The structure-function relationship of the lipases from Pseudomonas aeruginosa and Bacillus subtilis
| Autor: | Bauke W. Dijkstra, Onno Misset, Mieke Blaauw, Véronique Dartois, Karin Schanck, Karl-Erich Jaeger, Charles Colson, Stéphane Ransac, Gijs Gerritse, Ulrich Winkler, Emmanuel Lesuisse |
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| Přispěvatelé: | Groningen Biomolecular Sciences and Biotechnology |
| Jazyk: | angličtina |
| Rok vydání: | 1994 |
| Předmět: |
Models
Molecular Protein Folding 3-D STRUCTURE Molecular Sequence Data Triacylglycerol lipase Bioengineering Bacillus subtilis Crystallography X-Ray Biochemistry PSEUDOMONAS AERUGINOSA TRIAD FORMS Structure-Activity Relationship Bacterial Proteins MIEHEI TRIACYLGLYCERIDE LIPASE Hydrolase Escherichia coli Amino Acid Sequence Lipase Cloning Molecular GEOTRICHUM-CANDIDUM EXTRACELLULAR LIPASE Molecular Biology PANCREATIC LIPASE BACILLUS SUBTILIS Bacillaceae PURIFICATION biology Sequence Homology Amino Acid CRYSTAL Pseudomonas PRELIMINARY-X-RAY biology.organism_classification Bacillales Recombinant Proteins FLUORESCENS biology.protein Mutagenesis Site-Directed LIPASE CRYSTALLIZATION Biotechnology Pseudomonadaceae |
| Zdroj: | Protein Engineering, 7(4), 523-529 |
| ISSN: | 0269-2139 |
| DOI: | 10.1093/protein/7.4.523 |
| Popis: | Within the BRIDGE T-project on lipases we investigate the structure-function relationships of the lipases from Bacillus subtilis and Pseudomonas aeruginosa. Construction of an overproducing Bacillus strain allowed the purification of > 100 mg lipase from 30 I culture supernatant. After testing a large variety of crystallization conditions, the Bacillus lipase gave crystals of reasonable quality in PEG-4000 (38-45%), Na2SO4 and octyl-beta-glucoside at 22 degrees C, pH 9.0. A 2.5 Angstrom dataset has been obtained which is complete from 15 to 2.5 Angstrom resolution. P.aeruginosa wild-type strain PAC1R was fermented using conditions of maximum lipase production. More than 90% of the lipase was cell bound and could be solubilized by treatment of the cells with Triton X-100. This permitted the purification of similar to 50 mg lipase. So far, no crystals of sufficient quality were obtained. Comparison of the model we built for the Pseudomonas lipase, on the basis of sequences and structures of various hydrolases which were found to possess a common folding pattern (alpha/beta hydrolase fold), with the X-ray structure of the P.glumae lipase revealed that it is possible to correctly build the structure of the core of a protein even in the absence of obvious sequence homology with a protein of known 3-D structure. |
| Databáze: | OpenAIRE |
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