Effects of recombinant precursor pathway variations on poly[(R)-3-hydroxybutyrate] synthesis in Saccharomyces cerevisiae

Different recombinant R-3-hydroxybutyryl-CoA (3-HB) synthesis pathways strongly influenced the rate and accumulation of the biopolymer poly[(R)-3-hydroxybutyrate] (PHB) in Saccharomyces cerevisiae. It has been previously shown that expression of the Cupriavidus necator PHB synthase gene leads to PHB accumulation in S. cerevisiae [Leaf, T., Peterson, M., Stoup, S., Somers, D., Srienc, F., 1996. Saccharomyces cerevisiae expressing bacterial polyhydroxybutyrate synthase produces poly-3-hydroxybutyrate. Microbiology 142, 1169-1180]. This finding indicates that native S. cerevisiae expresses genes capable of synthesizing the correct stereochemical substrate for the synthase enzyme. The effects of variations of 3-HB precursor pathways on PHB accumulation were investigated by expressing combinations of C. necator PHB pathway genes. When only the PHB synthase gene was expressed, the cells accumulated biopolymer to approximately 0.2% of their cell dry weight. When the PHB synthase and reductase gene were co-expressed, the PHB levels increased approximately 18 fold to about 3.5% of the cell dry weight. When the beta-ketothiolase, reductase and synthase genes were all expressed, the strain accumulated PHB to approximately 9% of the cell dry weight which is 45 fold higher than in the strain with only the synthase gene. Fluorescent microscopic analysis revealed significant cell-to-cell heterogeneity in biopolymer accumulation. While the population average for the strain expressing three PHB genes was approximately 9% of the cell dry weight, some cells accumulated PHB in excess of 50% of their cell volume. Other cells accumulated no biopolymer. In addition, the recombinant strain was shown to co-produce ethanol and PHB under anaerobic conditions. These results demonstrate that the technologically important organism S. cerevisiae is capable of accumulating PHB aerobically and anaerobically at levels similar to some bacterial systems. The easily assayed PHB system also creates a convenient means of probing in vivo the presence of intracellular metabolites which could be useful for studying the intermediary metabolism of S. cerevisiae.     

Ethanol fermentation of mahula (<Emphasis Type="Italic">Madhuca latifolia</Emphasis>) flowers using free and immobilized bacteria <Emphasis Type="Italic">Zymomonas mobilis</Emphasis> MTCC 92

Mahula (Madhuca latifolia L.) is a deciduous tree commonly found in the tropical rain forests of Asian and Australian continent. Corolla, the edible part of its flowers, is rich in fermentable sugar (37 ± 0.23%; on dry weight basis). Batch fermentation of mahula flowers was carried out using Zymomonas mobilis MTCC 92 free cells and cells immobilized in calcium alginate matrix. The ethanol productions were 122.9 ± 0.972 and 134.6 ± 0.104 g/kg flowers on dry weight basis using free and immobilized cells, respectively, after 96 h of fermentation, which showed that cells entrapped in calcium alginate matrix yielded 8.7% more ethanol than free cells. Further, the immobilized cells were physiologically active up to three more cycles of fermentation producing 132.7 ± 0.095, 130.5 ± 0.09 and 128.7 ± 0.056 g ethanol per kg flower in first, second and third cycle, respectively.     

Poly(carbamoylsulphonate) as a matrix for whole cell immobilization - biological characterization

Summary Poly(carbamoylsulphonate) (PCS), a hydrogel matrix of low toxicity for the immobilization of microorganisms was characterized with respect to physiological properties. The survival rates of immobilized Paracoccus denitrificans in PCS were greater than 99 %, the initial division rate of the bacterium inside the gel was the same as of free suspended cells and decreases with increasing cell density within the first days. The polymer has good resistance to microbial degradation and excellent mechanical stability. First results of long term behaviour and kinetic data of nitrifying bacteria entrapped within PCS gel beads are shown.     

Flow behaviour of a POSS biopolymer solution

A non-biodegradable polyhedral oligomeric silsesquioxane (POSS) nanocomposite biopolymer has been developed for fabrication of medical devices and for tissue engineering human organs. The polymer in solution, containing 2 wt% of POSS, has been synthesized, characterized and investigated to determine its key rheological properties. Thus, the variation of shear stress and viscosity as a function of shear rate has been determined at ambient temperature to estimate yield stress and the index of pseudoplasticity, respectively. The temperature dependence of viscosity and the effect of ageing on the viscosity of the polymer have also been investigated. Results are compared with those of a conventional polycarbonate urethane (PCU) polymer solution. The POSS-PCU polymer solution shows near-Newtonian behaviour in the shear rate range to 1000 s(-1), having an apparent viscosity of approximately 3000 mPa s and a pseudoplasticity index of 0.90, decreasing slightly as the polymer solution is aged over 9 months. The temperature dependence of viscosity of the POSS polymer is extremely low and does not change with ageing but the yield strength increases from 2.7 Pa to 8.3 Pa.     

