Showing papers in "Biotechnology Techniques in 1994"
TL;DR: In this paper, the recovery of poly-3-hydroxybutyrate (PHB) in three chlorinated solvents with or without acetone pretreatment and degradation of extracted PHB (99% pure) in hot chloroform were studied.
Abstract: Recovery of poly-3-hydroxybutyrate (PHB) in three chlorinated solvents with or without acetone pretreatment and degradation of extracted PHB (99% pure) in hot chloroform were studied. When lyophilized Alcaligenes eutrophus biomass was used, the best results were obtained with acetone pretreatment and solvent reflux for 15 min in methylene chloride or chloroform. Recovered PHB had a 95% purity and molecular weights (Mw) of 1,050,000 and 930,000 g/mol respectively. Further heating resulted in a serious Mw, loss at reflux temperatures. Degradation of extracted PHB at 110°C in chloroform was due to random and chain-end scission, the former being predominant.
197 citations
TL;DR: Plasmid, pBI221, was introduced into protoplasts of Chlorella saccharophila c-211-1a prepared from the cells in the stationary phase by electroporation, where high membrane permeability to 70-kDa FITC-dextran was ascertained.
Abstract: Plasmid, pBI221, was introduced into protoplasts of Chlorella saccharophila c-211-1a prepared from the cells in the stationary phase by electroporation. Transient expression of the introduced plasmid was observed under a field strength of between 600 and 900 V/cm, and a pulse duration of around 400 ms, where high membrane permeability to 70-kDa FITC-dextran was ascertained.
68 citations
TL;DR: The accuracy and reproducibility of the gas-chromatographic method for the analysis of PHB and PHA in whole cells of Alcaligenes eutrophus H16 and Pseudomonas putida KT2442 were determined and it was found that for analysis of PHA the methanolysis time in the assay had to be increased to 4 h.
Abstract: The accuracy and reproducibility of the gas-chromatographic method for the analysis of PHB and PHA in whole cells of Alcaligenes eutrophus H16 and Pseudomonas putida KT2442 were determined. It was found that for analysis of PHA the methanolysis time in the assay had to be increased to 4 h. Accuracy of the PHB and PHA assay were 0.018 mg and 0.304 mg respectively, based on estimation of the measurement error.
57 citations
TL;DR: Using an ethanol solution of nile blue, an efficient method is developed to detect the colonies of poly(3-hydroxyalkanoic acids) (PHA) producing bacteria on the agar plate and fluorescence intensity increased with an increase in the PHA content of bacterial cells.
Abstract: Using an ethanol solution of nile blue, we have developed an efficient method to detect the colonies of poly(3-hydroxyalkanoic acids) (PHA) producing bacteria on the agar plate. When the bacterial colonies with PHA granules were stained with nile blue, the stained colonies fluoresced bright orange on the irradiation of UV light. In the fluoresce emission spectra, fluorescence intensity increased with an increase in the PHA content of bacterial cells.Alcaligenes eutrophus andA.latus colonies with poly(3-hydroxybutyric acid) (PHB) homopolymer exhibited an emission maximum at 580nm on the excitation at 490nm. On the other hand,Pseudomonas oleovorans andP.putida with medium-chain-length (mcl-) PHA copolymers of C6, C8 and C10 units exhibited an emission maximum at 570nm.
57 citations
TL;DR: Two most commonly used amino acid media supplements do not improve the specific rate of secretion of Schizosaccharomyces pombe acid phosphatase in S. cerevisiae relative to unsupplemented Synthetic Dextrose media.
Abstract: The two most commonly used amino acid media supplements do not improve the specific rate of secretion of Schizosaccharomyces pombe acid phosphatase in S. cerevisiae relative to unsupplemented Synthetic Dextrose (SD) media. New amino acid supplement formulations have been obtained which produce an 8-fold increase in total phosphatase secretion, and a 70% increase in specific phosphatase secretion.
51 citations
TL;DR: Endo-xylanase from T. viride has been purified 4.2 fold by precipitation with a commercially available enteric polymer Eudragit S-100 and the binding of the enzyme to the polymer was predominantly by electrostatic interaction.
Abstract: Endo-xylanase from T. viride has been purified 4.2 fold by precipitation with a commercially available enteric polymer Eudragit S-100. Electrophoretic analysis also indicated removal of contaminant proteins. The enzyme could be recovered in more than 89% yield. The binding of the enzyme to the polymer was predominantly by electrostatic interaction.
49 citations
TL;DR: The comparison of the behaviour of the two strains grown with or without ethanol enabled us to demonstrate the existing relationship between ethanol tolerance and membrane fluidity.
