Isolation and characterization of a xylose-glucose isomerase from a new strain Streptomyces thermovulgaris 127, var. 7-86.
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 gamma-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 degrees C at pH 7.0, using D-glucose, and up to 50-60 degrees C at pH 7.6, using D-xylose as substrates. The activation energy (Ea = 46 kJ x mol(-1)) and the critical temperature (Tc = 60 degrees C) were determined by fluorescence spectroscopy. Tc is in close coincidence with the melting temperature of denaturation (Tm = 59 degrees C), determined by circular dichroism (CD) spectroscopy. The free energy of stabilization in water after denaturation with Gdn.HCl was calculated to be 12 k x mol(-1). The specific activity (km values) for D-xylose-glucose isomerase at 70 degrees C toward different substrates, D-xylose, D-glucose, and D-ribose, were determined to be 4.4, 55.5, and 13.3 mM, respectively.
Raykovska
V
V
Institute of Microbiology, Bulgarian Academy of Sciences, Sofia.
Dolashka-Angelova
P
P
Paskaleva
D
D
Stoeva
S
S
Abashev
J
J
Kirkov
L
L
Voelter
W
W
D019747:Aldose-Ketose Isomerases / Q000302:isolation & purification* / Q000378:metabolism*; D000595:Amino Acid Sequence / Q000502:physiology; D004789:Enzyme Activation / Q000502:physiology; D004795:Enzyme Stability / Q000502:physiology; D005947:Glucose / Q000378:metabolism*; D019791:Guanidine / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D011489:Protein Denaturation / Q000187:drug effects / Q000502:physiology; D012266:Ribose / Q000378:metabolism*; D013045:Species Specificity; D013302:Streptomyces / Q000145:classification / Q000201:enzymology; D013696:Temperature; D014994:Xylose / Q000378:metabolism*
D012266:Ribose; D014994:Xylose; D019747:Aldose-Ketose Isomerases; C023298:xylose isomerase; D005947:Glucose; D019791:Guanidine