Production and Characterization of α-Amylase from an Extremely Halophilic Archaeon, Haloferax sp. HA10

Abstract

Haloarchaea are found at very high concentrations in salt-conditioned environments, hence produce enzymes which are able to catalyze reactions under harsh conditions, typical of many industrial processes. In the present study, culture conditions for extracellular amylase production from Haloarchaea isolated from a solar saltern were optimized and the purified enzyme was characterized. Haloferax sp. HA10 showed maximum amylase production at 3 M NaCl, 37 °C, pH=7 and 1% starch content. Purified α-amylase was a calcium-dependent enzyme with an estimated molecular mass of about 66 kDa and many industrially useful properties. It was found to be stable in a broad range of pH (from 5 to 9) and NaCl concentrations (from 0.5 to 3.0 M), retaining 48% activity even at 4 M. The optimal temperature for Haloferax sp. HA10 amylase activity was 55 °C (99% activity), and 57% activity was retained at 80 °C, which dropped to 44% with the increase of temperature to 90 or 100 °C. It was able to sustain various surfactants and detergents. To the best of our knowledge the detergent-stable α-amylases from halophilic archaeon have not been reported yet.

Keywords: Haloferax sp.; archaea; halophiles; solar saltern; α-amylase.

Fig. 1

Qualitative estimation of amylase produced by six haloarchaeal strains on starch agar plates

Fig. 2

Amylase activity of Haloferax sp. HA10 at different NaCl concentrations

Fig. 3

Effect of temperature and salt concentration on the growth of Haloferax sp. HA10 after 48 h

Fig. 4

Amylase production by Haloferax sp. HA10 at different temperatures and NaCl concentrations after 48 h

Fig. 5

Growth of and amylase production by Haloferax sp. HA10 at different pH values

Fig. 6

Growth of and amylase production by Haloferax sp. HA10 at different mass fractions of starch

Fig. 7

Growth of and amylase production by Haloferax sp. HA10 at different mass fractions of CaCl₂

Fig. 8

Relative activity of purified amylase from Haloferax sp. HA10 after incubation in a buffer at various pH values for 24 h

Fig. 9

Relative activity of purified amylase from Haloferax sp. HA10 after 30 min of incubation at various temperatures

Fig. 10

Relative activity of amylase from Haloferax sp. HA10 after 24 h of incubation in a buffer at pH=6.0 and different NaCl concentrations

Fig. 11

Effect of metal ions (5 mM) on the activity of purified amylase from Haloferax sp. HA10

Fig. 12

SDS-PAGE of purified amylase from Haloferax sp. HA10. Lanes 1, 2 and 3: samples; lanes 4 and 5: molecular mass marker in kDa

Fig. 13

Zymogram of amylase from Haloferax sp. HA10 on native-PAGE. Lanes 1 and 2: samples; lane 3: molecular mass marker in kDa