Abstract: Direct measurements of membrane fluidity by fluorescence anisotropy of protoplasts fromKloeckera apiculata andSaccharomyces cerevisiae, a low and a high ethanol tolerant strain respectively, are presented. The comparison of the behaviour of the two strains grown with or without ethanol enabled us to demonstrate the existing relationship between ethanol tolerance and membrane fluidity.
46 citations
TL;DR: Inoculation of large-scale plant root culture reactors can be carried out by briefly homogenizing bulk root tissue, followed by aseptic transfer as a slurry to the reactor, which is subsequently operated as a trickle bed reactor.
Abstract: Inoculation of large-scale plant root culture reactors can be carried out by briefly homogenizing bulk root tissue, followed by aseptic transfer as a slurry to the reactor. Uniform root distribution can be achieved in bioreactors by entrapment of the growing root inoculum onto process packing elements (e.g. distillation packings), randomly distributed within the reactor, in a bubble column operation. These two techniques have been successfully used to inoculate a 14 L pilot-scale reactor which is subsequently operated as a trickle bed reactor.
43 citations
TL;DR: The purification procedure includes Phenyl-Sepharose and Sephacryl HR 100 chromatographies and the enzymes obtained were pure according to criteria of specific activity and neutral sugar content.
Abstract: We describe a two-step method for the purification of two lipases (lipases A and B) from C. rugosa. The purification procedure includes Phenyl-Sepharose and Sephacryl HR 100 chromatographies. The enzymes obtained were pure according to criteria of specific activity and neutral sugar content.
40 citations
TL;DR: The nitrogen source and KH2PO4 concentration of the production medium had a marked effect on culture pH during the course of fermentation and, consequently, on xylanase activity.
Abstract: Endo-1,4-β-glucanase and xylanase were produced by Trichoderma reesei immobilized on polyurethane foam using lactose as the main carbon source. The most porous carrier was found to be the best of those tested. The nitrogen source and KH2PO4 concentration of the production medium had a marked effect on culture pH during the course of fermentation and, consequently, on xylanase activity. An increase in lactose concentration from 7 to 27 g/l resulted in an increase in endoglucanase activity (max. 730 U/ml), xylanase activity (max. 3350 U/ml) and filter paper activity (max. 3.0 FPU/ml).
38 citations
TL;DR: A photobioreactor using three concentric glass cylinders, with a light source mounted on the axis within them, with the culture of phototrophic microorganisms in the chamber between the outer cylinder and the middle one is described.
Abstract: A photobioreactor using three concentric glass cylinders, with a light source mounted on the axis within them is described. The space between two innermost cylinders is used as a waterjacket, while the culture of phototrophic microorganisms is in the chamber between the outer cylinder and the middle one. This chamber is also equipped with a stirrer. Rhodobacter capsulatus has been grown in the device at biomass concentration up to 550 mg/l without light limitation.
TL;DR: The authors showed that 1% (v/v) olive oil was as good as 0.1% polypropylene glycol when added to a yeast fermentation at 400 rpm, and 1% was better than 0.25% at 400rpm.
Abstract: Adding lard oil, olive oil or castor oil into a baffled and agitated fermentor decreased the volumetric oxygen transfer coefficient (KLa) in the low concentration range. However, KLa was increased when concentration reached 0.25% (v/v) at 400 rpm. Experimental results indicated that 1% (v/v) olive oil was as good as 0.1% (v/v) polypropylene glycol when added to a yeast fermentation.
TL;DR: The isolation of two Tuber magnatum Pico markers could be used as a probe in dot blot hybridization, allowing the development of a rapid test able to identify Tuer magnatum species.
Abstract: Different species of truffle were studied in order to identify species-specific markers. The isolation of two Tuber magnatum Pico markers is reported. One of these could be used as a probe in dot blot hybridization, allowing the development of a rapid test able to identify Tuber magnatum species.
TL;DR: Hairy roots of Catharanthus roseus were cultivated in three different types of bioreactors and the best growth and indole alkaloid production was achieved in an airsparged bioreactor with no other mixing.
Abstract: Hairy roots of Catharanthus roseus were cultivated in three different types of bioreactors. The best growth and indole alkaloid production was achieved in an airsparged bioreactor with no other mixing. In the stirred bioreactor or in the bioreactor with medium circulation the roots did not grow, suggesting that hairy roots of C. roseus are more sensitive to stress than root cultures of many other plant species.
TL;DR: The best isolation procedure to obtain the EPS of Volcaniella eurihalina was centrifugation at 36,000 x g for 60 min and precipitation with cold ethanol after tangential filtration with 100,000 D ultrafilters.
