טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
Ph.D Thesis
Ph.D StudentGihaz Shalev
SubjectStabilization of Lipase T6 from Geobacillus
Stearothermophilus as an Efficient Biocatalyst in
Organic Solvents
DepartmentDepartment of Biotechnology and Food Engineering
Supervisor Professor Ayelet Fishman
Full Thesis textFull thesis text - English Version


Abstract

Enhanced stability in organic solvents is a desirable feature for enzymes implemented under industrial conditions. Lipases potential as biocatalysts is mainly limited by denaturation in polar alcohols. This study focused on lipase from Geobacillus stearothermophilus T6 (LipT6) with the goal of improving its stability in methanol during biodiesel synthesis. The main approaches for LipT6 stabilization were immobilization and protein engineering.

Immobilization within a sol-gel matrix was performed with two matrix types; an 'aliphatic' matrix and an 'aromatic' matrix. Methanol-stable variant of LipT6 was entrapped within both matrices in crude and pure forms to evaluate its esterification activity in hexane. The influence of skim milk and soluble lysate proteins as bulking and stabilizing agents with sol-gel entrapment were investigated. E. coli lysate proteins were better stabilizing agents of LipT6 mutant than skim milk. Crude LipT6 in the aromatic matrix formed uniform, dense and non-aggregated particles imaged by SEM, which were recyclable for more than 20 consecutive cycles and had 80% biodiesel conversion yield from waste oil.

Three independent protein engineering methods were utilized to stabilize LipT6: solvent tunnels engineering, disulfide bond integration and iterative saturation mutagenesis (ISM).

Bulky aromatic residues were rationally incorporated to occupy solvent channels and induce new aromatic interactions. Out of 16 mutants which were screened in 60% methanol, 3 variants showed elevated stability up to 81-fold compared with WT. Double mutant A187F/L360F exhibited an increase in Tm of .0°C in methanol and a 3-fold increase in biodiesel yield from waste chicken oil. Seven crystal structures of different variants were solved disclosing new π-π or CH/π intramolecular interactions emphasizing their significance to improved solvent stability.

Computational disulfide bonds design was performed for LipT6 structure. Thirteen double mutants containing cysteine pairs were screened in 70% methanol. Superior stability was found with variant E251C/G332C (M13) having 5.5-fold higher activity and enhanced Tm of .9°C in 70% methanol, compared with WT. Moreover, M13 converted nearly 80% biodiesel from waste chicken oil, representing 2.4-fold improvement compared with WT. X-ray structural studies confirmed the existence of the engineered disulfide bonds shedding light on the interrelationship between the bond location and backbone architecture with its stabilization impact.

Lastly, ISM was applied on nine selected resides in LipT6 having the highest B-factors. After screening 3000 colonies, only four positions displayed moderate stabilizing effect and combined into a tetra mutant K103T/R104Q/G105E/Q388P.  The variant did not exhibit any thermal or solvent stability and had moderate improvement in biodiesel yield (.5%). X-ray structure of mutant K103T/R104Q/Q388P revealed that no significant intramolecular interactions were generated in the new variant. It is assumed that these flexible loop-located positions have inert role in stabilization of LipT6.

This work on LipT6 stabilization demonstrated the benefits and challenges of different methods to improve enzyme performance in organic solvents. The common technique of immobilization was found to be carried/matrix-dependent while robustness was not always obtained. Rational protein engineering was mostly beneficial depending on the utilized structural targets, however, in all approaches additional interactions were correlated with increased stability.