Kinetic parameters, thermal stability and inactivation kinetics of PPO

Kinetic parameters, thermal stability and inactivation kinetics of PPO from
S. lycocarpum fruits
Batista, K.A. 1; Di-Medeiros, M.C.B.1; Alves, G.L. 1; Fernandes, K.F. 1
1Labotatório de Química de Proteínas, Instituto de Ciências Biológicas, UFG,
Brazil
INTRODUCTION: Solanum lycocarpum is a common and abundant plant in the
Brazilian cerrado, whose fruits are an abundant and inexpensive source of
polyphenoloxidases (PPO). In this work, we studied the kinetic parameters,
thermal stability and inactivation kinetics of the partially purified PPO from
Solanum lycocarpum fruits. MATERIAL AND METHODS: The substrate
specificity of the PPO and the kinetic parameters were determined by
measuring its activity against catechol, 4-methylcatechol, pyrogallol. The effects
of the inhibitors ascorbic acid, citric acid, thiourea, sodium metabisulfite and
L-cysteine on Solanum lycocarpum PPO activity were studied. In addition,
thermal stability and thermal denaturation kinetic of PPO were determined.
RESULTS AND DISCUSSION: The purification procedure was based on a
single step freezing precipitation, resulting in a 6-fold purification. The partially
purified PPO presented high activity towards catechol (Vmax 3.42 U mL-1 and
Km 6.47 mM) being classified as a catecholase type polyphenol oxidase.
S.lycocarpum PPO was sensitive to various inhibitors, being L-cysteine the
most effective inhibitor, presenting a competitive inhibition. Results from
thermal inactivation of S.lycocarpum PPO at 30 ºC to 65 ºC evidenced half-life
(t1/2) varying from 2.71 to 0.8 h and D-values between 17.31 to 2.57 h. These
results evidenced that the partially purified PPO is a temperature-resistant
biocatalyst. The increase of ΔG values shows that there was no destabilization
of the protein increasing heat temperature. Also, ΔS values suggest a decrease
in disorder of the enzyme/solvent system upon denaturation, which leads to a
decrease of the molecular mobility and consequent thermal stability. The
thermodynamic parameters suggested that the inactivation process of the
partially purified PPO is related to aggregation of partially unfolded enzyme
molecules. CONCLUSION: S.lycocarpum fruits are a promising source of PPO
for biotechnological applications that demands a thermostable catecholase
activity.
Keywords: Solanum lycocarpum; polyphenol oxidase; thermal inactivation.
Fellowship: CAPES