טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
Ph.D Thesis
Ph.D StudentDror Adi
SubjectProtein Engineering of Lipase T6 from Geobacillus
Stearothermophilus for Improved Performance in
Biodiesel Production
DepartmentDepartment of Biotechnology
Supervisor Professor Ayelet Fishman
Full Thesis textFull thesis text - English Version


Abstract


In recent years, depletion in fossil fuel reserves, increase in crude oil price, and environmental concerns have led the world to search for alternative and renewable energy sources. One possible alternative energy source is biodiesel. Biodiesel is a non-petroleum-based diesel fuel consisting of monoalkyl-esters of long chain fatty acids derived from renewable feedstock and short chain alcohols. Enzymatic production of biodiesel by transesterification of triglycerides and alcohol, catalyzed by lipases, offers an environmentally-friendly, and efficient alternative to the chemically catalyzed process while using low-grade feedstock. Methanol is utilized frequently as the alcohol in the reaction due to its reactivity and low cost. However, one of the major drawbacks of the enzymatic system is the presence of high methanol concentrations which leads to methanol-induced unfolding and inactivation of the biocatalyst. Therefore, a methanol-stable lipase is of great interest for the biodiesel industry.

The goal of this study was to employ protein engineering methods to generate a lipase for enhanced stability in methanol in order to improve its performance in biodiesel production. Three protein engineering approaches were used: i) random mutagenesis, ii) structure-guided consensus and, iii) rational design. We chose to work with an unexplored lipase from the thermophilic bacterium Geobacillus stearothermophilus T6. A high throughput colorimetric screening assay was used to evaluate lipase activity after an incubation period in high methanol concentrations. All protein engineering approaches were successful in generating variants with elevated half-life values in 70% methanol. The best variant of the random mutagenesis library, Q185L, exhibited 22-fold improved stability yet its methanolysis activity was decreased by half. The best variant from the consensus library, H86Y/A269T, exhibited 56-fold improved stability in methanol along with elevated thermostability and enhanced methanolysis activity of soybean oil. In the rational design library based on surface modification, we identified variant R374W which exhibited 22-fold improved stability. This mutation was combined with variant H86Y/A269T to create triple mutant H86Y/A269T/R374W. The triple variant exhibited an additive stabilizing effect of 324 minutes in 70% methanol which reflects an 87-fold enhanced stability, elevated thermostability (°C) and 5.3-fold improved methanolysis activity compared to the wild type. This variants’ methanolysis activity of waste chicken oil, was comparable with commercial Lipolase 100L?, and much better than Novozyme? CALB.

Crystal structures of the wild type and the methanol-stable variant H86Y/A269T/R374W provided insights regarding structure-stability correlations. The most prominent features were the formation of new hydrogen bonds between surface residues directly or mediated by structural water molecules, and the stabilization of Zn and Ca binding sites. Mutation sites were also characterized by lower B-factor values indicating improved rigidity. On the other hand, we suggest that Q185L substitution encouraged a “closed lid” conformation which limited both the methanol and substrate excess into the active site.

In summary, in this study protein engineering was successfully used to design a lipase for enhanced stability in methanol and improved performance in biodiesel synthesis which makes it a potential biocatalyst for biodiesel production. In addition, crystal structures improved our understanding of the lipase structure-function correlations.