טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
Ph.D Thesis
Ph.D StudentMoran Brouk
SubjectDirected Evolution and Rational Mutagenesis of Toluene
Monooxygenases for Synthesis of Hydroxytyrosol
DepartmentDepartment of Biotechnology and Food Engineering
Supervisor Full Professor Fishman Ayelet
Full Thesis textFull thesis text - English Version


Abstract

The overall goal of this research was to design toluene-monooxygenases (TMOs) via protein engineering techniques in order to obtain enzymes with altered activity and selectivity, and to investigate the structure-function implications. Furthermore, special focus was placed on the biocatalytic production of the commercially-valuable hydroxytyrosol (HTyr), a potent antioxidant, from an inexpensive and abundant substrate, 2-phenylethanol. TMOs were previously shown to be versatile biocatalysts capable of oxidizing a large spectrum of substrates such as substituted aromatic and phenolic compounds. This research demonstrates that TMOs can be also utilized for the synthesis of antioxidants as HTyr. 

HTyr, an important phenol present in olives, stands out as a compound of high added value due to its exceptional antioxidant, antimicrobial and anticarcinogenic activities and beneficial-human health properties. The vast amount of data accumulated regarding the benefits of HTyr, together with its high bioavailability in humans make it a good candidate to serve as an antioxidant for either pharmaceutical or food preparations.

Wild-type TMOs were initially evaluated for their ability to oxidize numerous mono- and di-substituted aromatic compounds, providing further understanding of the factors responsible for controlling the regiospecificity of substrate hydroxylation. Attractive enzymes were further improved using rational and random protein engineering methods to generate mutants with altered substrate specificity and oxidation activity. It was shown that positions located at the orifice of the tunnel act as a gate, controlling the accessibility of substrates to the catalytic site. Additionally, it was discovered that increasing the size of the active-site pocket and enlarging its entrance enables HTyr formation, which wild-type is not capable of. The activity of the enzyme was further improved by utilizing a statistical model resulting with up-to 190-fold activity improvement compared to the wild-type enzyme. This highly improved activity on 2-phenylethanol is 2.6-fold higher than that of the wild-type on the natural substrate, toluene. Thus, by utilizing and combining different protein engineering approaches, we have successfully designed an improved enzyme capable of forming HTyr from 2-phenylethanol.

The design of a biocatalytic production process was next studied using several biochemical engineering tools. At first, the use of boric-acid beads was evaluated with the intention of in situ product removal of HTyr. The beads conjugated with boric acid were shown to be useful for the recovery, purification and stabilization of HTyr. One liter biotransformations were performed and different operational modes were considered, providing applicable insights into HTyr synthesis.