Characterization and expression in Pichia pastoris of a raw starch degrading glucoamylase (GA2) derived from Aspergillus flavus NSH9

Publication date: Available online 26 July 2019Source: Protein Expression and PurificationAuthor(s): Kazi Muhammad Rezaul Karim, Ahmad Husaini, Ngieng Ngui Sing, Tasmia Tasnim, Fazia Mohd Sinang, Hasnain Hussain, Md Anowar Hossain, Hairul RoslanAbstractA pH and thermostable glucoamylase encoding gene having starch-binding domain (SBD) from Aspergillus flavus NSH9 was successfully identified, isolated, and expressed in Pichia pastoris GS115 to produce recombinant glucoamylase (rGA2). The full-length glucoamylase gene (2039 bp) and cDNA (1839 bp) isolated was found to encode for 612 amino acid residues in the open reading frame with the first 19 amino acids presumed to be a signal peptide and an SBD at the C-terminal end. The deduced glucoamylase (GA2) exhibited the highest identity to glucoamylase from Aspergillus oryzae RIB40. The isolated GA2 cDNA was successfully expressed in Pichia pastoris GS115 to produce rGA2 at a yield of 8.23 U mL−1. The rGA2 was found to be a monomer with a size of about 80 kDa and exhibited optimum catalytic activity at pH 5.0, stable over a broad pH range (3.0–9.0) and at high temperature up to 70–80 °C. The enzyme also retained 58% of its activity even after 60 min of incubation at 70 °C. Metal ions such as Na+, K+, Ca++ and Mg++ were also found to enhance the enzyme activity. The raw starch degrading ability of rGA2 also increased in the presence of raw sago starch and prolonged incubation period causes larger and deeper holes t...
Source: Protein Expression and Purification - Category: Biochemistry Source Type: research