Abstract:
We have described a procedure for the isolation of mutants of Tetrahymena thermophila with hyperscretion of phospholipase A1 (PLA1). Using random chemical mutagenesis, uniparental cytogamy, genetic crossing and a new, fast and effective screening procedure, four PLA1-hypersecretory mutants were isolated. The screening procedure is based on the formation of a halo appearing around cylindrical holes in a lecithin-containing agar plate filled with cell-free supernatants. About 3,940 clones were tested with this procedure in primary screening for hypersecretory features, of which 60 putative hypersecretory mutants were isolated, subcloned and tested in a secondary screening. Of these, four selected mutants showed 1.8-2.2 more PLA1 activity in the cell-free supernatants compared to the wild-type strain CU 438.1. Hypersecretion was only observable for PLA1; no increased activity for two other lysosomal enzymes could be detected. These hypersecretory mutants of T. thermophila can be very useful for increasing the yield of PLA1 in fermentation processes. This is particularly relevant because, in contrast to other phospholipases, PLA1 is not available on the commercial market for fine chemicals and little is known about the role of PLA1 in cell signaling and metabolism.
Registro:
Documento: |
Artículo
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Título: | Screening for and characterization of phospholipase A1 hypersecretory mutants of Tetrahymena thermophila |
Autor: | Hartmann, M.; Guberman, A.; Florin-Christensen, M.; Tiedtke, A. |
Filiación: | Institut für Allgemeine Zoologie und Genetik, Westfälische Wilhelms-Universität, 48149 Münster, Germany Laboratory of Molecular Biology, Department of Biological Chemistry, Ciudad Universitaria, 1428 Buenos Aires, Argentina Institute of Neuroscience (INEUCI), Ciudad Universitaria, Pabellon 2, 1428 Buenos Aires, Argentina
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Palabras clave: | lysosome enzyme; phosphatidylcholine; phospholipase A1; article; bacterium isolation; bacterium mutant; chemical mutagenesis; enzyme activity; enzyme analysis; enzyme release; fermentation; molecular cloning; nonhuman; screening; strain difference; supernatant; Tetrahymena thermophila; Bacteria (microorganisms); Prokaryota; Protozoa; Tetrahymena thermophila; Tetrahymena thermophila |
Año: | 2000
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Volumen: | 54
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Número: | 3
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Página de inicio: | 390
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Página de fin: | 396
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DOI: |
http://dx.doi.org/10.1007/s002530000405 |
Título revista: | Applied Microbiology and Biotechnology
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Título revista abreviado: | Appl. Microbiol. Biotechnol.
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ISSN: | 01757598
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CODEN: | AMBID
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CAS: | phosphatidylcholine, 55128-59-1, 8002-43-5; phospholipase A1, 9043-29-2
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Registro: | https://bibliotecadigital.exactas.uba.ar/collection/paper/document/paper_01757598_v54_n3_p390_Hartmann |
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Citas:
---------- APA ----------
Hartmann, M., Guberman, A., Florin-Christensen, M. & Tiedtke, A.
(2000)
. Screening for and characterization of phospholipase A1 hypersecretory mutants of Tetrahymena thermophila. Applied Microbiology and Biotechnology, 54(3), 390-396.
http://dx.doi.org/10.1007/s002530000405---------- CHICAGO ----------
Hartmann, M., Guberman, A., Florin-Christensen, M., Tiedtke, A.
"Screening for and characterization of phospholipase A1 hypersecretory mutants of Tetrahymena thermophila"
. Applied Microbiology and Biotechnology 54, no. 3
(2000) : 390-396.
http://dx.doi.org/10.1007/s002530000405---------- MLA ----------
Hartmann, M., Guberman, A., Florin-Christensen, M., Tiedtke, A.
"Screening for and characterization of phospholipase A1 hypersecretory mutants of Tetrahymena thermophila"
. Applied Microbiology and Biotechnology, vol. 54, no. 3, 2000, pp. 390-396.
http://dx.doi.org/10.1007/s002530000405---------- VANCOUVER ----------
Hartmann, M., Guberman, A., Florin-Christensen, M., Tiedtke, A. Screening for and characterization of phospholipase A1 hypersecretory mutants of Tetrahymena thermophila. Appl. Microbiol. Biotechnol. 2000;54(3):390-396.
http://dx.doi.org/10.1007/s002530000405