Purification and characterization of α-amylase fromAspergillus flavus

Abstract

Aspergillus flavus produced approximately 50 U/mL of amylolytic activity when grown in liquid medium with raw low-grade tapioca starch as substrate. Electrophoretic analysis of the culture filtrate showed the presence of only one amylolytic enzyme, identified as an alpha-amylase as evidenced by (i) rapid loss of color in iodine-stained starch and (ii) production of a mixture of glucose, maltose, maltotriose and maltotetraose as starch digestion products. The enzyme was purified by ammonium sulfate precipitation and ion-exchange chromatography and was found to be homogeneous on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purified enzyme had a molar mass of 52.5 +/- 2.5 kDa with an isoelectric point at pH 3.5. The enzyme was found to have maximum activity at pH 6.0 and was stable in a pH range from 5.0 to 8.5. The optimum temperature for the enzyme was 55 degrees C and it was stable for 1 h up to 50 degrees C. The Km and V for gelatinized tapioca starch were 0.5 g/L and 108.67 mumol reducing sugars per mg protein per min, respectively.

Application of microbial α-amylase in industry - A review

“…Laboratory scale purification for α-amylase includes various combinations of ion exchange, gel filtration, hydrophobicity interactions and reverse phase chromatography. Alternatively, α-amylase extraction protocols using organic solvents such as ethanol, acetone and ammonium sulfate precipitation (25, 31, 44) and ultrafiltration have been proposed (53). These conventional multi-step methods requires expensive equipments at each step, making them laborious, time consuming, barely reproducible and may result in increasing loss of the desired product (5).…”

A thermostable maltose‐tolerant α‐amylase from Aspergillus tamarii

“…For this reason, the majority of commercial preparations of amylases are produced by bacterial sources, especially from Bacillus species, and an extensive screening, purification, and characterisation of bacterial amylases has already been reported (PANDEY et al 2000, GUZ-MÁN-MALDONADO andPAREDES-LÓPES 1995). In spite of among fungi the studies are more limited, attempts have been done to increase the production and to obtain homogeneous fractions of amylase among mesophilic and thermophilic fungi (GUZMÁN-MALDONADO and PAREDES-LÓPES 1995, JENSEN and OLSEN 1992, ABOUD-ZEID 1997, PARK et al 1995, CHANG et al 1995, PLANCHOT and COLONNA 1995, KHOO et al 1994, SIQUEIRA et al 1997, ODIBO and ULBRICH-HOFMANN 2001. In fungi, detailed studies on α-amylase purification have largely been limited to a few species of mesophilic fungi, particularly Aspergillus sp.…”

Purification and some properties of α-amylase from an ectomycorrhizal fungus, Tricholoma matsutake

“…On the other hand, there are reports of an α-amylase from Aspergillus oryzae having a stable pH, 4.0-5.0 (Chang et al 1995), pH 5.0-8.5 from A. flavus Link: Fries (Khoo et al 1994), and pH 4.5-7.5 from Schwanniomyces alluvis (Wilson and Ingledew 1982). As compared to α-amylase from other fungi, the α-amylase has a wide range of pH stability (pH 4.0-10.0).…”