短期连续剪切对光生物反应器内海带配子体细胞生长及其恢复能力的影响

以不同搅拌速率(0~1000r/min)对海带(Laminaria japonica)配子体细胞施加短期(0~60h)连续剪切,卸载剪切力之后细胞静止恢复23.5d,在此期间研究不同搅拌速率分别在连续剪切和恢复期间对细胞生长及其恢复能力的影响。研究结果表明,连续剪切期间,90r/min下细胞叶绿素浓度积累达到最大值2.36mg/L;中高速搅拌速率(270~1000r/min)下叶绿素浓度迅速下降,胞内氮磷池释放,1000r/min下细胞损伤率为静止对照样的18倍。恢复期间,所有组别细胞均呈现较强的恢复能力。此外,在连续剪切实验中,海带配子体细胞内叶绿素浓度比干重更能有效的表达生物量浓度,除细胞损伤率外,磷源释放可以作为判别细胞受损的参考指标之一。     

Collagen oligomers modulate physical and biological properties of three‐dimensional self‐assembled matrices

Elucidation of mechanisms underlying collagen fibril assembly and matrix‐induced guidance of cell fate will contribute to the design and expanded use of this biopolymer for research and clinical applications. Here, we define how Type I collagen oligomers affect in‐vitro polymerization kinetics as well as fibril microstructure and mechanical properties of formed matrices. Monomers and oligomers were fractionated from acid‐solubilized pig skin collagen and used to generate formulations varying in monomer/oligomer content or average polymer molecular weight (AMW). Polymerization half‐times decreased with increasing collagen AMW and closely paralleled lag times, indicating that oligomers effectively served as nucleation sites. Furthermore, increasing AMW yielded matrices with increased interfibril branching and had no correlative effect on fibril density or diameter. These microstructure changes increased the stiffness of matrices as evidenced by increases in both shear storage and compressive moduli. Finally, the biological relevance of modulating collagen AMW was evidenced by the ability of cultured endothelial colony forming cells to sense associated changes in matrix physical properties and alter vacuole and capillary‐like network formation. This work documents the importance of oligomers as another physiologically‐relevant design parameter for development and standardization of polymerizable collagen formulations to be used for cell culture, regenerative medicine, and engineered tissue applications. © 2010 Wiley Periodicals, Inc. Biopolymers 95: 77–93, 2011.     

Studies on fibre-entrapped whole microbial cells in urea hydrolysis

Microbial cells possessing high urease activity were entrapped in cellulose triacetate fibres, and their capability to hydrolyse urea was evaluated. The activity obtained using these fibres was found to be dependent on the amount of cells entrapped; the best cells-to-carrier ratio was 1:10. Optimum pH and optimum temperature were 7.2 and 60°C respectively, for both the free cells and the immobilized cells, which suggests that the cells are physically bound to the polymer matrix. The effect of flow rate on the extent of conversion and continuous hydrolysis of urea was examined. A flow rate corresponding to a residence time of 1.325 h was found to be satisfactory. The computed half-life of the system was 177.7 h. The potential industrial application is also discussed.     

以环氧乙烷为活性基的多孔颗粒状固定化青霉素酰化酶的制备

报道了用以环氧乙烷为活性基的多孔颗粒状载体（Eupergit-C）制备固定由巨大芽孢杆菌（B．megaterium）产生的青霉素酰化酶的研究。用已二胺,赖氨酸对载体进行化学修饰后制备固定化酶,获得了较好的固定结果。用未修饰的载体制备固化酶,经24h固定反应,酶活力达176．5IU／g（wet）,酶活力总叫率达53．7％,酶蛋白的固定量为19＝7mg/g（dr）,酶蛋白的固定效率达87．5％。游离酶的酶浓度对制备固定化酶的活力无显影响。当加酶量从312IU/g（dry）上升到6250IU／g（dry）时,固定化酶活力从89IU／g(wet）上升到475IU／g（wet）,总收率和固定化效率分别从99％和99％下降到26．5％和32．5％,酶蛋白的固定量从6．9mg/g（dry）上升到112mg/g（dry）,酶蛋白的固定效率从99％下降至80．5％。以酶活力为155IU/g(wet）,酶蛋白固定量为22mg/g(dry）的固定化酶水解青霉素G钾盐,经过20批循环水解后,剩余酶活力为92．5％。     

