Enzymatic synthesis of phenolic acid esters.

Abstract

As secondary plant metabolites phenolic acids are common components of daily human diet. It is well established that these compounds have several physiological activities, amongst which the antioxidant activity is the most studied one. Indications that degradation of commercial synthetic antioxidants leads to formation of toxic and carcinogenic compounds have made phenolic compounds even more attractive. Nevertheless, their hydrophilic nature limits their efficiency in non polar environments. One of the ways to overcome this obstacle is lipophilization of these compounds via esterification. Enzymatic synthesis seems to be eco-friendly way of producing lipophilic phenols, and the products obtained on this way are considered as green which is of utter importance for the products intended for human consumption. For enzymatic synthesis to prevail over the chemical synthesis, the process has to be efficient i.e. high ester yields should be produced using minimal amounts of enzyme. In order to achieve this it is necessary to optimize key process parameters (enzyme and substrate concentrations, temperature, mixing intensity and reaction time), that was set as the main goal of this thesis. The first aim was to find adequate biocatalyst which would synthesize phenolic esters with high yields. Commercial immobilized preparations of lipase B from Candida antarctica and lipase from Rhizomucor miehei, Novozyme 435 and Lipozyme RM IM, respectively, were tested along with several lipases isolated at the Department of Biochemical Engineering and Biotechnology, University of Belgrade. Amongst all tested enzymes, Novozyme 435 was proved as the superior biocatalyst for all synthesized esters. Further, it was shown that specificity of Novozyme 435 towards phenolic acids was highly dependent upon their structure. The highest conversions were achieved with p-hydroxyphenyl propionic and dihydrocaffeic acids, which both have saturated side chain. Double bond conjugated with aromatic ring of cinnamic acid caused slight decline in achieved conversions. Further, the introduction of electron donating groups in para position led to serious decline in achieved conversions. It was concluded that this inhibition was consequence of both electron and steric effects, with electron effect being the dominant one...

Fenolne kiseline su sekundarni metaboliti biljaka i kao takve čine deo svakodnevne čovekove ishrane. Poznato je da ova jedinjenja poseduju niz fizioloških svojstava koja ih čine atraktivnim u naučnom svetu, među kojima je antioksidativna aktivnost najbolje ispitana. Ovi prirodni antioksidansi još više dobijaju na značaju zbog indikacija da razgradnjom komercijalnih sintetskih antioksidanasa nastaju kancerogena jedinjenja. Međutim, zbog hidrofilne prirode njihova efikasnost u nepolarnim sredinama je ograničena, a lipofilizacija esterifikacijom je jedan od načina da se ovaj problem prevaziđe. Enzimska sinteza fenolnih estara predstavlja ekološki prihvatljiv način sinteze estara, a sami proizvodi dobijeni na ovaj način spadaju u zelene proizvode, što je od izuzetnog značaja kod proizvodnje namirnica namenjenih za ljudsku upotrebu. Kako bi enzimska sinteza estara bila konkurentna hemijskoj, odnosno kako bi se sintetisale velike količine estra uz male utroške enzima, neophodno je izvršiti optimizaciju ključnih reakcionih parametara (koncentracije enzima i supstrata, temperature, intenziteta mešanja i reakcionog vremena), što je bio i cilj ove disertacije. Pre svega pristupilo se pronalaženju adekvatnog biokatalizatora, koji bi sintetisao fenolne estre u visokom prinosu. Ispitani su komercijalni imobilisani preparati lipaze B iz Candida antarctica i lipaze iz Rhizomucor miehei, Novozyme 435 i Lipozyme RM IM, redom, kao i nekoliko lipaza izolovanih i prečišćenih na Katedri za Biohemijsko inženjerstvo i biotehnologiju. Od ispitanih enzima, Novozyme 435 se pokazao kao superioran u sintezi svih sintetisanih estara. U nastavku je pokazano da specifičnost ovog preparata zavisi od strukture fenolne kiseline i da najveći afinitet pokazuje prema fenolnim kiselinama sa zasićenim bočnim nizom, p-hidroksifenilpropionskom i dihidrokafenom kiselinom. Dvostruka veza konjugovana sa aromatičnim prstenom kod cimetne kiseline, dovela je do blagog pada prinosa reakcije, dok je uvođenje elektron donorskih grupa u para položaj prouzrokovalo drastičan pad u ostvarenim prinosima. Utvrđeno je da je elektronski efekat inhibicije dominantan u odnosu na sterni...