An immobilized cell culture system for berberine production by Thalictrum minus cells

Thalictrum minus cells immobilized in calcium-alginate beads were cultured in a newly devised bioreactor for the purpose of producing berberine. This system could prevent cells from leaking out of the beads, allowing them to release most of the berberine synthesized into the liquid medium. Furthermore, the biosynthetic capability of the immobilized cells was found to be comparable to that of freely suspended cells.     

Adsorption ofCatharanthus roseus cells on various support particles

Summary Adsorption ofC. roseus cells producing indole alkaloids on various support particles were investigated in an attempt to find a suitable support material for surface culture of plant cells. Five different support particles namely gelatin, agar, alginate, polypropylene and glass beads were tested. Gelatin was found to be the most effective adsorbent resulting in nearly 30% adsorption of cells initially present in suspension. Adsorption isotherm of cells on gelatin beads was represented by a three parameter expression due to sigmoidal shape of the isotherm. The constants of the adsorption isotherm were determined using the experimental data.     

Enzymic synthesis of o-succinylbenzoyl-CoA in cell-free extracts of anthraquinone producing Galium mollugo L. cell suspension cultures

o-Succinylbenzoic acid (OSB) is an intermediate in the biosynthesis of shikimatederived anthraquinones. The cell free activation of o-succinylbenzoic acid in extracts of anthraquinone producing cells of Galium mollugo L. is demonstrated for the first time. This activation depends on the presence of ATP, coenzyme A and Mg²⁺. The o-succinylbenzoic acid coenzyme A ester was identified by converting it to 1,4-dihydroxy-2-naphthoic acid by a bacterial enzyme, viz. naphthoatesynthase. It is thus demonstrated that the o-succinylbenzoic acid coenzyme A ester derived from bacteria and from Galium mollugo cells are identical.     

Alkaloid formation by Catharanthus roseus cells in a packed column biofilm reactor

Summary Catharanthus roseus cells producing indole alkaloids were grown on surfaces of Ca-alginate beads within the interspacial volume of a packed column. Production media was circulated through the packed column in an upflow mode. Growth and indole alkaloid formation were quantified and compared with suspension culture of cells. Final alkaloid concentration and alkaloid yield obtained in the packed bed was superior to those obtained in suspension culture. This is thought to be due to improved cell-cell contact and interaction in the packed column.     

Cytological changes associated with alkaloid production in cultured cells of Coptis japonica and Thalictrum minus

Cell structures were compared between alkaloid-producing and non-producing cell cultures of Coptis japonica and Thalictrum minus by electron microscopic observation. In alkaloid-producing cells of C. japonica, prior to the onset of alkaloid synthesis, the vacuoles showed a greater volume than in non-producing cells. These were characterized by a number of large starch grains in the cytoplasm. Furthermore, alkaloid-producing cells contained stacks of rough endoplasmic reticulum. Comparison of different cell lines suggested that there might be a negative correlation between accumulation of alkaloids and starch. Similar cytological differences were observed with T. minus cell cultures that release berberine into the culture medium. Alkaloid producing cells were found to contain an abundance of cytoplasmic vesicles (0.5 – 1 m in diameter).     

Butane 2,3-diol production by immobilizedAeromonas hydrophila

A technique for immobilizing cells ofAeromonas hydrophila on a titanium (IV) hydroxide matrix was developed. Immobilized cells were used to produce butane 2,3-diol from soluble starch. The influence of the addition of 1 g/l sodium acetate to the starch-based medium on diol production depended on the initial starch: acetate ratic.     

Release of periplasmic enzymes fromEscherichia coli using tangential flow filtration

Summary Escherichia coli cells producing the periplasmic enzymes nuclease, -lactamase and alkaline phosphatase were exposed to osmotic shock treatment in a closed system consisting of a tank, a pump and a filtration unit. The enzymes were released by circulating the cell suspensions in the filtration system and separated from cells and spheroplasts by filtration. This novel releasing method was shown to be equally effective as osmotic shock treatments performed by agitation and centrifugation. Since the extraction is performed in a closed system, aerosol formation is avoided. In addition the method may easily be scaled up.     

