Stability of native and covalently modified papain

Area of Science:

• Biochemistry
• Enzyme Engineering
• Polymer Chemistry

Background:

• Enzyme modification with polymers can enhance stability and activity.
• Papain, a cysteine protease, has industrial applications but limited stability.
• Understanding polymer effects on enzyme structure-function is crucial.

Purpose of the Study:

• To investigate the covalent coupling of papain with polymeric sucrose.
• To evaluate the impact of this modification on papain's catalytic activity, stability, and kinetic properties.
• To explore potential industrial applications of modified papain.

Main Methods:

• Covalent coupling of papain with polymeric sucrose (400 kDa) at varying ratios.
• Assessing retained catalytic activity, pH optima, and kinetic constants.
• Evaluating thermotolerance using T50 values and temperature optima.
• Determining the half-life of thermoinactivation and analyzing thermodynamic parameters.
• Investigating the effect of urea on modified papain activity.

Main Results:

• Modified papain derivatives retained over 80% of intrinsic catalytic activity.
• No significant changes in pH optima and kinetic constants were observed.
• Thermotolerance increased, with higher T50 values (6-10°C) and shifted temperature optima.
• Half-life of modified papain was prolonged 2- to 30-fold compared to native papain.
• Increased activation free energy and enthalpy indicated protein stabilization.
• Modified papain showed activation in the presence of urea.

Conclusions:

• Covalent modification of papain with polymeric sucrose significantly enhances its thermal stability and resistance to denaturation.
• The modification preserves intrinsic catalytic activity and key kinetic parameters, suggesting minimal disruption of the enzyme's tertiary structure.
• Enhanced stability and activity profile make polymeric sucrose-modified papain a promising candidate for various industrial applications requiring robust enzymes.