Isolation and characterization of a xylose-glucose isomerase from a new strainStreptomyces thermovulgaris127, var. 7-86

Abstract

A thermostable D-xylose–glucose isomerase was isolated from the thermophilic strain Streptomyces thermovulgaris 127, var. 7-86, as a result of mutagenic treatment by γ-irradiation of the parent strain, by precipitation and sequential chromatographies on DEAE–Sephadex A50, TSK-gel, FPLC-Mono Q/HR, and Superose 12™ columns. The N-terminal amino acid sequence and amino acid analysis shows 73–92% homology with xylose–glucose isomerases from other sources. The native molecular mass, determined by gel filtration on a Superose 12™ column, is 180 kDa, and 44.6 and 45 kDa were calculated, based on amino acid analysis and 10% SDS-PAGE, respectively. Both, the activity and stability of the enzyme were investigated toward pH, temperature, and denaturation with guanidine hydrochloride. The enzyme activity showed a clear pH optimum between pH 7.2 and 9.0 with D-glucose and 7.4 and 8.3 with D-xylose as substrates, respectively. The enzyme is active up to 60–85°C at pH 7.0, using D-glucose, and up to 50–60°C at pH 7.6, using D-xylose as substrates. The activation energy (Ea= 46 kJ·mol–1) and the critical temperature (Tc= 60°C) were determined by fluorescence spectroscopy. Tcis in close coincidence with the melting temperature of denaturation (Tm= 59°C), determined by circular dichroism (CD) spectroscopy. The free energy of stabilization in water after denaturation with Gdn.HCl was calculated to be 12 kJ·mol–1. The specific activity (kmvalues) for D-xylose-glucose isomerase at 70°C toward different substrates, D-xylose, D-glucose, and D-ribose, were determined to be 4.4, 55.5, and 13.3 mM, recpectively.Key words: D-xylose-glucose isomerase, protein sequencing, protein stability, protein denaturation.