while minimizing their weaknesses. In this study, a number of hybrid models have been compared for their ability to recover nearly noise-free stable oscillations of continuous flow Saccharomyces cerevisiae cultures from aberrant behavior caused by noise in the feed stream. Each hybrid filter had a different neural network in conjunction with an extended Kalman filter (EKF). The choice of the best configuration depended on the performance index. All hybrid filters were superior to both the EKF and purely neural filters. Along with previous studies of monotonic fermentations, the present results establish the suitability of hybrid neural filters for noise-affected bioreactors. IMTECH communication no.045/2006.     

微生物的遗传多样性

微生物是生物圈中不可或缺的重要组成部分, 维系着自然界生态平衡。随着分子生物学技术的发展, 微生物遗传多样性的研究从形态学水平、蛋白水平进入到了DNA水平。而高通量测序技术和宏基因组技术的发展, 不仅为我们理解微生物的遗传多样性提供了更加丰富的信息和有力的证据, 也对于合理利用生物资源、保护生态平衡等方面具有重要意义。文章就微生物遗传多样性研究的相关内容, 如物种的分离鉴定、微生物群体遗传结构、物种形成以及系统发育和进化等方面的研究进展进行综述。