Thermodynamic and Kinetic Parameters of Thermostable Alkaline Phosphatase from Geobacillus pallidus P26

Abstract

In this study, a thermophilic bacterium isolated from a thermal source in Turkey and identified as Geobacillus pallidus P26 was used to produce the alkaline phosphatase (ALP) enzyme. The enzyme was partially isolated by salting out the supernatant with saturated ammonium sulphate. Then, the enzyme was purified 23 times using ion-exchange (DEAE-Sepharose) and size-exclusion chromatography (Sephacryl S-200). Using p-nitrophenyl phosphate (pNPP) substrate, the maximum reaction velocity (V-max) of the purified enzyme was calculated as 0.255 mu mol/min/mg and Michaelis-Menten constant (K-m) for pNPP (p-nitrophenyl phosphate) as 3.92 mM. k(cat) (turnover number) and k(cat)/K-m were calculated as 0.812 s(-1) and 207.14 s(-1) M-1, respectively. The half-life of the enzyme (t(1/2)) purified from Geobacillus pallidus G26 was calculated as 38 hours at 60 degrees C, 14 hours at 70 degrees C and 1.5 hours at 80 degrees C. The thermodynamic parameters of ALP (Delta S-IN(#), Delta H-IN(#) and Delta G(IN)(#)) were also calculated. The enzymes extracted from thermophilic bacteria can maintain their stability at high temperatures for a longer period. This characteristic is favorable for implementations that require high temperatures.