A semi-continuous process for the production of human interferon-αc from E. coli using tangential-flow microfiltration and immuno-affinity chromatography

Summary A semi-continuous process for the production of human interferon-c (IFN) from E. coli is described. Harvesting of bacteria, removal of cell debris and concentration were performed by tangentialflow microfiltration (cross-flow microfiltration). Purification was done in a single step by a monoclonal-Ab column. In the process, purified material, 3–9×10⁸ units/mg protein was obtained with a 65% overall yield.     

Clonal propagation of Leptospermum spp. by tissue culture

Propagation by axillary and multiple axillary bud development was achieved in three native Leptospermum spp. when axillary buds derived from nodal tissues ex mature plants were placed in benzylaminopurine media (0.04–1.0 M) containing macro- and micro-nutrients, sucrose (0.06 M) and a vitamin/amino acid supplement. Reduction of agar concentration from 0.8 to 0.2% greatly stimulated axillary bud development and growth in L. flavescens and L. brachyandrum. Rooting of axillary shoots was stimulated by 2,4-dichlorophenoxyacetic acid and p-chlorophenoxy acetic acid in L. flavescens at concentrations of 5 and 1 M respectively. In L. petersonii ssp. root initiation and development was favoured by -naphthoxyacetic acid (1 M) and in L. brachyandrum indole butyric acid and -naphthalene acetic acid (1 M) were almost equally effective.     

Ester synthesis from α-substituted carboxylic acid catalyzed by polyethylene glycol-modified lipase fromCandida cylindracea in benzene

Summary Lipase fromCandida cylindracea was coupled with polyethylene glycol(PEG). In contrast to the previously used lipase fromPseudomonas fluorescens, the modified lipase catalyzed the ester synthesis in benzene at 25°C from short-chain alcohols and - or -substituted carboxylic acid. Using the modified lipase, following esters were synthesized; pentyl -methylpentanate, methyl benzoate, and methyl retinoate.     

In vitro plant regeneration of leguminous trees (Albizia spp)

Hypocotyl explants of three leguminous forest tree species, Albizia amara, A. lucida and A. richardiana, have differentiated shoot buds on B₅ basal medium. Maximum number of shoots per explant developed on basal medium augmented with 2,4-D (0.1 M) in A. amara (2) and BA (10 M) for both A. lucida (2) and A. richardiana (1.6). Higher concentrations of auxins in the medium, in general, enhanced rooting and callusing but cytokinins promoted the growth of green calli. BA enchanced the differentiation of shoots in the three species. The in vitro grown shoots of A. amara and A. richardiana, after subculturing on B₅+1 M IAA developed roots (up to 30–40%). These plants have been successfully transferred to the field.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - BA 6-benzylaminopurine - BM Gamborg's B₅ medium with 0.9% agar+3% sucrose - IAA Indole-3-acetic acid - IBA Indole-3-butyric acid - Kn Kinetin - NAA -naphthaleneacetic acid     

Reversible phosphorylation of tonoplast proteins involves tonoplast-bound calcium-calmodulin-dependent protein kinase(s) and protein phosphatase(s)

In highly purified tonoplast fractions from Acer pseudoplatanus cells, the in vitro reversible phosphorylation of proteins affected only a restricted set of polypeptides. The phosphorylation process has been shown to be dramatically stimulated by calcium via the mediation of calmodulin as the transducer. The protein kinase(s) was totally inhibited by micromolar concentrations of a calmodulin antagonist. Tonoplast appears to be potentially a good experimental system for the evaluation of the effects of protein phosphorylation on membrane properties in plants.Abbreviations CaM calmodulin - EGTA ethylene glycol-bis-(-amino ethylether)N,N,NN-tetraacetate - SDS-PAGE sodium dodecylsulphate polyacrylamide gel electrophoresis - TCA trichloroacetic acid     

Shoot and root organogenesis of Camellia sasanqua

In vitro-derived shoot tips, (10 mm) taken from primary cultures of Camellia sasanqua L., were evaluated for organogenesis when cultured on a half-strength MS medium supplemented with various concentrations of NAA, IBA, BA and GA₃. Maximum shoot proliferation and growth for juvenile and mature tissue was obtained when 0.54 M NAA, 8.8 M BA plus 14.4 to 28.9 M GA₃ was added to the culture media, with a pH between 4.5 and 5.0. In vitro-derived shoots (20 mm) from mature C. sasanqua Day Dream and juvenile C. sasanqua cultures initiated roots in vitro after immersion in 2.5 mM IBA for 30 min. Sixty percent of the mature shoots and 90% of the juvenile shoots initiated roots within 3 weeks of treatment with IBA.Abbreviations MS Murashige and Skoog (1962) - IBA lH-indole-3-butanoic acid - BA N-(phenyl-methyl)-lH-purine-6-amine - GA₃ gibberellic acid - kinetin N-(Z-furanyl-methyl)-lH-purine-6-amine - NAA l-naphthaleneacetic acid - L Linear - Q Quadratic     

