טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
Ph.D Thesis
Ph.D StudentFishman Ayelet
SubjectModification of Enzymes for Usage in Organic Solvents
DepartmentDepartment of Biotechnology and Food Engineering
Supervisor Professor Emeritus Uri Cogan


Abstract

In recent years, the employment of biocatalysts for organic synthesis has become an increasingly effective alternative to conventional chemical methods.  There is a special interest in biocatalysis in organic solvents for expanding the range and efficiency of practical applications.  Despite the many advantages of using organic media for biocatalysis, a major drawback is the fact that catalytic activity displayed by enzymes in organic solvents is far lower than in water.  Our objective was to improve enzymatic activity and stability in organic solvents using two different approaches: modification of lipases with surfactants, in particular fatty acids, and immobilization of a model enzyme, horseradish peroxidase (HRP), onto cellulose via cellulose-binding-domain (CBD) mediated binding.


The first approach used, was modification of various lipases by coating with surfactants, mainly fatty acids.  The fatty acid-coated hydrophobic enzymes, showed a 20-45 fold increase in activity in organic solvents, compared with the crude enzyme.  The results suggested, that the interactions within the enzyme-surfactant complex were based on hydrogen bonding and electrostatic interactions.  Fatty acids were also employed as templates to create bio-imprinted lipases.  In this technique, the enzyme was entrapped in an activated conformation when transferred from water to organic solvents.  The bio-imprinted enzymes exhibited activities of 3-10 times higher than the non-treated controls.  The combination of modification and immobilization was shown to be synergistic and effective with respect to enhancement of enzymatic activity and stability.


In a different approach, HRP was stabilized by immobilization onto cellulose beads, via linkage of a CBD.  The CBD-HRP fusion protein preserved both its binding capability, and its catalytic ability in mixed aqueous-organic solvent systems.  The immobilized enzyme was more stable than the native enzyme in increasing concentrations of acetone (0-92%).  Furthermore, it was more active in water-solvent mixtures containing acetone, tetrahydrofurane, acetone or ethanol.


Finally, we have developed a novel and efficient two-step resolution process for multi-kg scale production of both enantiomers of an important chiral molecule, namely, ethyl-3-hydroxybutyrate (HEB). Both enantiomers were obtained in high chemical and enantiomeric purity (>96%), with the total yield of the process being 73%.  The first reaction involved solvent-free acetylation of racemic HEB with vinyl acetate, to produce (S)-HEB. In the second reaction, the (R)-enriched ethyl-3-acetoxybutyrate fraction was subjected to alcoholysis with ethanol to afford the optically pure (R)-HEB.  The main features of the process are the exclusion of solvent (thus ensuring high process throughput), and the use of the same enzyme for both the acetylation and the alcoholysis steps in non-aqueous media.