Biomedical subjects

A Fiechter

Publications and source records attributed to A Fiechter.

At least 19 recordsLinked to original sources

PubMed1992

DNA transformations of Candida tropicalis with replicating and integrative vectors.

The alkane-assimilating yeast Candida tropicalis was used as a host for DNA transformations. A stable ade2 mutant (Ha900) obtained by UV-mutagenesis was used as a recipient for different vectors carrying selectable markers. A first vector, pMK16, that was developed for the transformation of C. albicans and carries an ADE2 gene marker and a Candida autonomously replicating sequence (CARS) element promoting autonomous replication, was compatible for transforming Ha900. Two transformant types were observed: (i) pink transformants which easily lose pMK16 under non-selective growth conditions; (ii) white transformants, in which the same plasmid exhibited a higher mitotic stability. In both cases pMK16 could be rescued from these cells in Escherichia coli. A second vector, pADE2, containing the isolated C. tropicalis ADE2, gene, was used to transform Ha900. This vector integrated in the yeast genome at homologous sites of the ade2 locus. Different integration types were observed at one or both ade2 alleles in single or in tandem repeats.

Alcohol Dehydrogenase↗

PubMed1992

Biosurfactants: moving towards industrial application.

Chemically synthesized surface-active compounds are widely used in the pharmaceutical, cosmetic, petroleum and food industries. However, with the advantages of biodegradability, and production on renewable-resource substrates, biosurfactants may eventually replace their chemically synthesized counterparts. So far, the use of biosurfactants has been limited to a few specialized applications because biosurfactants have been economically uncompetitive. There is a need to gain a greater understanding of the physiology, genetics and biochemistry of biosurfactant-producing strains, and to improve process technology to reduce production costs.

Amino Acid Sequence↗

PubMed1992

Biomass determination.

The reasons for and historical backgrounds of biomass determination are discussed under the aspects of theoretical and practical importance, usefulness and representativity. Off-line methods are evaluated and compared with on-line methods; constraints of applications and conclusiveness of results are rated. Special emphasis is given to the fact that mere knowledge of a bio-mass concentration is not sufficiently valuable to learn more about physiology nor to determine the effectiveness of a biotechnological process. A combination of several different alternative measuring principles in parallel as well as the exploitation of software sensors is proposed as a promising future solution.

Ecology↗

PubMed1992

On-line measurement in biotechnology: techniques.

Bioprocesses are generally ill controlled. This is due to the fact that the measurement of relevant variables is difficult. Therefore, fundamental knowledge of metabolic interrelations is, at least in vivo, limited. In this article, some of the most important measurement techniques are reviewed in order to provide an evaluation of their current state. Emphasis is given to the underlying principles and on-line capability which allow to judge their importance and potential for exploitation resulting in well (maybe entirely) controlled bioprocesses in the future.

Biosensing Techniques↗

PubMed1992

On-line measurement in biotechnology: exploitation, objectives and benefits.

Sound data biologically relevant are prerequisites when developing high-performance bioprocesses. Understanding of physiological regulation as well as sophisticated control strategies are highly dependent on the observability of the culture, i.e. the generation and exploitation of suited signals even under complex environmental measurement conditions. Against this background, the increasing number of analytical systems is very supportive and, accordingly, an appropriate handling of sensors and measured data is of decisive importance. This article reports on practical experience with routines for maintenance, service and calibration of hardware sensors which improve the quality of measurements significantly. Verification and validation of signals is outlined in order to make the value of data exploitation tools obvious. A method for the characterization of information is introduced by practical examples of Saccharomyces cerevisiae cultures when explaining the specific properties of extracting biological information from raw data. Finally, examples for advantageous exploitation of on-line data are given.

Biosensing Techniques↗

PubMed1992

The decisive role of the Saccharomyces cerevisiae cell cycle behaviour for dynamic growth characterization.