Comparison of different strains ofCellulomonas for production of cellulolytic and xylanolytic enzymes from biomass produced on saline lands

A comparison of the rate of carboxymethyl cellulase (CMCase), avicelase, xylanase, -glucosidase and -xylosidase production and rates of growth by 4 different strains ofCellulomonas revealed a wide range of behaviour; with some strains producing more CMCase, avicelase, xylanase, -glucosidase and -xylosidase from complex lignocellulosic (LC) biomass (from saline land) and CMC while some others producing small amounts of these enzymes. One strain,C. biazotea, was better with respect to enzyme production potential and growth behaviour than most of the other strains and has been chosen as a starting strain for genetic improvement for producing enzymes of the cellulase complex.     

On the structure and function of cytochrome b-559

A sumary of biochemical, biophysical, and molecular biological data is presented which led to the identification of two different polypeptides ( and , MW=9.16 and 4.27 kDa) in the cytochrome b-559 protein. The presence of a single His residue on each polypeptide, and the conclusion from spectroscopy that the heme coordination must be bis-histidine led to an obligatory requirement for coordination of a single heme through a heme cross-linked dimer. This structure does not have a precedent among soluble or membrane bound cytochromes. The possible participation of the cytochrome in the pathway of photoactivation is discussed.     

Plasmid stability during continuous culture in aSaccharomyces cerevisiae double mutant transformed by a plasmid carrying a eukaryotic gene

Summary An investigation of plasmid stability in aSaccharomyces cerevisiae double mutant has been performed. The host was a double recombinantura3 furl mutant containing a plasmid bearing the yeast URA3⁺ allele and an expression cassette for human ₁-antitrypsin. The mutant was grown in continuous culture employing a semi-defined medium containing added uracil to provide non selective growth conditions. After 150 generations of continuous growth, no cured cells had been detected: the specific expression level of ₁-antitrypsin remained constant throughout the experiment.     

The response ofDatura innoxia wild-type and auxotrophic cells to several enrichment agents

A number of growth-lethal agents were tested for their ability to discriminate between growing and non-growing cells of wild-type and several auxotrophic cell lines ofDatura innoxia P. Mill. Of these agents, 1--4-arabinofuranosylcytosine (ara-C), 5-FU and FUdR were shown to decrease the growth of an adenine-requiring auxotroph only slightly at concentrations at which wild-type cells were killed. Only nystatin and arsenate had a degree of discrimination with the adenine-requirer sufficient for counter-selection. However, neither of these agents could be used to differentiate between growing and non-growing cells of two otherDatura auxotrophs, an isoleucine-valine-and a pantothenate-requirer.The efficacy of general enrichment methods for plant cell auxotrophs is discussed.Abbreviations Ara-C 1--4-arabinofuranosylcytosine - FPGA filter-paper-growth-assay - 5-FU 5-fluorouridine - FUdR 5-fluoro-2-deoxyuridine - GDW glass-distilled water     

Influence of succinic acid on the extracellular α-amylase production by chemostat-crow Bacillus licheniformis

Summary An 8-fold increase in -amylase production by pulsing of succinic acid to a chemostat culture ofBacillus licheniformis has been shown. The -amylase concentration was found to be at the highest value two doubling times after the addition, indicating that the effect may be due to regulatory control.     

High productivity ethanol fermentations with Zymomonas mobilis using continuous cell recycle

Summary Cell recycle studies have been carried out with a strain of Zymomonas mobilis selected for its improved ethanol tolerance and faster rates of glucose uptake and ethanol production. As part of the investigation a capilliary cross-flow microfiltration unit with polyamide membranes has been evaluated in view of its potential advantages (low cost and ability to withstand repeated cleaning with caustic soda). The results demonstrate that ethanol concentrations of 60–65g/l can be sustained at productivities ranging from 120–200g/l/h.     

