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

Ali I, Akbar A, Anwar M, Prasongsuk S, Lotrakul P, Punnapayak H - Biomed Res Int (2015)

Bottom Line: 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).

View Article: PubMed Central - PubMed

Affiliation: Plant Biomass Utilization Research Unit, Department of Botany, Faculty of Science, Chulalongkorn University, Bangkok 10330, Thailand ; Institute of Biochemistry, University of Balochistan, Quetta 87300, Pakistan.

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.

No MeSH data available.


Related in: MedlinePlus

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.
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fig1: 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.

Mentions: The initial 90% saturation (NH4)2SO4 cut gave a 2.5-fold increased enzyme specific activity for a 32% yield loss and a 76% total protein reduction (Table 1). Following Sephadex G100 gel filtration, the eluted fraction showing the highest amylase activity was then evaluated for its apparent purity and molecular weight by SDS-PAGE resolution and coomassie blue staining. Only a single protein band was visualized suggesting the apparent enrichment of the enzyme to homogeneity (Figure 1). Overall, the enzyme activity was increased 6.96-fold following the 90% saturation (NH4)2SO4 precipitation and Sephadex G100 gel filtration for a loss of 56% activity. The specific activity of the enriched α-amylase was estimated to be 118.4 ± 1.2 U·mg−1 (Table 1), with an estimated molecular weight of ~42 kDa by SDS-PAGE analysis (Figure 1).


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

Ali I, Akbar A, Anwar M, Prasongsuk S, Lotrakul P, Punnapayak H - Biomed Res Int (2015)

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.
© Copyright Policy - open-access
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4477103&req=5

fig1: 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.
Mentions: The initial 90% saturation (NH4)2SO4 cut gave a 2.5-fold increased enzyme specific activity for a 32% yield loss and a 76% total protein reduction (Table 1). Following Sephadex G100 gel filtration, the eluted fraction showing the highest amylase activity was then evaluated for its apparent purity and molecular weight by SDS-PAGE resolution and coomassie blue staining. Only a single protein band was visualized suggesting the apparent enrichment of the enzyme to homogeneity (Figure 1). Overall, the enzyme activity was increased 6.96-fold following the 90% saturation (NH4)2SO4 precipitation and Sephadex G100 gel filtration for a loss of 56% activity. The specific activity of the enriched α-amylase was estimated to be 118.4 ± 1.2 U·mg−1 (Table 1), with an estimated molecular weight of ~42 kDa by SDS-PAGE analysis (Figure 1).

Bottom Line: 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).

View Article: PubMed Central - PubMed

Affiliation: Plant Biomass Utilization Research Unit, Department of Botany, Faculty of Science, Chulalongkorn University, Bangkok 10330, Thailand ; Institute of Biochemistry, University of Balochistan, Quetta 87300, Pakistan.

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.

No MeSH data available.


Related in: MedlinePlus