טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
Ph.D Thesis
Ph.D StudentMeshulam-Simon Galit
SubjectIsolation and Characterization of Lipases from Thermophilic
Bacteria for the Preparation of Optically Active
Compounds
DepartmentDepartment of Biotechnology and Food Engineering
Supervisors Professor Yuval Shoham
Dr. Jonah Gavrieli


Abstract

The overall goal of this research was to develop methods for obtaining highly active, novel lipases (triacylglycerol acylhydrolase, EC 3.1.1.3) from thermophilic bacteria, that will be suitable for industrial-scale preparation of optically active compounds.  Over ten thermophilic bacteria were isolated from nature.  Growth characteristics and lipase production from these strains, as well as from previously characterized thermophiles were studied.  The lipase of Bacillus stearothermophilus T-6 was most promising, exhibiting high extracellular lipolytic activity and stereoselectivity in a model reaction.    An lEMBL-3 phage genomic library of strain T-6 was screened and several clones exhibiting lipolytic activity were isolated.  One of the clones, lTB1, contained the 1257 bp lipase gene (lip).  The lip gene encodes for a mature lipase of 388 amino acid residues, corresponding to a molecular weight of 42.6 kDa.  The protein share over 80% identity with other thermophilic Bacillus lipases.  The lip gene was cloned via PCR into a T-7 expression vector and was overexpressed in E. coli resulting in over of 1 gr of enzyme per liter culture.  The recombinant enzyme was purified by heat treatment and hydrophobic interaction chromatography.  Crude lyophilized lipase T-6 powder shows activity in acetylation reactions of primary and secondary alcohols with preference to bulky, aromatic sec-alcohols over primary and aliphatic sec-alcohols.  It also synthetizes a fatty acid ester of middle-chain fatty acid.   It is likely that lipase T-6 can be used as a biocatalyst for the preparation of optically active compounds.