The dynamic behaviour of the cell cycle and the physiology of Saccharomyces cerevisiae was monitored in transient experiments. Frequent flow cytometric analyses of the DNA (nuclear phase state) and the cell size enabled us to characterize the proliferation properties of yeast cells under well controlled and undisturbed cultivation conditions. Preliminarily, the correlation between flow cytometric light scattering measurements and the cell size was attested for yeasts. These flow cytometric results are compared with the physiological behaviour of the culture that was detected by high resolution on-line analyses and off-line measurements. The presented results focus on the importance of the yeast cell cycle behaviour for the dynamic growth characterization. Any kind of transients in yeast cultures induced partial synchronization. The characteristics and the time course of the yeast cell cycle were found to be strongly dependent on the physiological environment.

Cell Cycle↗

PubMed1992

Identification and characterization of additional members of the cytochrome P450 multigene family CYP52 of Candida tropicalis.

Using different DNA probes from the first two previously described alkane-inducible cytochrome P450 genes of the Candida tropicalis CYP52 gene family, we isolated five independent additional members by screening a genomic library under low-stringency conditions. These genes are not allelic variants and, when taken gogether, constitute the largest gene family known in this organism. The seven members of this gene family are located on four different chromosomes and four of them are tandemly arranged on the C. tropicalis genome. The products of the seven genes, alk1 to alk7, were compared to each other and revealed a high degree of divergence: the two most diverged proteins exhibit a sequence identity of only 32%. Six of the seven genes were shown to be induced by a variety of different aliphatic carbon sources but repressed when the organism was grown on glucose. Three of the five additional CYP52 genes could be successfully expressed in Saccharomyces cerevisiae and display different substrate specificities in in vitro assays with model substrates: alk2 and alk3 exhibit a strong preference for hexadecane, while alk4 and alk5 preferentially hydroxylate lauric acid.

Amino Acid Sequence↗

PubMed1992

Purification and characterization of two alpha-L-arabinofuranosidases from Streptomyces diastaticus.

A nonsporulating strain of Streptomyces diastaticus producing alpha-L-arabinofuranosidase activity (EC 3.2-1.55) was isolated from soil. Two alpha-L-arabinosidases were purified by ion-exchange chromatography and chromatofocusing. The enzymes had molecular weights of 38,000 (C1) and 60,000 (C2) and pIs of 8.8 and 8.3, respectively. The optimum pH range of activity for both enzymes was between 4 and 7. The apparent Km values with p-nitrophenyl arabinofuranoside as the substrate were 10 mM (C1) and 12.5 mM (C2). C1 retained 50% of its activity after 8 h of incubation at 25 degrees C, while C2 retained 80% activity. After 3 h of incubation at 50 degrees C, C1 lost 90% of its initial activity while C2 lost only 40%. The purified enzymes hydrolyzed p-nitrophenyl alpha-L-arabinofuranoside and liberated arabinose from arabinoxylan and from a debranched beta-1,5-arabinan.

Glycoside Hydrolases↗

PubMed1992

Phenol hydroxylase from Trichosporon cutaneum: gene cloning, sequence analysis, and functional expression in Escherichia coli.

A cDNA clone encoding phenol hydroxylase from the soil yeast Trichosporon cutaneum was isolated and characterized. The clone was identified by hybridization screening of a bacteriophage lambda ZAP-based cDNA library with an oligonucleotide probe which corresponded to the N-terminal amino acid sequence of the purified enzyme. The cDNA encodes a protein consisting of 664 amino acids. Amino acid sequences of a number of peptides obtained by Edman degradation of various cleavage products of the purified enzyme were identified in the cDNA-derived sequence. The phenol hydroxylase cDNA was expressed in Escherichia coli to yield high levels of active enzyme. The E. coli-derived phenol hydroxylase is very similar to the T. cutaneum enzyme with respect to the range of substrates acted upon, inhibition by excess phenol, and the order of magnitude of kinetic parameters in the overall reaction. Southern blot analysis revealed the presence of phenol hydroxylase gene-related sequences in a number of T. cutaneum and Trichosporon beigelii strains and in Cryptococcus elinovii but not in Trichosporon pullulans, Trichosporon penicillatum, or Candida tropicalis.

Amino Acid Sequence↗

PubMed1991

Characterization of a second alkane-inducible cytochrome P450-encoding gene, CYP52A2, from Candida tropicalis.

