טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
M.Sc Thesis
M.Sc StudentShira Raikin
SubjectIsolation and Characterization of Bacterial Lipases with
Unique Activity
DepartmentDepartment of Biotechnology and Food Engineering
Supervisor Full Professors Shoham Yuval


Abstract

The aim of this work was to isolate bacterial lipases showing specificity to the sn-2 position in triglycerides.  Lipases (triacylglycerol acylhydrolases EC 3.1.1.3) catalyze the hydrolysis of triglycerides and can be used in organic solvents for chemical synthesis.

Lipases with specificity towards the sn-2 position can facilitate the introduction of unique fatty acids into the sn-2 position and thus are of great interest for organic synthesis.  An enrichment culture technique was used to enrich for bacteria possessing lipase sn-2 activity.  The enrichment was based on a unique energy and carbon source, named 2-palmitoyl-1,3-dihexadecyloxy propane, in which only the fatty acid at sn-2 position can be hydrolyzed. The two other carbon chains are attached to the glycerol backbone by ether bonds.   Only bacteria expressing lipases that are active on the sn-2 position are able to utilize this substrate as a sole carbon source. Following the enrichment culture, over fifty bacteria species were isolated.  Two strains, identified as Acinetobacter sp., exhibited high sn-2 preference as was shown by the hydrolysis products of triolein. In each of these isolated strains, two lipases genes, lip1 and lip2, were identified and cloned.   The genes were expressed in E. coli (DE3)BL21 cells. The Lip1 protein was not active on the sn-2 position of the substrates. Lip2 was expressed but was mostly insoluble in the form of inclusion bodies.   Different methods were tested to improve the solubility of Lip2, including unfolding and refolding with urea and in vitro folding in the presence of specific foldase. None of tested procedures resulted in soluble and active Lip2.