Abstract: The best isolation procedure to obtain the EPS of Volcaniella eurihalina was centrifugation at 36,000 x g for 60 min and precipitation with cold ethanol after tangential filtration with 100,000 D ultrafilters. Ion chromatography showed that this EPS contained glucose, rhamnose and mannose in a molar ratio of 3.2: 1.1:1, respectively.
TL;DR: Image analysis has been used to investigate the effect of spore inoculum concentration on the subsequent morphology of Penicillium chrysogenum grown in both shake flasks and a 6L agitated bioreactor.
Abstract: Image analysis has been used to investigate the effect of spore inoculum concentration on the subsequent morphology of Penicillium chrysogenum grown in both shake flasks and a 6L agitated bioreactor. Even with the same inoculum concentration, differences were found between the two systems in the eventual morphology of both the freely dispersed mycelia and the mycelial aggregates or “clumps”.
TL;DR: The adsorption technique was superior to the entrapment technique, and it was possible to obtain bioparticles capable of keeping their activity for more than 25 days.
Abstract: Aspergillus niger was immobilized by adsorption and entrapment in polyurethane foams and the efficiency of retention capacity, citric acid productivity and the operational stability of a fluidized bed reactor were then compared. The adsorption technique was superior to the entrapment technique, and it was possible to obtain bioparticles capable of keeping their activity for more than 25 days.
TL;DR: A 130 litre packed-bed bioreactor was used for pectinase production with Aspergillus niger using absorbed substrate fermentation techniques, and pectinolytic enzyme activity and relative CO2 production were used as indicators of metabolic activity.
Abstract: A 130 litre packed-bed bioreactor was used for pectinase production with Aspergillus niger using absorbed substrate fermentation techniques. Pectinolytic enzyme activity and relative CO2 production were used as indicators of metabolic activity. Absorbed substrate fermentation is an efficient process for pectinase production and is also an interesting model because the culture medium, water, nutrients and specific inducers, can be designed at the desired concentrations.
TL;DR: In this paper, the enzyme was almost completely resistant to deactivation by acetaldehyde, which is liberated as unavoidable byproduct in acyl-transfer reactions with vinyl esters, and the enantioselectivity was enhanced up to threefold.
Abstract: Immobilization of Candida rugosa lipase (Amano AY-30) by adsorption onto Celite 545 led to a markedly improved performance of the enzyme: Firstly, the enzyme was almost completely resistant to deactivation by acetaldehyde, which is liberated as unavoidable by-product in acyl-transfer reactions with vinyl esters, and secondly, the enantioselectivity was enhanced up to three-fold.
TL;DR: In this paper, near-infrared spectroscopy was used to determine biomass and glycerol concentrations in E.coli whole-broth fermentation samples and a standard error of prediction (SEP) of 0.2 g/L and correlation coefficient (r) was obtained.
Abstract: Near-infrared spectroscopy was used to determine biomass and glycerol concentrations in E.coli whole broth fermentation samples. For dry cell weight, a standard error of prediction (SEP) of 0.2 g/L and correlation coefficient (r) of 0.991 were obtained. The SEP and r for glycerol, carbon nutrient, were 0.3 g/L and 0.979. respectively. Off-line analysis was accomplished within 2 minutes of sampling and therefore provides the opportunity to monitor fermentations quickly enough to permit in-process development and troubleshooting.
TL;DR: In this paper, optimal conditions for handling biomass samples recovered from acid and alkaline pretreatments prior to traditional compositional analysis were developed and methods for processing slurries from simultaneous saccharification and fermentation (SSF) were also determined.
Abstract: The successful implementation of simultaneous saccharification and fermentation (SSF) technology for biomass conversion to ethanol requires competent analysis of complex biomass process streams, often obtained at extremes of pH. In this study, optimal conditions for handling biomass samples recovered from acid and alkaline pretreatments prior to traditional compositional analysis were developed. Methods for processing slurries from SSF were also determined. In both cases, a mixed wastepaper feedstock was used to test improved handling procedures and to document recommended performance.
TL;DR: An ultrasonication protocol for Escherichia coli recombinant cells is developed and optimized, adapted to laboratory-scale release of β-galactosidase fusion proteins, which allows the fusion proteins to maintain the active form throughout the process.
Abstract: In this work, we have developed and optimized an ultrasonication protocol for Escherichia coli recombinant cells, adapted to laboratory-scale release of β-galactosidase fusion proteins. After a single sonication treatment of 15 minutes, about 30% of recombinant protein present in the sample remains still associated to cellular debris, and it can not be removed by increasing the sonication time. After a clarification step a second sonication treatment of the resuspended cell debris again releases only a 70% of the remaining product. Therefore, the application of two short, consecutive sonication treatments permits a global recovery yield of about 90%. The use of a new disruption buffer to stabilize β-galactosidase allows the fusion proteins to maintain the active form throughout the process.