A second alkane-inducible cytochrome P450-encoding gene (CYP52A2) from the yeast Candida tropicalis was sequenced and characterized. CYP52A2 is located 1 kb upstream from CYP52A1, the previously characterized P450 gene [Sanglard and Loper, Gene 76 (1989) 121-136] and shows the same orientation. Like CYP52A1, CYP52A2 is induced by growth on alkane. Both promoter regions share repeats of the sequence CATGTGAA that could be of importance for the induction of the two genes. At the amino acid level, alk2 shows an overall identity of 68.2% and an overall similarity of 81.6% to alk1. Regions of high homology between the two proteins are found in the distal and proximal heme binding sites which contain the highly conserved cysteine residue as the fifth ligand to the heme iron. However, marked differences between the two proteins exist at their N-terminal end, which includes the transmembrane domain, and at the putative substrate-binding domain. Upon expression of CYP52A2 in Saccharomyces cerevisiae, alk2 was shown to hydroxylate hexadecane, but had no hydroxylation activity towards lauric acid, whereas alk1 showed both activities. Comparative immunoblots demonstrate that neither alk1 nor alk2 expressed in S. cerevisiae corresponds to the main cytochrome P450 present in C. tropicalis when grown on alkane.

Alkanes↗

PubMed1991

The characterisation of immobilised lignin peroxidase by flow injection analysis.

Immobilised lignin peroxidase has been investigated using a flow system in the steady state and by flow injection analysis (FIA). In the steady state, the extreme sensitivity of the enzyme towards inactivation by H2O2 resulted in a stable response only in the presence of saturating levels of organic substrate and at very low (10 microM) peroxide concentrations. By contrast, the low contact time during FIA led to a stable response to injections of 100 microM H2O2. At higher peroxide concentrations a reproducible inactivation was observed, allowing a study of factors affecting both activity and stability. Lignin peroxidase substrates that undergo at least semi-reversible oxidation/reduction, including high-molecular-weight lignin fractions, could be detected by electrochemical reduction of the oxidation products. With this detection system it was possible to demonstrate the role of veratryl alcohol as mediator. This mediated oxidation of lignin functioned only when all components were present simultaneously, and was not observed when lignin was separated from the site of veratryl alcohol oxidation.

Acetonitriles↗

PubMed1991

Genetic transformation of auxotrophic mutants of the filamentous yeast Trichosporon cutaneum using homologous and heterologous marker genes.

A transformation system for the filamentous yeast Trichosporon cutaneum based on auxotrophic markers is presented and techniques for the induction, isolation and characterization of mutants are described. A number of auxotrophic mutants were isolated and characterized by using biosynthetic precursors and/or inhibitors. A mutant unable to grow in the presence of ornithine could be complemented successfully by spheroplast transformation experiments using the cloned Aspergillus nidulans ornithine transcarbamoylase gene (argB gene) as selection marker with an efficiency of 5-100 transformants per microgram of DNA. In these transformants the heterologous argB gene was present in multiple tandem copies and the transforming DNA was found to remain stable after more than 50 generations in non-selective media. The same mutant could be complemented by a T. cutaneum cosmid gene library and a complementary cosmid was subsequently isolated from this library by a sib-selection strategy. This cosmid transformed T. cutaneum spheroblasts with an efficiency of 50-200 colonies per microgram of DNA. Southern blot analyses were consistent with the view that the transforming sequences became stably integrated into the host genome at the homologous site.

Arginine↗

PubMed1991

Biosurfactants from Bacillus licheniformis: structural analysis and characterization.

The surface-active compounds of the strain Bacillus licheniformis were isolated and their structure was elucidated. The high surface-active capacity of the crude extract was basically due to traces of long-chain saturated fatty acids, especially of palmitic and stearic acids, to a mixture of small amounts of hydrocarbons with chain lengths of 20 and 22 carbons, and mainly to appreciable amounts of four slightly different lipopeptides. The lipopeptides were found to be a mixture of four closely related compounds. The lipophilic part consisting of i-, n-C14 or i-, ai-C15 beta-OH fatty acids was linked to the hydrophilic peptide moiety, which contained seven amino acids (Glu, Asp, Val, three Leu and Ile) by a lactone linkage. Fifteen milligrams per litre of the purified lipopeptide product decreased the surface tension of water from 72 mN m-1 to 27 mN m-1, characterizing the product as a powerful surface-active agent that compares favourably to other (bio)surfactants. Antibiotic activity was demonstrated against bacteria and yeasts.