Immobilization of baker's yeast using plaster of Paris

Summary Plaster of Paris was used as a matrix to bind radiation-killed cells ofSaccharomyces cerevisiae. Immobilized cells retained invertase activity for over 1 month of continuous operation and were about 20% more active than cells immobilized in polyacrylamide.     

Production of HBsAg by growth rate control with recombinantSaccharomyces cerevisiae in fed-batch

Summary To maintain a constant specific growth rate for a recombinantS.cerevisiae in fed-batch, the medium feeding rate has been controlled with respect to the hourly calculated glucose uptake rate. The recombinant yeast producing HBsAg showed the exponential production trend in proportion to the exponential cell growth. Total cell yield in fed-batch was about 0.402 g cells/g glucose, and HBsAg was produced about ten times more than in batch. Decrease of growth rate by HBsAg produced was not shown.     

Bioconversion of hydrogen sulfide by free and immobilized cells ofChlorobium thiosulfatophilum

Summary The transformation of hydrogen sulfide into elementary sulfur and sulfate was investigated in a photo-bioreactor using autotropic bacteriaChlorobium thiosulfatophilum. The accumulations of sulfur and sulfate in the reactor were found to be dependent on the light energy and the feed rate of H₂S. The optimum operation lines were established to limit sulfide or sulfate. Immobilization of the whole cells in strontium-alginate matrix enhanced the conversion more than with the free cells.     

Kinetics of phosphorus uptake by immobilizedChlorella

Summary Alginate-entrappedChlorella demonstrate rapid uptake of phosphorus from synthetic growth medium in batch culture. Rates of phosphorus uptake demonstrated by immobilized algae were found to be much lower than those of non-immobilized cells. Uptake was dependent upon matrix stocking density, cell preculture conditions and cell viability, but not upon cell growth.     

Relationships between growth,cyclic amp and tylosin production in two mutants of Streptomyces fradiae

Summary A mutant of S. fradiae producing higher amounts of tylosin than its parent also showed higher intracellular cAMP and DNA. Similarly the addition of chloroquine to producing cultures of the parent strain significantly increased the production of tylosin, cAMP, and DNA. The most likely hypothesis is that cAMP acts on tylosin production through a stimulation of the synthesis of DNA, which may prevent aging of the producing cells and lead to higher overvall antibiotic production.     

Shikimate pathway activity in shake and fermenter cultures of Cinchona succirubra

Cell suspension cultures of Cinchona succirubra were cultivated in shake cultures and for the first time in airlift fermenters. Under both conditions L-tryptophan exerts a stimulatory effect on alkaloid formation. In this context the regulatory pattern of some shikimate pathway enzymes was investigated in non-supplemented and tryptophan supplemented Cinchona cell cultures. A remarkable increase of tryptophan decarboxylase (TDC) activity was observed in Cinchona cells under the influence of tryptophan. Apparently, like in some other indole alkaloid producing cell cultures, a high TDC activity is a prerequisite for alkaloid formation. Growth pattern and some enzyme activities of C. succirubra fermenter cultures at controlled and non-regulated pH levels were followed. Optimum growth and alkaloid formation were recorded under non-regulated (normal) pH conditions.Abbreviations TDC tryptophan decarboxylase - try L-tyrosine - phe L-phenylalanine - DAHP 3-deoxy-D-arabino-heptulosonic acid-7-phosphate - trp L-tryptophan - E-4-P erythrose-4-phosphate - PEP phosphoenolpyruvate - MDH malate dehydrogenase - G-6-PDH glucose-6-phosphate dehydrogenase - 6-PG-DH 6-phosphogluconate dehydrogenase - Ch-mutase chorismate mutase - AS-synthase anthranilate synthase - n.d. not determined     

Characteristics of yeast β-galactosidase immobilized on calcium alginate gels

Summary Saccharomyces anamensis having -galactosidase activity, has been immobilized in calcium alginate gel matrix that retained 78.6% enzyme activity to that of native cells. Optimum pH(7.0) was negligibly affected by immobilization. K_m values for immobilized and native cells were 119 mM and 102 mM respectively. Protective agents like dithioerythritol, bovine serum albumin, enhance the enzyme activity when added prior to immobilization. Immobilized cells can be stored in refrigeration(4°C) for 42 days without a significant loss of enzyme activity.     