The distribution of nuclear and cytoplasmic proteins in the blastoderm of the loach, Misgurnus fossilis L

The concentration of dry substance (protein) and the dry weight of nuclei, cytoplasm and cells from different blastoderm regions at the early blastula and midgastrula stages were determined by interferentional microscopy. It was shown that at the early blastula stage the dry weight of cells in the basal layer is higher than that in the outer layer. Although the protein concentration in the basal layer cells appears to be somewhat higher, differences in their dry weight are due primarily to the big volume of cytoplasm of the basal layer cells. By the midgastrula stage, the total (nucleus + cytoplasm) protein concentration increases (by 17% in the basal layer cells and by 9% in the outer layer cells) due to the increase of nuclear protein concentration. At the same time dry weight of these cells markedly decreases due to the decrease of their volumes in the process of cell divisions. At the midgastrula stage the epiblast cells have the highest dry weight due to the highest protein concentration in the cytoplasm and the biggest cell volume. The results obtained are discussed with respect to the data on the pattern of accumulation of newly synthesized protein in nuclei and cytoplasm with special reference to the duration of individual cell cycle phases.     

Removal of heavy metals from aqueous solution using Rhizopus delemar mycelia in free and polyurethane-bound form

This study assesses the ability of mycelia of Rhizopus delemar (both free and immobilized on polyurethane foam) to remove heavy metals from single-ion solutions as well as from a mixture of them. All experiments were conducted using 0.5-5 mm solutions of CuSO4 x 5H2O, CoCl2-6H2O and FeSO4 7H2O. Mycelia immobilized on polyurethane foam cells showed some times increase in uptake compared with that of free cells. Metal ions accumulation from a mixed solution was decreased slightly for cobalt and iron and considerable for copper ions. Heavy metal uptake was examined in the immobilized column experiments and more than 92% heavy metal removal (mg heavy metals removed/mg heavy metals added) from a mixed solution was achieved during the 5 cycles. During these experiments, the dry weight of the immobilized cells was decreased by only 2%. These results showed that immobilized mycelia of Rhizopus delemar can be used repeatedly for removal of heavy metals from aqueous solutions.     

Taxol (paclitaxel) production using free and immobilized cells of Taxus cuspidata

The production characteristics for Taxol (paclitaxel) using free and immobilized cells of Taxus cuspidata were investigated in a perfusion culture bioreactor. Although the cell growth was inhibited by higher dilution rates, the specific production rate of Taxol was increased by perfusion compared with that using batch operation. Perfusion cultures using a nylon-mesh cell separator for free suspension cells showed similar production profiles to those obtained using immobilized cells. Continuous Taxol production was successfully obtained at an approximate specific production rate of 0.3 mg/g DCW (dry cell weight) per day for up to 40 days. (c) 1997 John Wiley & Sons, Inc.     

Chromium accumulation by living yeast at various environmental conditions

Yeast tolerance to Cr (III) and Cr (VI) as well as chromium accumulation potential were shown to depend on treatment time, metal concentration, biomass density and the phase of growth. Kinetic studies as exemplified by Pichia guilliermondii ATCC 201911 revealed a biphasic mode of Cr (III) uptake: a rapid sorption phase was followed by a slow process of accumulation, in which the contribution of the cell-bound Cr fraction increased, while the total cellular Cr level remained constant. Cr (VI) uptake was characterized by a time-dependent increase of total Cr and by a constant fractional contribution of the cell-adsorbed chromium, which suggests that the amount of cell-accumulated Cr also tended to increase over time. The resistance to Cr and metal accumulation levels were substantially elevated for a given strain when cultures were treated at high initial biomass densities (1 mg dry weight/ml) of exponentially proliferating cells. Maximum accumulation capabilities ranged between 4.0 and 13 mg Cr (III)/g dry weight and 2-6.7 mg Cr (VI)/g dry weight. The total cell-accumulated Cr contained 29.3% and 52.3% of organically bound chromium for the treatment of P. guilliermondii with Cr (III) and Cr (VI), respectively. Selected yeast strains, under specified physiological conditions, can be applied for bioremediation of environmental Cr contamination, and might be useful too for attempts to obtain chromium-enriched biomass containing biostabilized and nontoxic Cr forms for nutritional applications.     

