Optimization of conditions for the production of pullulan and high molecular weight pullulan by Aureobasidium pullulans

Aureobasidium pullulans had a maximum yield coefficient of pullulan (Y _p/s=0.24) with an initial pH of the culture broth of 6.5 in a shake-flask culture. In a batch culture, the maximum pullulan yield coefficient of 0.30 was obtained at the aeration rate of 0.5 vvm. A yeast-like form and mycelial form of cells were found at the culture broth with pH controlled at 4.5 with a maximum yield coefficient of pullulan of 0.27. However, a high portion (35%) of high molecular weight pullulan (M _w>2 000 000) was produced at pH 6.5 with a yeast-like morphology of the cells.     

普鲁兰的特性及其在食品和医学上的应用

普鲁兰是出芽短梗霉菌株分泌到胞外的一种水溶性葡聚糖。独特的联接模式赋予了普鲁兰及其衍生物与众不同的特性,具有胶粘性,成型性的同时,可以形成纤维以及很强的阻氧性等。这些性质决定了其广泛的应用价值。在介绍其结构和性质后重点论述了其在食品、生物医学以及其他工业上的应用。     

Production of high molecular weight pullulan by Aureobasidium pullulans using glucosamine

Pullulan productivity was optimized in Aureobasidium pullulans ATCC 42023 with 54 g glucose l^–1. Pullulan with its higher molecular weight (>1000000) was produced using 2% (w/v) glucose and 3% (w/v) glucosamine together. The maximum concentration of pullulan was 8 g l^–1 at 140 h with shake-flask culture.     

二价阳离子对短梗霉多糖发酵的影响

就二价阳离子对短梗霉多糖和黑色素的影响进行了分析和研究。结果表明 ,二价阳离子对短梗霉多糖的合成和黑色素的形成均有较大的影响。通过对培养基中二价阳离子含量和种类的控制不仅可以抑制细胞黑色素的形成 ,而且还保持了很高的多糖发酵水平 ,在 30L生物反应器中短梗霉多糖的产量和转化率分别达到了 59 8g/L和 61 5%。     

Hydrodynamic characteristics of the Oosterschelde in recent decades

Hydrodynamic conditions in the Oosterschelde have changed as a result of the Delta Project. Engineering works were carried out between 1960 and 1970 in the northern branch of the Oosterschelde. The Grevelingen dam was built in 1964 and the Volkerak dam was completed in the spring of 1969. With the completion of these dams, the northern branch of the Oosterschelde was cut off from other estuaries, and river flow was then regulated by a sluice complex in the Volkerak dam. Between 1980 and 1987 a storm-surge barrier was constructed across the mouth of the Oosterschelde, and two compartmentalisation dams were built in the eastern part of the basin. The storm surge barrier decreased the effective cross-sectional area at the mouth from 80 000 m² to 17 900 m². The compartmentalisation dams reduce the surface area of the Oosterschelde basin from 452 km² to 351 km². The hydraulic impact of the works is demonstrated with data from field measurements and data from model calculations.     

Hydrodynamic properties of mushroom tyrosinase

The hydrodynamic properties of mushroom tyrosinase were determined at pH 6.5 using a Sephadex G-200 column. From the comparison of its gel-filtration behaviour with those of standard proteins, the following parameters were calculated: MW (122 500 ± 1%), Stokes' radius (42.75 × 10^?8 cm²/sec), diffusion coefficient (5.048 × 10^?7 cm²/sec) and frictional ratio (1.26). These values suggest a globular conformation of this enzyme.     

Pretreatment of beet molasses to increase pullulan production

Pretreatment of beet molasses with cation exchange resin, sulphuric acid, tricalcium phosphate, potassium ferrocyanide, and ethylenediaminetetraacetic acid and disodium salt (EDTA) to increase the production of pullulan was investigated. Among the above techniques used for the removal of heavy metals, sulphuric acid treatment gave better results regarding polysaccharide concentration, polysaccharide yield, and sugar utilization. Aureobasidium pullulans grown on beet molasses produced a mixture of pullulan and other polysaccharides. The pullulan content of the crude polysaccharide was 30–35%. The addition of nutrients improved the production of polysaccharide. A maximum polysaccharide concentration (32·0±1·0 g litre⁻¹) was achieved in molasses solution (70 g litre ¹ initial sugar concentration, pH 6·5–7·5) treated with sulphuric acid and supplemented with K₂HPO₄ 0·5%, -glutamic acid 1%, olive oil 2·5% and Tween 80 0·5%. In this case, the highest values of biomass dry weight (33·8±1·0 g litre⁻¹), polysaccharide yield (63·5±2·5%), and sugar utilization (97·5±1·5%) were obtained at pH 6·5, 3·5, and 4·5–7·5, respectively.     