Isolation of Bacillus licheniformis mutants for stable production profiles of alkaline protease

Summary A phenotypic requirement for cysteine was introduced inBacillus licheniformis producing alkaline protease to facilitate its isolation from poor or nonproducerBacillus species. This facilitated purification of the strain in cases of cross-contamination, preparation of good inocula for commercial production and stabilization of alkaline protease harvest values, alleviating economic losses incurred through cross-contamination.     

Metalloprotease ADAMTS-1 decreases cell migration and invasion modulating the spatiotemporal dynamics of Cdc42 activity

ADAMTSs (A Disintegrin And Metalloproteinase with ThromboSpondin motifs) are secreted proteases dependent on Zn²⁺/Ca²⁺, involved in physiological and pathological processes and are part of the extracellular matrix (ECM). Here, we investigated if ADAMTS-1 is required for invasion and migration of cells and the possible mechanism involved. In order to test ADAMTS-1's role in ovarian cancer cells (CHO, NIH-OVCAR-3 and ES2) and NIH-3 T3 fibroblasts, we modified the levels of ADAMTS-1 and compared those to parental. Cells exposed to ADAMTS-1-enriched medium exhibited a decline in cell migration and invasion when compared to controls with or without a functional metalloproteinase domain. The opposite was observed in cells when ADAMTS-1 was deleted via the CRISPR/Cas9 approach. The decline in ADAMTS-1 levels enhanced the phosphorylated form of Src and FAK. We also evaluated the activities of cellular Rho GTPases from cell lysates using the GLISA® kit. The Cdc42-GTP signal was significantly increased in the CRISPR ADAMTS-1 ES-2 cells. By a Förster resonance energy transfer (FRET) biosensor for Cdc42 activity in ES-2 cells we demonstrated that Cdc42 activity was strongly polarized at the leading edge of migrating cells with ADAMTS-1 deletion, compared to the wild type cells. As conclusion, ADAMTS-1 inhibits proliferation, polarization and migration.     

A rapid screening ofStreptomyces avermitilis production strains

Summary A simple biological test based on paralysis of movement of a free-living nematodeCaenorhabditis elegans, suitable for screening of mutant strains ofS.avermitilis producing a complex of avermectins is described.     

A simple and rapid method for selection of high diosgenin yielding clones ofDioscorea deltoidea callus

Summary Antimony pentachloride has been used to detect high diosgenin producing callus and cell clones ofDioscorea deltcidea grownin vitro. Using this method high yielding cultures, with a potential to produce up to 1.86% diosgenin, were selected.     

Phenylalanine ammonia-lyase activity in suspension cultures of Ulmus pumila and U. campestris treated with spores of Ceratocystis ulmi

Summary Cell suspension cultures of a Ceratocystis ulmi-resistant (Ulmus pumila) and a -susceptible elm (U.campestris) were established from leaf callus tissue. Treatment of cultures with spores of C.ulmi induced a large increase in the activity of phenylalanine ammonialyase, only in the cells of the resistant species U.pumila with a maximum after 24 h. Inoculated U.pumila cells also excreted a red unidentified chemical into the culture medium. Neither responses were induced in inoculated U.campestris cultures. The results are discussed in relation to the development of the elm cell culture system as a model for studying the differential biochemical mechanisms of disease resistance in elms.     

Fusion of Agrobacterium and E. coli spheroplasts with Nicotiana tabacum protoplasts — Direct gene transfer from microorganism to higher plant

Spheroplasts of Agrobacterium tumefaciens strains and E. coli were fused with protoplasts of Nicotiana tabacum. Fusion products were cultured in the presence of antibiotics to eliminate remaining bacterial spheroplasts. On hormone free medium, tobacco protoplasts treated with wild type Agrobacterium-strains formed colonies with an average frequency of 10^–4. Opine synthesis was detected in the tissues. Some calli derived from protoplasts treated with A. tumefaciens C58C1pRi15834 formed typical hairy roots. Kanamycin resistant calli were obtained after fusion with A. tumefaciens containing pLGVTi23 neo (frequency=10^–3). Fusion of E. coli spheroplasts containing a virulent pTiB6S3::RP4 co-integrate with tobacco protoplasts yielded two hormone independent growing calli producing octopine out of 10⁵ microcalli.Abbreviations PEG Polyethylene glycol - PVA Polyvinyl alcohol