| Autor: |
Farah Qoura, Thomas Brück, Skander Elleuche, Torben Rehn, Garabed Antranikian, Ute Lorenz |
| Rok vydání: |
2015 |
| Předmět: |
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| Zdroj: |
Journal of Molecular Catalysis B: Enzymatic. 116:70-77 |
| ISSN: |
1381-1177 |
| DOI: |
10.1016/j.molcatb.2015.03.001 |
| Popis: |
An activity-based screening approach led to the identification of a novel glycoside hydrolase family-13 pullulanase gene (pul13A) from a psychrophilic bacterium. The recombinant enzyme exhibited a deduced peptide sequence of 1440 amino acid residues and was produced in a heterologous host in Escherichia coli. Purification from inclusion bodies was achieved by a six-step dialysis protocol enabling mild refolding of the urea-denaturated protein followed by affinity- and size-exclusion chromatography under native conditions. Pul13A is a type-I pullulanase, which is capable of hydrolysing α-1,6-glycosidic bonds in pullulan to produce maltotriose, while maltose and intermediate oligosaccharides are produced from soluble starch and amylopectin. The recombinant enzyme exhibited typical properties of cold-adapted proteins including low thermostability at elevated temperatures. It showed a temperature optimum at 35 °C, while at 10 °C residual activity (25%) remained. The optimal pH was in the range of 6.0–7.0, with Pul13A being stable at neutral and basic pH, but not in the acidic range. Catalytic activity was increased in the presence of divalent cations calcium and cobalt and both metal ions were also able to restore catalytic activity of EDTA-chelated enzyme samples. Pul13A represents the first type-I pullulanase from a psychrophile that has been produced in recombinant form. Moreover, its favourable enzymatic properties make this enzyme a potential candidate for industrial applications such as starch degradation for ethanol based biofuel production. |
| Databáze: |
OpenAIRE |
| Externí odkaz: |
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Full Text from ScienceDirect