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Purification and Characterization of Thermostable and Detergent-Stable α-Amylase from Anoxybacillus sp. AH1

 

Ömer Acer1*, Fatma Matpan Bekler1, Hemşe Pirinççioğlu1, Reyhan Gül Güven2 and Kemal Güven1

 

1Molecular Biology and Genetic Department, Faculty of Science, Dicle University, TR-21280 Diyarbakır, Turkey
2Division of Science Teaching, Ziya Gökalp Faculty of Education, Dicle University, TR-21280 Diyarbakır, Turkey



Article history:
Received May 12, 2015
Accepted September 11, 2015


Key words:
detergent-stable α-amylase, Anoxybacillus sp. AH1, enzyme activity inhibition, enzyme purification



Summary:
A thermostable and detergent-stable α-amylase from a newly isolated Anoxybacillus sp. AH1 was purified and characterized. Maximum enzyme production (1874.8 U/mL) was obtained at 24 h of incubation. The amylase was purified by using Sephadex G-75 gel filtration, after which an 18-fold increase in specific activity and a yield of 9 % were achieved. The molecular mass of the purified enzyme was estimated at 85 kDa by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). The optimum pH and temperature values of the enzyme were 7.0 and 60 °C, respectively. The enzyme was highly stable in the presence of 30 % glycerol, retaining 85 % of its original activity at 60 °C within 120 min. Km and vmax values were 0.102 μmol and 0.929 μmol/min, respectively, using Lineweaver-Burk plot. The enzyme activity was increased by various detergents, but it was significantly inhibited in the presence of urea. Mg2+ and Ca2+ also significantly activated α-amylase, while Zn2+, Cu2+ and metal ion chelators ethylenediaminetetraacetic acid (EDTA) and 1,10-phenanthroline (phen) greatly inhibited the enzyme activity. α-Amylase activity was enhanced by β-mercaptoethanol (β-ME) and dithiothreitol (DTT) to a great extent, but inhibited by p-chloromercuribenzoic acid (PCMB). Iodoacetamide (IAA) and N-ethylmaleimide (NEM) had a slight, whereas phenylmethylsulfonyl fluoride (PMSF) had a strong inhibitory effect on the amylase activity.


 

 

*Corresponding author:  email3  oacer21@gmail.com
                                      tel3  +904 (0)122 488 550
                                      fax2  +904 (0)122 488 300
                                     

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