Download The link between Darwin and antioxidants from olives

The link between Darwin and antioxidants from olives
Moran Brouk (Dr. Ayelet Fishman-supervisor)
Department of Biotechnology and Food Engineering, Technion-Israel Institute of Technology,
Haifa, 32000, Israel
Hydroxytyrosol (HTyr), one of the most important phenols present in olives, stands out as a
compound of high added value due to its exceptional antioxidant, antimicrobial and anti-carcinogenic
activities. It is believed to be the antioxidant with the highest free radical scavenging capacity: double that
of quercetin and more than three times that of epicatechin. It has been demonstrated that HTyr inhibits
human low-density lipoprotein (LDL) oxidation, scavenges free radicals, inhibits platelet aggregation and
confers cell protection. In addition, it was found that a low dose of HTyr reduces oxidative stress caused
by smoking and it was identified also as an HIV-1 inhibitor. The vast amount of data accumulated
regarding the benefits of HTyr, together with its high bioavailability in human, make it a good candidate
to serve as an antioxidant for either pharmaceutical or food preparations (i.e. functional foods). Despite
the great potential of HTyr, it is presently not commercially available, therefore its production will have
important industrial applications.
The goal of this research is to engineer oxidizing enzymes, namely toluene monooxygenases (TMOs),
for the biosynthesis of commercially-valuable HTyr, from a cheap and abundant substrate, 2phenylethanol (PEA). This enzymatic hydroxylation is a novel and promising method to synthesize HTyr
in a low cost single-step reaction, with high selectivity while utilizing an environmentally friendly
process.
Eschericia coli cells manipulated to express TMOs are capable of oxidizing a wide range of
substituted aromatic and phenolic compounds with high regiospecificity. Despite the resemblance of PEA
to the natural substrate, toluene, it was found to be a very poor substrate for the wild-type enzymes. In this
research, by employing several protein engineering approaches, the substrate specificity and oxidation
activity were dramatically improved. The non-rational approach of directed evolution, also named
"Darwinian evolution in the test tube", led to the discovery of a new important position, distant from the
active site, which affects the enzyme’s activity. Another valuable residue, located at the entrance of the
channel leading to the active site, was found based on rational design. Furthermore, a statistical model was
developed to give predictions to which mutations should be combined to give further rise in activity. One
triple mutant suggested by this model, had a 200-fold improvement in activity compared to the wild-type
enzyme. It was concluded that increasing the size of the active site pocket and the channel entrance,
enables for the first time, HTyr formation, which the wild-type enzyme was not capable of producing.