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A New Screening Method for the Directed Evolution of Thermostable Bacteriolytic Enzymes

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Improving thermostability of papain through structure-based protein engineering.

Debi Choudhury¹, Sampa Biswas, Sumana Roy

¹Crystallography and Molecular Biology Division, Saha Institute of Nuclear Physics, 1/AF Bidhannagar, Kolkata 700064, India.

Protein Engineering, Design & Selection : PEDS

|March 23, 2010

Summary

Engineered papain mutants show enhanced thermostability. A triple mutant (K174RV32SG36S) significantly improved thermal stability and enzymatic activity, making it ideal for industrial applications.

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Area of Science:

Biochemistry
Protein Engineering
Enzymology

Background:

Papain is an industrially important plant cysteine protease.
Its two-domain structure has a catalytic cleft at the domain interface.
Improving thermostability is crucial for industrial enzyme applications.

Purpose of the Study:

To enhance papain's thermostability using structure-based rational design.
To introduce mutations in the interdomain region without affecting enzymatic activity.
To utilize Ervatamin C as a template for thermostable mutant generation.

Main Methods:

Generated single (K174R), double (K174RV32S), and triple (K174RV32SG36S) papain mutants.
Assessed thermostability through half-life (t(1/2)) and temperature profiles (T(max), T(50)).
Employed molecular modeling for in silico stability analysis.

Main Results:

The triple mutant exhibited maximum thermostability, with t(1/2) extended by 94 min at 60°C and 45 min at 65°C.
T(max) and T(50) increased by 15°C and 4°C, respectively, for the triple mutant.
Molecular modeling confirmed highest stability for the triple mutant due to increased interdomain H-bonds/salt-bridges and reduced flexibility.

Conclusions:

Rational design successfully improved papain's thermostability.
The triple mutant demonstrates significant potential for industrial enzyme applications.
Further studies on enzyme inactivation rates beyond T(max) are warranted.