Direct visualization of the binding and internalization of a ferritin conjugate of epidermal growth factor in human carcinoma cells A-431

We have prepared a conjugate of epidermal growth factor (EGF) and ferritin that retains substantial binding affinity for cell receptors and is biologically active. Glutaraldehyde-activated EGF was covalently linked to ferritin to produce a conjugate that contained EGF and ferritin in a 1:1 molar ratio. The conjugate was separated from free ferritin by affinity chromatography using antibodies to EGF. Monolayers of human epithelioid carcinoma cells (A-431) were incubated with EGF:ferritin at 4 degrees C and processed for transmission electron microscopy. Under these conditions, approximately 6 X 10(5) molecules of EGF:ferritin bound to the plasma membrane of each cell. In the presence of excess native EGF, the number of bound ferritin particles was reduced by 99%, indicating that EGF:ferritin binds specifically to cellular EGF receptors. At 37 degrees C, cell-bound EGF:ferritin rapidly redistributed in the plane of the plasma membrane to form small groups that were subsequently internalized into pinocytic vesicles. By 2.5 min at 37 degrees C, 32% of the cell-bound EGF:ferritin was localized in vesicles. After 2.5 min, there was a decrease in the proportion of conjugate in vesicles with a concomitant accumulation of EGF:ferritin in multivesicular bodies. By 30 min, 84% of the conjugate was located in structures morphologically identified as multivesicular bodies or lysosomes. These results are consistent with other morphological and biochemical studies utilizing 125I-EGF and fluorescein-conjugated EGF.     

Dielectric spectroscopy as a novel and convenient tool for the study of the shear sensitivity of plant cells in suspension culture.

Plant cell suspensions of different species and different age were subjected to hydrodynamic stress while following the decline in the volume fraction of intact cells by measuring the permittivity of the cell suspension at radio frequencies. Results were compared with the fresh weight, dry weight, packed cell volume and cell number of the suspensions. At first a rapid decline is seen as the most shear-sensitive cells are broken up, followed by a slower decline as less sensitive cells are broken up. The sensitivity of the cells to shear stress depended strongly on the cell line used but only slightly on their age, older cells being more sensitive. The dependence of the shear sensitivity on the cell line might be an effect of the species investigated, the culturing conditions of the cell line, or both. It was found that cells that grow in a finely dispersed suspension are much less prone to shear stress than is often assumed.     

Cooperative binding of concanavalin A to thymocytes at 4 degrees C and micro-redistribution of concanavalin A receptors.

The mode of binding of 125I-labelled concanavalin A and succinyl-concanavalin A to rat thymocytes at 4 degrees C was investigated. Simultaneously, the free binding sites of the cell-bound lectin molecules were quantified by horseradish peroxidase binding. Concanavalin A showed cooperative binding while succinyl-concanavalin A did not. The number of molecules of concanavalin A bound to the cell surface when it was saturated was twice the number of molecules of succinyl-concanavalin A. We interpret these results as showing that the binding of native concanavalin A to thymocytes at 4 degrees C brings about a cooperative modification of the membrane which leads to appearance of new receptors. Divalent succinyl-concanavalin A has no such effect. Horseradish peroxidase binding to cell-bound lectin was shown to be related to the immobilization of membrane receptors; the more they are immobilized, the more receptor-associated lectin can bind horseradish peroxidase. This allowed us to establish that post-binding events, which we called micro-redistribution, occurred at 4 degrees C when either concanavalin A or succinyl-concanavalin A binds to cells. A cooperative restriction of the micromobility of cell receptors is produced by increasing concentrations of concanavalin A. Succinyl-concanavalin A does not restrict cell receptor mobility at any concentration tested. The results are discussed in terms of cell stimulation and cell agglutination.     