Amino Acid Sequence↗

PubMed1991

Optimization of Candida tropicalis cytochrome P450alk gene expression in Saccharomyces cerevisiae with continuous cultures.

The cytochrome P450alk gene (P45alk) from Candida tropicalis ATCC 750 was expressed in Saccharomyces cerevisiae GRF18 under control of the alcohol dehydrogenase I (ADHI) promoter. To achieve stable expression over long time periods, a 2-microns derived replicative and an integrative expression system were tested in continuous culture. The 2-microns derived replicative system could not be maintained in cells over high generation numbers. In continuous culture, the instability was more pronounced at high dilution rates (D) and high histidine concentration, for which the yeast is auxotrophic. The nature of the instability was probably due to a gene conversion event between the plasmid and the yeast chromosome. In contrast, the integrative expression system was stably maintained in cells over prolonged cultivation times. Since this work focused on the production of large quantities of P450 by heterologous expression in yeast over prolonged time periods, the integrant was used to optimize P450alk expression by varying continuous culture parameters. The P450alk expression was shown to be dependent on the D applied to the culture. The highest P450alk expression levels were obtained at high D, when cell metabolism was shifted to partial glucose oxidation, yielding ethanol as a major metabolite in the culture supernatant. In contrast, when glucose was completely oxidized at low D, the ADHI-dependent P450alk expression was reduced and followed by a corresponding decrease in heterologous protein.

Alcohol Dehydrogenase↗

PubMed1991

Automatic bioprocess control. 2. Implementations and practical experiences.

Our improved implementation for bioprocess control allows flexible responses to many process needs. It is based on computer equipment consisting of three hierarchically ordered levels. On the lowest level, a DDC slave computer handles setpoints and simple tasks generating the chemical and physical environment for the cells. It can be designed manually by the user or automatically by the supervisory computer on the second level. This provides for raw data organization, analysis and interpretation either to support personnel on line in decision making, to select predefined control strategies, or even to search for others. In the coordinating computer on the third level, common tasks of different supervisory computers (bioprocesses) are shared, saving money for the equipment. Tasks and concepts as well as experimental experiences are described to outline the capabilities of the configuration.

Biotechnology↗

PubMed1991

Automatic bioprocess control. 1. A general concept.

Automation of bioprocesses is presented and discussed. A general concept is applied to laboratory scale reactors as well as to large scale production facilities consisting of many unit operations with a hierarchical and highly modular structure. The implementation of non-dedicated and intelligent analytical subsystems is foreseen. Hard- and software requirements are discussed in view of the functional requirements of both scientific research and production engineering. Some practical experience is reported using several different components in parallel installations.

Biotechnology↗

PubMed1991

On-line determination of glucose in biotechnological processes: comparison between FIA and an in situ enzyme electrode.

Two different analysis techniques for on-line monitoring of glucose in biotechnological processes have been tested: an in situ enzyme electrode and a flow injection analysis system (FIA). The measuring ranges, detection limits, response times and the reliabilities of each system have been compared during monitoring of batch and continuous cultures of Saccharomyces cerevisiae.

Computers↗

PubMed1991

Degradation of organochlorine compounds in spent sulfite bleach plant effluents by actinomycetes.

Actinomycetes isolated from different soil samples were tested for their abilities to utilize spent sulfite bleach effluents from a paper mill. Degradation and dechlorination of the chlorinated compounds in the effluents of the first two bleaching stages, i.e., chlorination stage [(C + D)red.] and alkaline extraction stage (E1O), were monitored by determining total organic carbon (TOC) and activated-carbon-adsorbable organic-bound halogen (AOX). The isolates showed increased degradation rates after repeated incubations in the effluent-containing medium. Separation of the culture supernatants by ultrafiltration into three fractions of different molecular weights revealed substantial AOX and TOC reductions in the low-molecular-weight fraction. The AOX values of the higher-molecular-weight fractions were also reduced. Extracellular peroxidase and cell wall-bound catalase activities were produced during growth of the microorganisms on bleach effluents.

Actinomycetales↗