Characterization and expression in Pichia pastoris of a raw starch degrading glucoamylase (GA2) derived from Aspergillus flavus NSH9
| Autor: | Ngieng Ngui Sing, Hasnain Hussain, Kazi Muhammad Rezaul Karim, Fazia Mohd Sinang, Hairul Azman Roslan, Tasmia Tasnim, Anowar Hossain, Ahmad Husaini |
|---|---|
| Rok vydání: | 2019 |
| Předmět: |
0106 biological sciences
Starch Aspergillus flavus 01 natural sciences Pichia Pichia pastoris 03 medical and health sciences chemistry.chemical_compound Transformation Genetic Aspergillus oryzae 010608 biotechnology Complementary DNA Amino Acid Sequence Cloning Molecular Phylogeny 030304 developmental biology chemistry.chemical_classification 0303 health sciences biology food and beverages Hydrogen-Ion Concentration biology.organism_classification Recombinant Proteins Amino acid Biochemistry chemistry Glucan 1 4-alpha-Glucosidase Sequence Alignment Biotechnology Starch binding |
| Zdroj: | Protein expression and purification. 164 |
| ISSN: | 1096-0279 |
| Popis: | The Aspergillus flavus NSH9 gene, encoding a pH and thermostable glucoamylase with a starch binding domain (SBD), was expressed in Pichia pastoris to produce recombinant glucoamylase (rGA2). The full-length glucoamylase gene (2039 bp), and cDNA (1839 bp) encode a 612 amino acid protein most similar to glucoamylase from Aspergillus oryzae RIB40; the first 19 amino acids are presumed to be a signal peptide for secretion, and the SBD is at the C-terminal. The cDNA was successfully secreted by Pichia at 8.23 U mL-1, and the rGA2 was found to be: a 80 kDa monomer, stable from pH 3.0-9.0, with optimum catalytic activity at pH 5.0, active at temperatures up to 80°C (rGA2 retained 58% of its activity after 60 min of incubation at 70°C), and metal ions such as Na+, K+, Ca++ and Mg++ enhanced rGA2 enzyme activity. The starch degrading ability of rGA2 was also observed on raw sago starch and where prolonged incubation generated larger, deeper, holes on the starch granules, indicating rGA2 is an excellent candidate for industrial starch processing applications. |
| Databáze: | OpenAIRE |
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