Optimization of the microbial synthesis of dihydroxyacetone in a semi-continuous repeated-fed-batch process by in situ immobilization of Gluconobacter oxydans

The effect of in situ immobilization of Gluconobacter oxydans on a novel carrier material in a repeated-fed-batch operated packed-bed bubble-column bioreactor for the production of the fine chemical dihydroxyacetone was investigated experimentally. The carrier material were biocompatible, durable, coated Ralu-rings. The coating was a porous silicone matrix with satisfactory wetting characteristics. Settling of cells was relatively rapid. The cells were protected from abrasion caused by shear forces. A sufficiently high oxygen supply rate to the immobilized cells was provided due to the high oxygen permeability of the silicone matrix. The immobilized biomass was estimated to be about 65% of the total biomass contained in the bioreactor after 18 days of operation. The observed space-time yield was approx. 76% higher compared to a similar process which was performed without an optimized fermentation medium. Compared to previous experiments with a trickle-bed bioreactor, the space-time yield was approx. 3.7 times higher. A typical time course of the immobilization process was observed: after an induction phase, a transition phase followed which later on gave way to a nearly linear accumulation phase. A stationary phase with regard to the amount of immobilized active cells, however, was not reached. Hence, a higher bioreactor performance than observed could be expected at longer operation times.     

聚氨酯泡沫固定产脂肪酶粗状假丝酵母（Candidav alida T2）细胞的研究

对聚氨酯泡沫固定产脂肪酶粗状假丝酵母（Candida validaT2）细胞的固定化条件进行了研究。实验结果表明,聚氨酯泡沫颗粒经密度和粒径筛选,酸碱处理,以及固液比优化,载体固定细胞干质量比达到157lmg／g,细胞脂肪酶的酶活为每克干细胞1415U。电镜图片显示粗状假丝酵母菌（Candida validaT2）在载体孔隙内和脊壁上缠绕充盈,生长良好,固定结构稳定。固定化细胞连续催化水解桐籽油5批次,细胞相对水解酶活保持率达73％,固定细胞的损失率为12．5％。固定化细胞颗粒显示出良好的操作稳定性和酶活保持率,为进一步的应用研究提供了实验基础。     

Polyamine Titer in the Embryonic Axis and Cotyledons of Glycine max (L.) during Seed Growth and Maturation

Active polyamine metabolism occurs in Glycine max (L.) seeds during development. Most (≥97%) of putrescine (Put), spermidine (Spd), spermine (Spm), and cadaverine (Cad) are present as free forms in the growing embryo. In the cotyledon or embryonic axis, Put decreases to a nearly undetectable level, while Spd level sharply increases as seed dry weight accumulation progresses. Spm level in the axis also increases along with the Spd level. There is little change in Spm level in the cotyledons. Maturation and dehydration results in a slight reduction of Spd level in the cotyledons. Cad is present in relatively large quantities (5.5-12 micromoles per gram dry weight) in the axes of mature soybean seeds. Only traces of Cad, as expressed on a dry weight basis, are found in the developing or mature cotyledons. The synthesis and accumulation of Cad in the axis begins at the time when the axis or the seed accumulates 30 to 50% of its maximum dry weight. The Cad accumulation (0.8 nanomole per axis per day) proceeds until the later stages of dehydration. When soybean plants are subjected to complete defoliation and shade during the midpoint of seed maturation, Cad accumulation in the axis and seed dry weight accumulation ceased almost immediately. The treatment, however, does not affect the viability of soybean seeds.     

Phytate degradation by immobilized<Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> phytase in soybean-curd whey

Saccharomyces cerevisiae CY phytase-producing cells were immobilized in calcium alginate beads and used for the degradation of phylate. The maximum activity and immobilization yield of the immobilized phytase reached 280 mU/g-bead and 43%, respectively. The optimal pH of the immobilized cell phytase was not different from that of the free cells. However, the optimum temperature for the immobilized phytase was 50°C, which was 10°C higher than that of the free cells; pH and thermal stability were enhanced as a consequence of immobilization. Using the immobilized phytase, phytate was degraded in a stirred tank bioreactor. Phytate degradation, both in a buffer solution and in soybean-curd whey mixture, showed very similar trends. At an enzyme dosage of 93.9 mU/g-phytate, half of the phytate was degraded after 1 h of hydrolysis. The operational stability of the immobilized beads was examined with repeated batchwise operations. Based on 50% conversion of the phytate and five times of reuse of the immobilized beads, the specific degradation (g phytate/g dry cell weight) for the immobilized phytase increased 170% compared to that of the free phytase.     

产糖化酶黑曲霉的固定化研究

采用多孔聚酯材料作为固定化载体,考察并比较了载体吸附固定化黑曲霉菌丝细胞的条件,当菌丝体细胞与载体预培养的条件为pH值5.0、孢子浓度为105个/ml、固液比为1/75时,有利于菌丝体的生长、吸附固定及发酵产酶.在产糖化酶的发酵过程中,与游离菌丝体细胞相比,发酵过程持续产酶时间有一定程度的延长,产糖化酶活力始终高于游离菌丝体.