Gene cloning and expression of a detergent stable alkaline protease from Aspergillus clavatus ES1 |
| |
| Affiliation: | 1. Laboratoire de Génie Enzymatique et de Microbiologie, Ecole Nationale d’Ingénieurs de Sfax, Route Soukra Km 4, B.P. “W” 3038 Sfax, Tunisia;2. Laboratoire Pathogènes et Environnement, Université de Montpellier 2, CC093, place Eugène Bataillon, 34095 Montpellier Cedex 5, Tunisia;1. School of Chemistry and Chemical Engineering, Jiangsu University, Zhenjiang, Jiangsu Province, 212013, PR China;2. School of Food and Biological Engineering, Jiangsu University, Zhenjiang, Jiangsu Province, 212013, PR China;1. Institut des Sciences de la Vie, Université Catholique de Louvain, Croix du Sud 2, B-1348 Louvain-la-Neuve, Belgium;2. Louvain Drug Research Institute, Université Catholique de Louvain, Avenue Mounier 73, B-1200 Woluwé-Saint-Lambert, Belgium;3. Laboratoire d’Ecologie Animale et Ecotoxicologie, Université de Liège, Allée du 6 août 15, B-4000 Liège, Belgium;1. Centre for Plant Biotechnology and Molecular Biology, Kerala Agricultural University, Thrissur, 680 656, India;2. Department of Vegetable Science, Kerala Agricultural University, Thrissur, 680 656, India;3. ICAR-National Bureau of Plant Genetic Resources, Regional Station, Thrissur, 680 656, India;4. Department of Plant Breeding and Genetics, College of Agriculture, Kerala Agricultural University, Thrissur, 680 656, India |
| |
| Abstract: | The genes, cDNA alpES1 and alpES1, encoding Aspergillus clavatus ES1 alkaline protease were amplified from complementary DNA (cDNA) and genomic DNA, respectively, cloned in pCR®II-TOPO plasmid and then sequenced. Sequence analysis of the cDNA alpES1 gene revealed an open reading frame (ORF) of 1212 bp encoding a pre–pro-protein of 403 amino acid residues consisting of a 21-aa signal peptide, a 100-aa pro-peptide and a 282-aa mature protein with a calculated molecular weight of 28.5 kDa. Compared to the cDNA alpES1 gene, the alpES1 gene contained three introns, which had 53, 57 and 54 bp, respectively. The cDNA alpES1 gene was then sub-cloned in pET-30b(+) and expressed in Escherichia coli BL21 (λDE3). The purified recombinant protease had a molecular weight of about 32 kDa estimated by SDS-PAGE. Kinetic parameters, Km and kcat values of the recombinant AlpES1 for casein, were 0.23 mM and 12.38 min−1, respectively. The catalytic efficiency (kcat/Km) was 53.82 min−1 mM−1. |
| |
| Keywords: | |
| 本文献已被 ScienceDirect 等数据库收录！ |
|
