Računalna i eksperimentalna karakterizacija esteraze iz Rhodopseudomonas Palustris za razgradnju polimliječne kiseline

master's thesis

• Title (english): Computational and experimental characterization of esterase from Rhodopseudomons palustris for the degradation of polylactic acid
• Author: Davor Mendeš
• Mentor: Aleksandra Maršavelski (mentor)
• Mentor: Marko Močibob (mentor)
• Committee member: Aleksandra Maršavelski (predsjednik povjerenstva)
• Committee member: Marko Močibob (član povjerenstva)
• Committee member: Ivan Kodrin (član povjerenstva)
• Committee member: Tomica Hrenar (član povjerenstva)
• Committee member: Đani Škalamera (član povjerenstva)
• Granter: University of Zagreb; Faculty of Science; (Department of Chemistry); Zagreb
• Defense date and country: 2024-02-23, Croatia
• Scientific / art field, discipline and subdiscipline: NATURAL SCIENCES; Chemistry

Abstract

U sklopu diplomskog rada okarakterizirana je α/β hidrolaza RPA1511 iz bakterije Rhodopseudomonas palustris s hidrolitičkom aktivnosti prema polimliječnoj kiselini. Konstruirana je mutirana inačica Arg243Lys, a konstrukcija mutanta motivirana je prethodnim in silico istraživanjem. Hidrolaze su heterologno eksprimirane u bakteriji Escherichia coli, potom su eksprimirani proteini pročišćeni afinitetnom kromatografijom. Aktivnost hidrolize hidrolaza polimliječne kiseline ispitana je na suspenziji polimliječne kiseline uklopljene u agarozi te pomoću turbidimetrijskog testa. Dinamika RPA1511 i njene mutirane inačice ispitana je pomoću simulacija molekularne dinamike, te je procijenjena entalpija vezanja modelnog supstrata dekamera mliječne kiseline. Nadalje, istražen je pojedinačni doprinos aminokiselina entalpije vezanja dekamera mliječne kiseline.

Abstract (english)

In this work, an α/β hydrolase RPA1511 from Rhodopseudomonas palustris with activity towards polylactic acid was characterized experimentally and computationally by means of turibidmetric assays and molecular dynamics simulations. Motivated by in silico studies perfomed on RPA1511, a mutant Arg243Lys with potentially higher hydrolitic acitvity towards polylactic acid was also characterized. Both hydrolazes were expressed heterologously in Escherichia coli and purified by affinity chromatography. Their hydrolitic acitivity towards polylactic acid was assessed on polyester suspension solidified with agarose and by turbidimetric suspension assay. On the other hand, their dynamical properties were studied in silico utilizing molecular dynamics simulations modelled with the decamer of lactic acid. The binding enthalpy was calculated with the use of an end-point method. To assess the importance of amino acid residues involved in binding decameric lactic acid, the per-residue enthalpy decomposition was calculated.

• Keywords: hidrolaze; enzimska razgradnja bioplastike; polimliječna kiselina; simulacijske molekularne dinamike; testovi hidrolitičke aktivnosti
• Keywords (english): hydrolazes; biodegradation; molecular dynamics simulations; polylactic acid; turbidimetric assays
• Language: croatian
• URN:NBN: urn:nbn:hr:217:245970
• Study programme: Title: Graduate university studies in chemistry; specializations in: Graduate university studies in chemistry; Course: Graduate university studies in chemistry; Study programme type: university; Study level: graduate; Academic / professional title: sveučilišni/a magistar/magistra kemije (sveučilišni/a magistar/magistra kemije)
• Type of resource: Text
• File origin: Born digital
• Access conditions: Open access
• Created on: 2024-03-08 09:16:49