Optimization and characterization of pullulan production by a newly isolated high-yielding strain Aureobasidium melanogenum

Abstract

Pullulan is an extracellular water-soluble polysaccharide with wide applications. In this study, we screened strains that could selectively produce high molecular weight pullulan for application in industrial pullulan production. A new fungus strain A4 was isolated from soil and identified as Aureobasidium melanogenum based on colony characteristics, morphology, and internally transcribed spacer analysis. Thin-layer chromatography, Fourier-transform infrared spectroscopy, and nuclear magnetic resonance analysis suggested that the dominant exopolysaccharide produced by this strain, which presented a molecular weight of 1.384?×?10⁶ Dalton in in-gel permeation chromatography, was pullulan. The culture conditions for A. melanogenum A4 were optimized at 30?°C and 180?rpm: carbon source, 50?g/L maltose; initial pH 7; and 8?g/L Tween 80. Subsequently, batch fermentation was performed under the optimized conditions in a 5-L stirred-tank fermentor with a working volume of 3?L. The fermentation broth contained 303?g/L maltose, which produced 122.34?g/L pullulan with an average productivity of 1.0195?g/L/h and 82.32?g/L dry biomass within 120?h. The conversion efficiency of maltose to pullulan (Y%) and specific production rate (g/h/g dry cells) (Qs) reached 40.3% and 0.0251?g/L/g dry cells, respectively. The results showed strain A4 could be a good candidate for industrial production.     

Hydrodynamic properties of rigid macromolecules composed of ellipsoidal and cylindrical subunits.

A procedure is devised for the calculation of hydrodynamic properties of rigid macromolecules composed subunits that are modeled as ellipsoids of revolution and cylinders. Owing to the axial symmetry of these shapes, smooth shell models can be constructured for the subunit structure. The bead shell model so constructed is employed for the calculation of the properties. A computer program, HYDROSUB, has been written implementing both the model building and the hydrodynamic calculation. A detailed example of the use of this methodology is presented for the case of the solution properties of the human antibody molecule immunoglobulin G3 (IgG3). Finally, hints are given on other uses and applications of the procedure.     

Conjugation of heparin into carboxylated pullulan derivatives as an extracellular matrix for endothelial cell culture

A carboxylated pullulan, for use as a structural material for a number of tissue engineering applications, was synthesized and conjugated with heparin. By immobilization of heparin to pullulan, endothelial cells (ECs) attached on the heparin-conjugated pullulan were more aggregated than when attached to other pullulan derivatives. Attachments were 50, 45, 49, and 90% for a polystyrene dish, pullulan acetate, carboxylated pullulan, and heparin-conjugated pullulan, respectively. Heparin-conjugated pullulan inhibited the proliferation of smooth muscle cells (SMCs) in vitro. Heparin-conjugated pullulan material can thus be used for the proliferation of vascular ECs and to inhibit the proliferation of SMCs.     

Hydrodynamic characteristics of immobilized cell beads in a liquid-solid fluidized-bed bioreactor

This study examined the hydrodynamic characteristics of a liquid-solid fluidized-bed bioreactor using elastic particles (PVA gel beads) of various diameters as carriers. The drag coefficient-Reynolds number, velocity-voidage, and expansion index-Reynolds number relationships observed during fluidization of PVA gel beads in a fluidized bed in our experiments were compared with the published results. Predictions made from previous correlations were examined with our new experimental findings, revealing the inadequacy of most of these correlations. Thus, new correlations describing the above-mentioned relationships are suggested. The drag coefficient of immobilized cell beads is larger than that of free cell ones at the same Reynolds number because the surface of the immobilized cell beads is rougher. For multiparticle systems, the correction factor, f(epsilon), is a function of the falling gel bead properties (Reynolds number) as well as the fluidized gel bead properties (Archimedes number), and depend strongly on the bed voidage (epsilon). A new simple relation was developed to predict easily the epsilon value from 0.5-0.9 at 4,986 < A(r) < 40,745 or 34 < Re(t) < 186. For all the immobilized cell beads used in this study, the prediction error of the bed voidage was less than 5% at epsilon > 0.5. The prediction equations in this study can be further applied to analyzing the hydrodynamic characteristics of a fluidized-bed reactor using similar entrapped elastic particles as carriers.     

