| Autor: |
Bielen, A, Abramić, M, Pigac, J, Franekić, J, Vujaklija, D |
| Jazyk: |
angličtina |
| Rok vydání: |
2007 |
| Předmět: |
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| Popis: |
GDS(L) lipases are interesting group of hydrolytic enzymes, often with multifunctional properties and potential for use in biotechnology. A putative lipase gene from Streptomyces coelicolor showed high amino acid sequence homology (66%) with cloned and characterised GDS(L) lipase from Streptomyces rimosus. The gene with corresponding RBS, was PCR-amplified with the addition of restriction sites and 6 histidine codons at the 3’ terminus. Amplifed DNA fragment was ligated into the pET-15b vector, resequenced, and subsequently ligated into the Streptomyces expression plasmid pANT849. This construct was transformed into heterologous, lipase defficient host Streptomyces lividans. The time-course of lipase production was monitored spectrofotometrically following degradation of p-nitrophenyl palmitate and the cultivation was stopped after the increase of lipase activity ceased. A simple three step protein purification procedure was developed. Firstly ammonium sulphate precipitation followed by Ni-NTA metal-affinity chromatography were applied. Obtained sample contained lipase (approximately 80%) and a contaminant, lipase-activity deficient protein of higher molecular mass. Therefore, gel-filtration was applied as the final purification step gaining > 95% purified lipase. Basic biochemical features of the enzyme were determined and will be disscused. |
| Databáze: |
OpenAIRE |
| Externí odkaz: |
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