Purification, kinetic and thermodynamic characterization of soluble acid invertase from sugarcane (Saccharum officinarum L.).

We report for the first time kinetic and thermodynamic properties of soluble acid invertase (SAI) of sugarcane (Saccharum officinarum L.) salt sensitive local cultivar CP 77-400 (CP-77). The SAI was purified to apparent homogeneity on FPLC system. The crude enzyme was about 13 fold purified and recovery of SAI was 35%. The invertase was monomeric in nature and its native molecular mass on gel filtration and subunit mass on SDS-PAGE was 28 kDa. SAI was highly acidic having an optimum pH lower than 2. The acidic limb was missing. Proton transfer (donation and receiving) during catalysis was controlled by the basic limb having a pKa of 2.4. Carboxyl groups were involved in proton transfer during catalysis. The kinetic constants for sucrose hydrolysis by SAI were determined to be: k(m)=55 mg ml(-1), k(cat)=21s(-1), k(cat)/k(m)=0.38, while the thermodynamic parameters were: DeltaH*=52.6 kJ mol(-1), DeltaG*=71.2 kJ mol(-1), DeltaS*=-57 J mol(-1) K(-1), DeltaG*(E-S)=10.8 kJ mol(-1) and DeltaG*(E-T)=2.6 kJ mol(-1). The kinetics and thermodynamics of irreversible thermal denaturation at various temperatures 53-63 degrees C were also determined. The half -life of SAI at 53 and 63 degrees C was 112 and 10 min, respectively. At 55 degrees C, surprisingly the half -life increased to twice that at 53 degrees C. DeltaG*, DeltaH* and DeltaS* of irreversible thermal stability of SAI at 55 degrees C were 107.7 kJ mol(-1), 276.04 kJ mol(-1) and 513 J mol(-1) K(-1), respectively.