Purification of an alpha amylase from Aspergillus flavus NSH9 and molecular characterization of its nucleotide gene sequence

Abstract

In this study, an alpha-amylase enzyme from a locally isolated Aspergillus flavus NSH9 was purified and characterized. The extracellular α-amylase was purified by ammonium sulfate precipitation and anion-exchange chromatography at a final yield of 2.55-fold and recovery of 11.73%. The molecular mass of the purified α-amylase was estimated to be 54 kDa using SDS-PAGE and the enzyme exhibited optimal catalytic activity at pH 5.0 and temperature of 50 °C. The enzyme was also thermally stable at 50 °C, with 87% residual activity after 60 min. As a metalloenzymes containing calcium, the purified α-amylase showed significantly increased enzyme activity in the presence of Ca 2+ ions. Further gene isolation and characterization shows that the α-amylase gene of A. flavus NSH9 contained eight introns and an open reading frame that encodes for 499 amino acids with the first 21 amino acids presumed to be a signal peptide. Analysis of the deduced peptide sequence showed the presence of three conserved catalytic residues of α-amylase, two Ca 2+ -binding sites, seven conserved peptide sequences, and several other properties that indicates the protein belongs to glycosyl hydrolase family 13 capable of acting on α-1,4-bonds only. Based on sequence similarity, the deduced peptide sequence of A. flavus NSH9 α-amylase was also found to carry two potential surface/secondary-binding site (SBS) residues (Trp 237 and Tyr 409) that might be playing crucial roles in both the enzyme activity and also the binding of starch granules.

Microbial Alpha-Amylase Production: Progress, Challenges and Perspectives

“…In iodine method, Lugol interacts with starch and forms complexes and alpha-amylase degrades starches and reduces UV absorbance at 580 nm. 31 Figure 2 shows measurement of the alpha-amylase activity by iodine method. Also, While Nelson -Somogyi method is used for measuring of α-amylase activity.…”

“…30 Also, other fungi species have been reported to possess some unique features making them suitable for industrial goals; for example, an alpha-amylase produced by Aspergillus flavus NSH9 is thermally stable at 50°C. 31 Table 1 shows different microbial sources for alpha-amylase production.…”

Production, Partial Purification and Characterization of Two α-Amylase Isoforms from Saccharomyces cerevisiae strain YOP 1/2-2 Isolated from Tchapalo (Côte d’Ivoire)

“…α-Amylase [α- (1,4)-D-glucan glucanohydrolase (E.C.3.2.1.1)] is an endoenzyme that belongs to family 13 of glycoside hydrolases (GH13) [1,2]. It randomly hydrolyzes osidic bonds of amylose, amylopectin, starch, glycogen and other polysaccharides containing three or more α-(1,4)-D-glucose bonds [3].…”

“…It randomly hydrolyzes osidic bonds of amylose, amylopectin, starch, glycogen and other polysaccharides containing three or more α-(1,4)-D-glucose bonds [3]. Amylases can be found in several sources (plants, animals and microorganisms) and are among the most important enzyme classes having approximately 25-30% of the world enzyme market [2,4]. However, those of microbial origin (bacterial and fungal) have found their application in biotechnology and into a wide range of industrial processes: food industry, pharmaceutical, textile, distilling, fermentation, paper and detergent [2,5].…”

“…Amylases can be found in several sources (plants, animals and microorganisms) and are among the most important enzyme classes having approximately 25-30% of the world enzyme market [2,4]. However, those of microbial origin (bacterial and fungal) have found their application in biotechnology and into a wide range of industrial processes: food industry, pharmaceutical, textile, distilling, fermentation, paper and detergent [2,5]. With the increasing interest of fungal amylases, several investigations were carried out on extracellular amylolytic enzymes secreted by species such as Aspergillus [1,2], Rhizopus [3], Talaromyces pinophilus [6], Lipomyces, Saccharomycopsis and Schwanniomyces [7].…”