Continuous ethanol production by immobilized cells of Zymomonas mobilis

Summary Studies have been carried out with a highly productive strain of Zymomonas mobilis in an immobilized cell reactor using both Ca alginate and -carrageenan as supporting matrices. Productivities above 50 g/l/h have been found at ethanol concentrations in excess of 60 g/l. With immobilized cells of Z. mobilis, there was a decline of approximately 30s% in activity after 800 h operation.     

Immobilization of glucoamylase on naphthyl cotton cloth

Summary Aspergillus niger glucoamylase was adsorbed to -naphthyl cotton cloth by hydrophobic interaction. The adsorbed enzyme was cross-linked with glutaraldehyde. The immobilized glucoamylase exhibited greater pH dependence though the optimal pH did not change. The immobilized glucoamylase in a packed bed column completely hydrolysed 5% soluble starch at a specific velocity of about 4. Used naphthyl cloth could be regenerated by heating in 2 N NaOH at 100°C for 1 hour.     

Glucose production using immobilized mycelial-associated β-glucosidase of Trichoderma E58

Summary The immobilization of the mycelial-associated -glucosidase of Trichoderma E-58 has been carried out by encapsulating, in calcium alginate beads, the fungal mycelium obtained durinq liquid culture. The activity of this immobilized -glucosidase was found to vary with culture age and to be more thermally stable than the extracellular -glucosidase produced by this organism. The activity of the immobilized enzyme was successfully demonstrated in both static and shake-flask batch reaction mixtures at 50°C using both cellobiose and salicin as substrates.     

Comparative studies of the phytochrome system in cell cultures from different plants

Phytochrome contents have been assayed in vivo in cell suspension cultures of Petroselinum hortense, Daucus carota and Glycine max. After transferring the cells to fresh medium phytochrome increased in parallel with the increase in cell number, whereas the amount of phytochrome per cell remained constant. The rate of phytochrome reaccumulation after pretreatment with 15 h red light was very similar in all three systems (2.8–3.6 (e) 10^–5/h). Dark reversion and a fast and slow P_fr destruction were observed in all systems. The rate constants of these reactions varied strongly between the systems. The phytochrome systems of the cell cultures were compared with those of etiolated and light-grown seedlings and it was concluded that the cell suspension cultures of Petroselinum hortense and Daucus carota behaved similarly to light-grown seedlings. In contrast, those of Glycine max behaved similarly to a dark grown seedling.Abbreviations P_r'fr red, far-red absorbing forms of phytochrome - P_tot P_r+P_fr total amount of phytochrome - fwt fresh weight     

Callus formation from Malus x domestica cv. ‘Jonathan’ protoplasts

Protoplasts could be successfully isolated and cultured from callus and suspension cultures of Malus xdomestica cv. Jonathan. Protoplast-derived colonies were recovered when the osmoticum (glucose) was gradually reduced in semi-solid 8p medium or by the use of feeder plates. Formation of embryo-like structures was induced from the protoplast-derived callus on media supplemented with IAA and BA. These structures formed roots but plants failed to develop. Protoplasts could be isolated from leaves, but not from stems or petioles. The leaf protoplasts failed to divide.List of abbreviations BA benzyladenine - 2,4-D 2,4-dichlorophenoxyacetic acid - ABA abscisic acid - IAA indole acetic acid     

High resolution optical fibre thermometer: Applications to biotechnology

Summary The temperature rise on addition of glucose to a cell suspension of Pseudomonas fluorescens has been monitored using monomode optical fibre interferometric techniques. A temperature rise of -0.5°C is measured, however the technique is shown to be capable of ultimate resolution in the K–mK range.     

Dynamic mechanical analysis as a technique for the study of fungal degradation of wood

Summary Dynamic mechanical analysis has been used to follow changes in the properties of wood (Pinus sylvestris) subject to attack by Coniophora puteana. Shear storage modulus fell steadily over the 42 day observation period and correlated with weight loss. Tan shows relatively little change implying uniform degradation of the structural polymers.     

Characterization of two β-glucosidase genes fromClostridium thermocellum

Summary Two -glucosidase genes, designatedbglA andbglB, were isolated from a gene bank ofClostridium thermocellum DSM 1237. The coding sequences forbglA andbglB were located on non-homologous DNA fragments of 3.2– and 3.4-kb, respectively. Both genes direct inEscherichia coli the synthesis of cytoplasmic -glucosidases, which differ with respect to substrate specificity and temperature profile. The properties of thebglA-encoded -glucosidase A closely resemble that of a -glucosidase previously isolated fromC. thermocellum cultures.