Purification and Characterization of a Polyextremophilic α -Amylase from an Obligate Halophilic Aspergillus penicillioides Isolate and Its Potential for Souse with Detergents

Abstract

An extracellular α-amylase from the obligate halophilic Aspergillus penicillioides TISTR3639 strain was produced and enriched to apparent homogeneity by ammonium sulfate precipitation and Sephadex G100 gel filtration column chromatography. The mass of the purified amylase was estimated to be 42 kDa by SDS-PAGE. With soluble starch as the substrate it had a specific activity of 118.42 U · mg(-1) and Vmax and Km values of 1.05 µmol · min(-1) · mg(-1) and 5.41 mg · mL(-1), respectively. The enzyme was found to have certain polyextremophilic characteristics, with an optimum activity at pH 9, 80 °C, and 300 g · L(-1) NaCl. The addition of CaCl2 at 2 mM was found to slightly enhance the amylase activity, while ZnCl2, FeCl2, or EDTA at 2 mM was strongly or moderately inhibitory, respectively, suggesting the requirement for a (non-Fe(2+) or Zn(2+)) divalent cation. The enzyme retained more than 80% of its activity when incubated with three different laundry detergents and had a better performance compared to a commercial amylase and three detergents in the presence of increasing NaCl concentrations up to 300 g · L(-1). Accordingly, it has a good potential for use as an α-amylase in a low water activity (high salt concentration) and at high pH and temperatures.

Figure 1

SDS-PAGE analysis of the enriched α-amylase from A. penicillioides TISTR 3639. Lane 1: molecular mass ladder; lane 2 the enriched α-amylase, showing a single band at approximately 42 kDa.

Figure 2

Effect of the (a) pH (at 30°C) and (b) temperature (at pH 9.0) on the activity of the enriched α-amylase from A. penicillioides TISTR 3639. Data are shown as the mean relative activity (%) ±1 SD (error bars), derived from three repeats. Means with a different lowercase superscript letter (a, b, and c) are significantly different (ANOVA and DMRT of the transformed data, P < 0.05).

Figure 3

Effect of the NaCl concentration (g·L⁻¹) on the activity of the enriched α-amylase from A. penicillioides TISTR 3639 at pH 9 and 80°C. Data are shown as the mean relative activity (%) ±1 SD (error bars), derived from three repeats. Means with a different lowercase superscript letter (a, b, and c) are significantly different (ANOVA and DMRT of the transformed data, P < 0.05).

Figure 4

Lineweaver-Burk plot for the determination of the V _max⁡ and K _m values of the enriched α-amylase from A. penicillioides TISTR 3639, at optimum conditions (pH 9 and 80°C), in the presence of different concentrations of soluble starch.

Figure 5

Performance comparison of a commercial amylase, detergents A, B, and C, and the enriched α-amylase (control) from A. penicillioides TISTR 3639 at pH 7 and 30°C in the presence of different concentrations of NaCl. The results are expressed as the relative activity (%) to that of the enriched α-amylase from A. penicillioides TISTR 3639 without NaCl and are shown as the mean relative activity (%) ±1 SD (error bars), derived from three repeats. Means with a different lowercase superscript letter (a, b, and c) are significantly different (ANOVA and DMRT of the transformed data, P < 0.05).