Screening for and characterization of phospholipase A1 hypersecretory mutants of Tetrahymena thermophila
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 isol...
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| Acceso en línea: | http://hdl.handle.net/20.500.12110/paper_01757598_v54_n3_p390_Hartmann |
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todo:paper_01757598_v54_n3_p390_Hartmann2023-10-03T15:07:55Z Screening for and characterization of phospholipase A1 hypersecretory mutants of Tetrahymena thermophila Hartmann, M. Guberman, A. Florin-Christensen, M. Tiedtke, A. 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 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. Fil:Guberman, A. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina. Fil:Florin-Christensen, M. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina. JOUR info:eu-repo/semantics/openAccess http://creativecommons.org/licenses/by/2.5/ar http://hdl.handle.net/20.500.12110/paper_01757598_v54_n3_p390_Hartmann |
| institution |
Universidad de Buenos Aires |
| institution_str |
I-28 |
| repository_str |
R-134 |
| collection |
Biblioteca Digital - Facultad de Ciencias Exactas y Naturales (UBA) |
| topic |
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 |
| spellingShingle |
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 Hartmann, M. Guberman, A. Florin-Christensen, M. Tiedtke, A. Screening for and characterization of phospholipase A1 hypersecretory mutants of Tetrahymena thermophila |
| topic_facet |
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 |
| description |
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. |
| format |
JOUR |
| author |
Hartmann, M. Guberman, A. Florin-Christensen, M. Tiedtke, A. |
| author_facet |
Hartmann, M. Guberman, A. Florin-Christensen, M. Tiedtke, A. |
| author_sort |
Hartmann, M. |
| title |
Screening for and characterization of phospholipase A1 hypersecretory mutants of Tetrahymena thermophila |
| title_short |
Screening for and characterization of phospholipase A1 hypersecretory mutants of Tetrahymena thermophila |
| title_full |
Screening for and characterization of phospholipase A1 hypersecretory mutants of Tetrahymena thermophila |
| title_fullStr |
Screening for and characterization of phospholipase A1 hypersecretory mutants of Tetrahymena thermophila |
| title_full_unstemmed |
Screening for and characterization of phospholipase A1 hypersecretory mutants of Tetrahymena thermophila |
| title_sort |
screening for and characterization of phospholipase a1 hypersecretory mutants of tetrahymena thermophila |
| url |
http://hdl.handle.net/20.500.12110/paper_01757598_v54_n3_p390_Hartmann |
| work_keys_str_mv |
AT hartmannm screeningforandcharacterizationofphospholipasea1hypersecretorymutantsoftetrahymenathermophila AT gubermana screeningforandcharacterizationofphospholipasea1hypersecretorymutantsoftetrahymenathermophila AT florinchristensenm screeningforandcharacterizationofphospholipasea1hypersecretorymutantsoftetrahymenathermophila AT tiedtkea screeningforandcharacterizationofphospholipasea1hypersecretorymutantsoftetrahymenathermophila |
| _version_ |
1807314595609575424 |