Protein rigidity and thermophilic adaptation

We probe the hypothesis of corresponding states, according to which homologues from mesophilic and thermophilic organisms are in corresponding states of similar rigidity and flexibility at their respective optimal temperatures. For this, the local distribution of flexible and rigid regions in 19 pairs of homologous proteins from meso- and thermophilic organisms is analyzed and related to activity characteristics of the enzymes by constraint network analysis (CNA). Two pairs of enzymes are considered in more detail: 3-isopropylmalate dehydrogenase and thermolysin-like protease. By comparing microscopic stability features of homologues with the help of stability maps, introduced for the first time, we show that adaptive mutations in enzymes from thermophilic organisms maintain the balance between overall rigidity, important for thermostability, and local flexibility, important for activity, at the appropriate working temperature. Thermophilic adaptation in general leads to an increase of structural rigidity but conserves the distribution of functionally important flexible regions between homologues. This finding provides direct evidence for the hypothesis of corresponding states. CNA thereby implicitly captures and unifies many different mechanisms that contribute to increased thermostability and to activity at high temperatures. This allows to qualitatively relate changes in the flexibility of active site regions, induced either by a temperature change or by the introduction of mutations, to experimentally observed losses of the enzyme function. As for applications, the results demonstrate that exploiting the principle of corresponding states not only allows for successful thermostability optimization but also for guiding experiments in order to improve enzyme activity in protein engineering.     

黄土高原关中地区植草沟径流水力学特性

植草沟被广泛应用于海绵城市建设中,可有效改善城市生态环境。为探究植草沟径流调控作用的机理,通过放水模拟试验,研究了5个坡度(1%、2%、3%、4%、5%)、5个放水流量(20、30、40、50、60 L·min^-1)组合条件下植草沟径流的水力学特性与断面流速分布变化规律。结果表明: 植草沟径流水力学特性主要表现为,随流量和坡度的增加,流速、雷诺数和佛汝德数均逐渐增加,曼宁糙率系数与Darcy-Weisbach阻力系数逐渐减小。流速(V)可以用流量(Q)与坡度(S)的幂函数V=0.3387Q^0.555S^0.6601来表示。雷诺数和佛汝德数变化范围分别为1160.95~6596.82和0.17~1.21,径流流态均为紊流,流型受坡度影响较大。流量和坡度较小时,两者对阻力系数的影响较大,试验条件下水流阻力系数与雷诺数呈负相关关系。植草沟径流断面流速分布表现出关于中心两侧对称分布,最大流速点位于中心水面处,断面流速等值线随流量和坡度的增加而逐渐变密,流速变化梯度增大。本研究可为黄土区海绵城市建设中植草沟的设计应用与水力计算提供理论基础,也可以通过分析植草沟径流水力学特性以探索其径流调控机理。     

Hydrodynamic stress and lethal events in sparged microalgae cultures

The effect of high superficial gas velocities in continuous and batch cultures of the strains Dunaliella tertiolecta, Chlamydomonas reinhardtii wild-type and cell wall-lacking mutant was studied in bubble columns. No cell damage was found for D. tertiolecta and C. reinhardtii (wild-type) up to superficial gas velocities of 0.076 and 0.085 m s(-1), respectively, suggesting that high superficial gas velocities alone cannot be responsible for cell death and, consequently, bubble bursting cannot be the sole cause for cell injury. A death rate of 0.46 +/- 0.08 h(-1) was found for C. reinhardtii (cell wall-lacking mutant) at a superficial gas velocity of 0.076 m s(-1), and increased to 1.01 +/- 0.29 h(-1) on increasing superficial gas velocity to 0.085 m s(-1). Shear sensitivity is thus strain-dependent and to some extent the cell wall plays a role in the protection against hydrodynamic shear. When studying the effect of bubble formation at the sparger in batch cultures of D. tertiolecta by varying the number of nozzles, a death rate of 0.047 +/- 0.016 h(-1) was obtained at high gas entrance velocities. D. tertiolecta was cultivated in a pilot-plant reactor under different superficial gas velocities of up to 0.026 m s(-1), with relatively low gas entrance velocities and no cell damage was observed. There is some indication that the main parameter causing cell death and damage was the gas entrance velocity at the sparger.     

Hydrodynamic and molecular characteristics of polyelectrolyte complexes between sodium dextransulfate and chitosan hydrochloride

Hydrodynamic and molecular characteristics of particles of polyelectrolyte complexes (PEC) between sodium dextransulfate and chitosan hydrochloride were studied by various physicochemical methods (high-rate sedimentation, viscosimetry, turbidimetry, and diffusion). As was shown, the complex formation is accompanied by increase in the average sizes with simultaneous changes in the shape of the particles of the investigated PEC. According to the proposed polycomplexes model, side by side aggregation of the taken macromolecules could cause disordering of adjacent helical parts of polyanion-matrix that facilitates the formation of sphere-like polycomplexes particles.     

