Evaluacija nitrilaza kao biokatalizatora za sintezu (R)-4-kloro-3-hidroksimaslačne kiseline

master's thesis

• Title (english): Evaluation of nitrilases as biocatalysts for the synthesis of (R)-4-chloro-3-hydroxybutyric acid
• Author: Ana Antolović
• Mentor: Zvjezdana Findrik Blažević (mentor)
• Committee member: Zvjezdana Findrik Blažević (predsjednik povjerenstva)
• Committee member: Marija Vuković Domanovac (član povjerenstva)
• Committee member: Martina Sudar (član povjerenstva)
• Granter: University of Zagreb; Faculty of Chemical Engineering and Technology; Zagreb
• Defense date and country: 2022-09-30, Croatia
• Scientific / art field, discipline and subdiscipline: TECHNICAL SCIENCES; Chemical Engineering

Abstract

Enzimi nitrilaze kataliziraju reakcije hidrolize nitrila u karboksilne kiseline i amonijak, bez stvaranja slobodnih amidnih međuprodukata. Iz tog razloga nitrilaza se može koristiti kao biokatalizator u reakcijama pretvorbe (R)-4-klor-3-hidroksibutironitrila (KHB) u (R)-4-klor-3-hidroksibutansku kiselinu, pri čemu dolazi do spontane ciklizacije produkta u (R)-3-hidroksi-γ-butirolakton (HBL). HBL se koristi u sintezi različitih polimera, otapala i farmaceutika kao kiralni građevni blok. Enzimski katalizirani procesi sve se češće primjenjuju za sintezu HBL-a jer koriste blage reakcijske uvjete, postižu visoku selektivnost te mogu pružiti ekonomski isplativ postupak sinteze ove vrijedne kemikalije. Brojni, već postojeći kemijski putevi sinteze HBL-a, pokušavaju se što manje koristiti jer ih većina ima nedostatke poput skupih ili opasnih reaktanata, intenzivnih procesnih uvjeta, te niskih iskorištenja i selektivnosti, što uzrokuje poskupljenje procesa izdvajanja produkta. U ovom radu provedena je evaluacija 24 nitrilaze u reakciji sinteze HBL-a. Tijekom ispitivanja korišteni su isti početni uvjeti, koncentracija supstrata (KHB) i masena koncentracija nitrilaza te je reakcija praćena 6 sati. Enzim s najvećim iskorištenjem na produktu izabran je za sljedeći korak, kinetičko istraživanje. Određeni su kinetički parametri, nitrilazom katalizirane sinteze (R)-4-klor-3-hidroksibutanske kiseline iz (R)-4-klor-3-hidroksibutironitrila, iz kojih je razvijen matematički model. Sastoji se od kinetičkog modela i bilanci tvari u reaktoru. Utvrđeno je da enzim ima nizak afinitet prema supstratu te da reakcija prati Michaelis-Menteničinu kinetiku uz inhibicijski učinak supstrata i produkta. Model je uspješno validiran provedbom reakcije u šest različitih kotlastih reaktora s različitim početnim uvjetima u reaktoru. Uzorci reakcijske smjese su analizirani na tekućinskom kromatografu visoke djelotvornosti i plinskom kromatografu. Iz navedenih eksperimenata procijenjena je konstanta deaktivacije te je zaključeno da početni uvjeti u reaktoru utječu na stabilnost enzima.

Abstract (english)

Nitrilases catalyze nitrile hydrolysis reactions to carboxylic acids and ammonia, without the formation of free amide intermediates. For this reason, nitrilases can be used as a biocatalyst in conversion reactions (R)-4-chlorine-3-hydroxybutyronitrile (KHB) into (R)-4-chlorine-3-hydroxybutane acid, where spontaneous cycling of the product occurs in (R)-3-hydroxy-γ-butyrobactone (HBL). HBL is used in the synthesis of various polymers, solvents and pharmaceuticals as a chiral building block. Enzyme catalyzed processes are increasingly used for the synthesis of HBL because of the mild reaction conditions, high selectivity and can provide an economically viable synthesis process for this valuable chemical. While numerous, already existing chemical pathways of HBL synthesis are attempted to be used as little as possible because most of them have defects such as expensive or dangerous reactants, intense process conditions, and low utilization and selectivity, which causes the product extraction process to increase. In this work, a panel of 24 nitrilases was evaluated. During the study, the same initial conditions were used, the concentration of the substrate (KHB) and the mass concentration of nitrilase, while the reaction was monitored for 6 hours. The enzyme with the highest obtained product yield was chosen for the next step, i.e., kinetic research. Kinetic parameters of nitrilase catalyzed synthesis (R)-4-chlorine-3-hydroxybutanic acid from (R)-4-chlorine-3-hydroxybutironitrile were determined, from which the mathematical model was developed. It consists of a kinetic model and a balance of substances in the reactor. It was found that the enzyme has a low affinity for the substrate and that the reaction accompanies Michaelis-Menten's kinetics and the inhibition effect of the substrate and the product. The model was successfully validated by the implementation of the reaction in six different batch reactors with different initial conditions in the reactor. Samples of the reaction mixture were analyzed on a high-efficiency liquid chromatograph and a gas chromatograph. From these experiments, the deactivation constant was evaluated and it was concluded that the initial conditions in the reactor affect the stability of the enzyme.

• Keywords: nitrilaza; kinetički model
• Keywords (english): nitrilase; kinetic model
• Language: croatian
• URN:NBN: urn:nbn:hr:149:769393
• Study programme: Title: Environmental Engineering - Graduate study; Study programme type: university; Study level: graduate; Academic / professional title: magistar/magistra inženjer/inženjerka ekoinženjerstva (magistar/magistra inženjer/inženjerka ekoinženjerstva)
• Type of resource: Text
• File origin: Born digital
• Access conditions: Embargoed access; Embargo expiration date: 2024-10-01
• Created on: 2023-03-31 09:45:15