title: Heterologous, Expression, and Characterization of Thermostable Glucoamylase Derived from Aspergillus flavus NSH9 in Pichia pastoris.
abstract: A novel thermostable glucoamylase cDNA without starch binding domain (SBD) of Aspergillus flavus NSH9 was successfully identified, isolated, and overexpressed in Pichia pastoris GS115. The complete open reading frame of glucoamylase from Aspergillus flavus NSH9 was identified by employing PCR that encodes 493 amino acids lacking in the SBD. The first 17 amino acids were presumed to be a signal peptide. The cDNA was cloned into Pichia pastoris and the highest expression of recombinant glucoamylase (rGA) was observed after 8 days of incubation period with 1% methanol. The molecular weight of the purified rGA was about 78 kDa and exhibited optimum catalytic activity at pH 5.0 and temperature of 70°C. The enzyme was stable at higher temperature with 50% of residual activity observed after 20 min at 90°C and 100°C. Low concentration of metal (Mg(++), Fe(++), Zn(++), Cu(++), and Pb(++)) had positive effect on rGA activity. This rGA has the potential for use and application in the saccharification steps, due to its thermostability, in the starch processing industries.
authors: Department of Molecular Biology, Faculty of Resource Science and Technology, Universiti Malaysia Sarawak, 94300 Kota Samarahan, Sarawak, Malaysia; Institute of Nutrition and Food Science, University of Dhaka, Dhaka 1000, Bangladesh.
authors: Kazi Muhammad Rezaul
authors: KM
authors: Karim
authors: Department of Molecular Biology, Faculty of Resource Science and Technology, Universiti Malaysia Sarawak, 94300 Kota Samarahan, Sarawak, Malaysia.
authors: Ahmad
authors: 0000-0002-6559-5602
authors: A
authors: Husaini
authors: Department of Biochemistry and Molecular Biology, University of Rajshahi, Rajshahi 6205, Bangladesh.
authors: Md Anowar
authors: MA
authors: Hossain
authors: Department of Molecular Biology, Faculty of Resource Science and Technology, Universiti Malaysia Sarawak, 94300 Kota Samarahan, Sarawak, Malaysia.
authors: Ngieng Ngui
authors: NN
authors: Sing
authors: Department of Molecular Biology, Faculty of Resource Science and Technology, Universiti Malaysia Sarawak, 94300 Kota Samarahan, Sarawak, Malaysia.
authors: Fazia
authors: F
authors: Mohd Sinang
authors: Department of Molecular Biology, Faculty of Resource Science and Technology, Universiti Malaysia Sarawak, 94300 Kota Samarahan, Sarawak, Malaysia.
authors: Mohd Hasnain Md
authors: MH
authors: Hussain
authors: Department of Molecular Biology, Faculty of Resource Science and Technology, Universiti Malaysia Sarawak, 94300 Kota Samarahan, Sarawak, Malaysia.
authors: Hairul Azman
authors: HA
authors: Roslan
mesh_terms: D000595:Amino Acid Sequence; D001231:Aspergillus flavus / Q000145:classification / Q000201:enzymology* / Q000235:genetics; D003001:Cloning, Molecular / Q000379:methods; D004789:Enzyme Activation; D004795:Enzyme Stability; D005087:Glucan 1,4-alpha-Glucosidase / Q000096:biosynthesis* / Q000737:chemistry* / Q000235:genetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010843:Pichia / Q000235:genetics* / Q000378:metabolism*; D015202:Protein Engineering / Q000379:methods*; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D013045:Species Specificity; D013379:Substrate Specificity; D013696:Temperature
chemical_list: D011994:Recombinant Proteins; D005087:Glucan 1,4-alpha-Glucosidase