Expression of a Bacillus Phytase C Gene in Pichia pastoris and Properties of the Recombinant Enzyme
| Autor: | Lilí Rodríguez-Blanco, Juan A Gallegos-López, José M. Viader-Salvadó, Martha Guerrero-Olazarán, J. Gerardo Carreon-Treviño |
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| Rok vydání: | 2010 |
| Předmět: |
Glycosylation
Genetic Vectors Coenzymes Gene Expression Bacillus subtilis Applied Microbiology and Biotechnology Pichia law.invention Pichia pastoris Calcium Chloride Bacterial Proteins law Enzyme Stability medicine Trypsin Cloning Molecular Enzymology and Protein Engineering Thermostability chemistry.chemical_classification 6-Phytase Ecology biology Protein Stability Temperature Hydrogen-Ion Concentration biology.organism_classification Recombinant Proteins Molecular Weight Enzyme chemistry Biochemistry Recombinant DNA Phytase Food Science Biotechnology medicine.drug |
| Zdroj: | Applied and Environmental Microbiology. 76:5601-5608 |
| ISSN: | 1098-5336 0099-2240 |
| DOI: | 10.1128/aem.00762-10 |
| Popis: | The cloning and expression of a native gene encoding a Bacillus subtilis phytase using Pichia pastoris as the host is described. In addition, the influence of N-glycosylation on the biochemical properties of the B. subtilis phytase, the influence of pH on the thermostability of the recombinant and native B. subtilis phytases, and the resistance of both phytases to shrimp digestive enzymes and porcine trypsin are also described. After 48 h of methanol induction in shake flasks, a selected recombinant strain produced and secreted 0.82 U/ml (71 mg/liter) recombinant phytase. This phytase was N-glycosylated, had a molecular mass of 39 kDa after N-deglycosylation, exhibited activity within a pH range of 2.5 to 9 and at temperatures of 25 to 70°C, had high residual activity (85% ± 2%) after 10 min of heat treatment at 80°C and pH 5.5 in the presence of 5 mM CaCl 2 , and was resistant to shrimp digestive enzymes and porcine trypsin. Although the recombinant Bacillus phytase had pH and temperature activity profiles that were similar to those of the corresponding nonglycosylated native phytase, the thermal stabilities of the recombinant and native phytases were different, although both were calcium concentration and pH dependent. |
| Databáze: | OpenAIRE |
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