Abstract
Problem statement: This study reported the purification and characterization of a novel highly thermostable alkaline amylase from a newly isolated Bacillus strain HUTBS71. Approach: The enzyme was purified using ammonium sulfate precipitation, ion exchange and gel filtration chromatography. Results: Maximum amylase activity (72 U mL-1) was obtained at 100°C after 10 min of incubation. The enzyme was purified 24 fold with 12.5% yield and showed a monomer band with a molecular weight of 58.8 kDa by SDS-PAGE. This enzyme exhibited maximum activity at pH and temperature, 7.8 and 100°C, respectively. It performed stability over a broad range of pH and temperature, 5.2-10.0 and 80-115°C, respectively. The half-life of the enzyme at 90 and 100°C was estimated to be 3 h. The activation energy of denaturation of purified enzyme was 2.53 kJ moL-1. The enzyme was activated by 5 mM of CoCl2, MgSO4, MnCl2, ZnSO4 and MnSO4 (relative activity was 133, 126, 133, 106.6 and 103%, respectively). It was strongly inhibited by CuSO4 and CdCl2 but less affected by NaCl, CaCl2, FeCl3, ZnCl2 and EDTA. Conclusion: The present purified amylase therefore could be defined as a highly thermostable, extremely hyperthermophilic and alkalitolerant with new properties make the present enzyme applicable for many starch processing and food industries.
Highlights
The discovery of hyperthermophilic bacteria has provided a valuable tool for the analysis of protein stability
Horikoshi[2] first reported an alkaline amylase of an alkalophilic Bacillus sp. strain A-40-2
The obtained data showed that the fractions from 60% ammonium sulfate saturation correlated with high proteolytic and specific activities compared with the crude amylase and other concentrations
Summary
The discovery of hyperthermophilic bacteria has provided a valuable tool for the analysis of protein stability. The intrinsic thermal stability of the enzymes isolated from these sources makes it possible to study the molecular mechanisms governing structure and function in a system adapted for elevated temperatures. The thermostability exhibited by these enzymes is maintained without any components unique to thermophiles, suggesting that the increase in molecular stability is accomplished through the same stereochemical interactions found in their mesophilic counterparts. Through analysis of these enzymes it should be possible to determine the stabilizing interactions by which the enzymes maintain activity at extreme temperatures[1]. Horikoshi[2] first reported an alkaline amylase of an alkalophilic Bacillus sp.
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