The stability and thermodynamic parameters of a very thermostable and calcium-independent α-amylase from a newly isolated bacterium, Anoxybacillus beppuensis TSSC-1

Abstract

Anoxybacillus beppuensis TSSC-1 (GenBank Number, EU710556), a thermophilic bacterium isolated from a hot spring reservoir, was found to optimally secrete a monomeric α-amylase at 55°C and pH 7. The enzyme was purified to homogeneity by a single-step purification on phenyl sepharose 6FF, achieving a 58% yield, 10,000U/mg specific activity and 19.5 fold purification. The molecular weight, Km and Vmax were 43kD, 0.5mgml−1 and 3571.42μmolml−1m−1, respectively. The enzymatic catalysis of soluble starch was optimum at 80°C and pH 7. The thermodynamic parameters, Kd, t1/2, ΔH*, ΔS*, E and ΔG*, were consistent. The very compact structure of the enzyme and the transitional enzyme–substrate complex resisted denaturation at extreme temperatures and alkaline pH. The Kd and t1/2 measurements were consistent with the high thermostability and pH tolerance observed. The structural stability of the enzyme was also reflected by the values of ΔH*, ΔS*, E and ΔG*. While the enzyme did not exhibit metal ion dependency, it was resistant to chemical denaturation. The broad thermo- and pH-tolerance of this enzyme suggests potential commercial opportunities.