Effect of equilibrium pH on the structure and properties of bleach-damaged human hair fibers

Hair proteins are significantly affected by environmental pH. This impact tends to increase with prior hair damage. To understand how pH affects bleached hair properties, we utilized a number of techniques allowing for the determination of hair thermal properties, swelling and water sorption, and dry and wet tensile properties. At pH 5, hair proteins had the best structural integrity, as determined by differential scanning calorimetry and the highest tensile modulus. At pH 10, protein cross-linking density decreased, water content and hair cross-sectional diameter increased. Alkaline treatment, when compared with pH 5, did not reduce intermediate filament conditions (evaluated via enthalpy measurement) nor mechanical property performance in the wet state. In contrast to alkaline-treated hair, bleached hair equilibrated at pH 3 behaved very differently: it contained two different crosslink density zones, was the least stiff in dry and stiffest in wet conditions. Additionally, it absorbed less water and had the lowest diameter because of reduced water binding by protonated carboxyl groups. The pH 3 to 10 did not affect the mechanical strength of bleached hair in dry or wet conditions.     

RhoA is required for cortical retraction and rigidity during mitotic cell rounding

Mitotic cell rounding is the process of cell shape change in which a flat interphase cell becomes spherical at the onset of mitosis. Rearrangement of the actin cytoskeleton, de-adhesion, and an increase in cortical rigidity accompany mitotic cell rounding. The molecular mechanisms that contribute to this process have not been defined. We show that RhoA is required for cortical retraction but not de-adhesion during mitotic cell rounding. The mitotic increase in cortical rigidity also requires RhoA, suggesting that increases in cortical rigidity and cortical retraction are linked processes. Rho-kinase is also required for mitotic cortical retraction and rigidity, indicating that the effects of RhoA on cell rounding are mediated through this effector. Consistent with a role for RhoA during mitotic entry, RhoA activity is elevated in rounded, preanaphase mitotic cells. The activity of the RhoA inhibitor p190RhoGAP is decreased due to its serine/threonine phosphorylation at this time. Cumulatively, these results suggest that the mitotic increase in RhoA activity leads to rearrangements of the cortical actin cytoskeleton that promote cortical rigidity, resulting in mitotic cell rounding.     

Temperature dependence of the flexural rigidity of single microtubules

Although the flexural rigidity of a microtubule has previously been estimated by various methods, its temperature dependence has never been systematically examined. Here, we measured the flexural rigidity of a single taxol-stabilized microtubule from thermal fluctuation of the free end of a microtubule, the other end of which was fixed, at different temperatures; the results showed that the flexural rigidity is 2.54 × 10⁻²⁴ N m² independent of temperature in the range of 20-35 °C. Next, we applied temperature pulse microscopy (TPM) [K. Kawaguchi, S. Ishiwata, Thermal activation of single kinesin molecules with temperature pulse microscopy. Cell Motil. Cytoskeleton 49 (2001) 41-47; H. Kato, T. Nishizaka, T. Iga, K. Kinosita Jr., S. Ishiwata, Imaging of thermal activation of actomyosin motors. Proc. Natl. Acad. Sci. USA 96 (1999) 9602-9606], which created the temperature gradient (1-2 °C/μm) along a microtubule gliding on kinesins in the presence of ATP. As a result, the gliding microtubule was buckled between two interacting kinesin molecules, when the microtubule had been propelled faster by the rear kinesin (higher temperature) and slower by the front one (lower temperature). By estimating the critical force to induce buckling of a microtubule, the flexural rigidity of a microtubule was estimated to be (2.7-7.8) × 10⁻²⁴ N m², which was in good agreement with the value determined above. We discuss the buckling process based on the temperature dependence of the force-velocity relationship of kinesin motility.     

Length-dependence of flexural rigidity as a result of anisotropic elastic properties of microtubules

Unexplained length-dependence of flexural rigidity and Young's modulus of microtubules is studied using an orthotropic elastic shell model. It is showed that vibration frequencies and buckling load predicted by the accurate orthotropic shell model are much lower than that given by the approximate isotropic beam model for shorter microtubules, although the two models give almost identical results for sufficiently long microtubules. It is this inaccuracy of the isotropic beam model used by all previous researchers that leads to reported lower flexural rigidity and Young's modulus for shorter microtubules. In particular, much lower shear modulus and circumferential Young's modulus, which only weaken flexural rigidity of shorter microtubules, are responsible for the observed length-dependence of the flexural rigidity. These results confirm that longitudinal Young's modulus of microtubules is length-independent, and the observed length-dependence of the flexural rigidity and Young's modulus is a result of strongly anisotropic elastic properties of microtubules which have a length-dependent weakening effect on flexural rigidity of shorter microtubules.