TL;DR: A large scale plasmid isolation technique is described for the isolation of plasmids from exopolysaccharide producing strains of Lactobacillus spp.
Abstract: A large scale plasmid isolation technique is described for the isolation of plasmids from exopolysaccharide producing strains of Lactobacillus spp. Plasmids of 1.9 to 56 kb were isolated which were pure enough to be used for restriction analysis and cloning experiments.
TL;DR: An electroporation-induced transformation system was developed for Leuconostoc oenos and of the three plasmids tested, only pGK13 was expressed, and transformation efficiencies of 1 X 103 were obtained.
Abstract: An electroporation-induced transformation system was developed forLeuconostoc oenos. Of the three plasmids tested (pGK13, pAMS100 and pIL253), only pGK13 was expressed. Several parameters were studied with possible influence on transformation efficiency. Electroporation conditions of 2000 V/cm, 25μF and 200 ohm proved successful. Transformation efficiencies (number of cells/μg plasmid DNA) of 1 X 103 were obtained.
TL;DR: In this paper, a H+-selective liquid membrane microelectrode was used to measure the pH profile evolution during colonization of gel beads immobilizing Lactobacillus helveticus in a whey permeate medium.
Abstract: A H+-selective liquid membrane microelectrode was prepared and used to measure the pH profile evolution during colonization of gel beads immobilizing Lactobacillus helveticus in a whey permeate medium. A large pH gradient was observed in a highly active periferal layer thickness that decreased from 500 to 300 μm for an immobilized cell population that increased from 5.8 × 109 to 3.1 × 1010 CFU/g. The flat pH profile (pH 4.4) in the central part of the bead was attributed to a high concentration of the inhibitory undissociated form of lactic acid.
TL;DR: A continuous perforated rotating disc contactor was used for the extraction of a recombinant cutinase from an aqueous solution to a reversed micellar phase of AOT in isooctane with protein yield of 78% after 70 minutes of operation.
Abstract: A continuous perforated rotating disc contactor was used for the extraction of a recombinant cutinase from an aqueous solution to a reversed micellar phase of AOT in isooctane. Cutinase was extracted to the organic phase with protein yield of 78% after 70 minutes of operation.
TL;DR: A novel cultivation method using a microporous membrane (membrane-surface liquid culture) was developed, in which moulds are grown on the membrane surface with its opposite side being in contact with a liquid medium.
Abstract: A novel cultivation method using a microporous membrane (membrane-surface liquid culture) was developed, in which moulds are grown on the membrane surface with its opposite side being in contact with a liquid medium. The amount of neutral protease from Aspergillus oryzae produced was more than 10 times higher than that produced by the conventional liquid culture.
TL;DR: In all cases, short-term or long-term uptake and single ion solution or metal mixtures, whole cells showed the highest metal accumulation while hcw adsorbed more metal ions than mcw.
Abstract: Short-term uptake of cadmium, copper and lead by whole cells, mother cell walls (mcw) and cell walls from cell homogenates (hcw) of Chlorella fusca were similar for all biosorbents: Adsorption equilibrium was reached five minutes after metal addition. Analogous, over 90% of the readily adsorbed metal ions could be desorbed by a 1 minute EDTA treatment. In all cases, short-term or long-term uptake and single ion solution or metal mixtures, whole cells showed the highest metal accumulation while hcw adsorbed more metal ions than mcw. The role of the cell wall for metal ion adsorption is discussed.
TL;DR: A Flow Injection Analysis for glucose using glucose oxidase (E.C.3.1.4) was developed and was able to measure glucose at levels from 5 to 500mM with a response time of 2min using 250μl of sample.
Abstract: A Flow Injection Analysis (FIA) for glucose using glucose oxidase (E.C.1.1.3.4) was developed. The enzyme was immobilised on polyaniline chemically synthetized on internal surface of silicon tube (2.0 mm of diameter and length from 1 to 5 meters), using glutaraldehyde as a bifunctional agent. The system was able to measure glucose at levels from 5 to 500mM with a response time of 2min using 250μl of sample. The system has been operated satisfactorily for one month with more than 300 assays with loss of 60% of activity.
TL;DR: The polyethylene glycol-albumin hydrogel showed high protein retention and a very good swelling capacity and a new and rapid method for preparing a protaneous-polymeric hydrogels is described.
Abstract: A new and rapid method for preparing a protaneous-polymeric hydrogel is described. The gel matrix is obtained by covalent cross-linking between preactivated polyethylene glycol of 3350 molecular mass and bovine serum albumin in aqueous solution at basic pH. The polyethylene glycol-albumin hydrogel showed high protein retention and a very good swelling capacity.