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Enzyme immobilization strategies for the synthesis of perdeuterated molecules

Bogojevic, Oliver LU (2018) KBKM05 20181
Pure and Applied Biochemistry
Computational Chemistry
Abstract
This thesis covers established procedures for the successful and attempted immobilization of lipases and phospholipases to various hydrophobic support materials, utilizing a rotating bed reactor, magnetic stirrer and tube-rotator. The design of a robust platform was established with great instrumental knowledge and proper conditioning for the creation of efficient immobilizations and activity assays. Influencing factors as substrate amount, pH, temperature, solvent selection, solubility, RPM, Co-factors, support material, protein loading and water activity have all been considered and assessed, although not thoroughly evaluated due to the purpose of solely finding an efficient and working system for immobilization. The success of the... (More)
This thesis covers established procedures for the successful and attempted immobilization of lipases and phospholipases to various hydrophobic support materials, utilizing a rotating bed reactor, magnetic stirrer and tube-rotator. The design of a robust platform was established with great instrumental knowledge and proper conditioning for the creation of efficient immobilizations and activity assays. Influencing factors as substrate amount, pH, temperature, solvent selection, solubility, RPM, Co-factors, support material, protein loading and water activity have all been considered and assessed, although not thoroughly evaluated due to the purpose of solely finding an efficient and working system for immobilization. The success of the immobilization experiments has primarily been validated utilizing the NanoDrop- instrument, for protein concentration estimations, and a variety of activity assays. The activity assays are performed utilizing both already commercially immobilized lipases and self-immobilized (in-house) lipases and phospholipases. The conversion rate, established from the activity assays were analyzed utilizing Gas Chromatography Flame Ionization Detector (GC-FID), Nuclear Magnetic Resonance (NMR) and Thin Layer Chromatography (TLC). Lipase from Rhizopus oryzae (ROL) was successfully immobilized, with the proper conditions and working procedures, via adsorption to various hydrophobic support materials. ROL effected a 94% conversion of the lauric acid in the esterification reaction of lauric acid to propyl laurate, and a maintained catalytic activity of 99% after two cycles. No confirmed immobilization of the sn2- specific phospholipase A2 from Porcine pancreas (PLA2) could be presented during the thesis, although a deeper knowledge for the reaction settings and conditions were acquired, resulting in promising opportunities for future development. (Less)
Popular Abstract (Swedish)
Användandet av nya strategier för immobilisering av enzymer (proteiner) möjliggör
för stora framsteg inom neutron forskning. Genom noggranna tester upprättades väl
valda villkor för användandet av enzymer, med ambitionen att effektivisera
framställning av så kallade ”tunga molekyler”. Något som i sin tur är värdefullt för att förstå hur celler är strukturerade samt signalerar och interagerar med varandra.
Please use this url to cite or link to this publication:
author
Bogojevic, Oliver LU
supervisor
organization
course
KBKM05 20181
year
type
H2 - Master's Degree (Two Years)
subject
keywords
Applied Chemistry, Lipases, Phospholipases, Rhizopus oryzae
language
English
id
8951126
date added to LUP
2022-06-30 10:50:41
date last changed
2022-06-30 10:50:41
@misc{8951126,
  abstract     = {{This thesis covers established procedures for the successful and attempted immobilization of lipases and phospholipases to various hydrophobic support materials, utilizing a rotating bed reactor, magnetic stirrer and tube-rotator. The design of a robust platform was established with great instrumental knowledge and proper conditioning for the creation of efficient immobilizations and activity assays. Influencing factors as substrate amount, pH, temperature, solvent selection, solubility, RPM, Co-factors, support material, protein loading and water activity have all been considered and assessed, although not thoroughly evaluated due to the purpose of solely finding an efficient and working system for immobilization. The success of the immobilization experiments has primarily been validated utilizing the NanoDrop- instrument, for protein concentration estimations, and a variety of activity assays. The activity assays are performed utilizing both already commercially immobilized lipases and self-immobilized (in-house) lipases and phospholipases. The conversion rate, established from the activity assays were analyzed utilizing Gas Chromatography Flame Ionization Detector (GC-FID), Nuclear Magnetic Resonance (NMR) and Thin Layer Chromatography (TLC). Lipase from Rhizopus oryzae (ROL) was successfully immobilized, with the proper conditions and working procedures, via adsorption to various hydrophobic support materials. ROL effected a 94% conversion of the lauric acid in the esterification reaction of lauric acid to propyl laurate, and a maintained catalytic activity of 99% after two cycles. No confirmed immobilization of the sn2- specific phospholipase A2 from Porcine pancreas (PLA2) could be presented during the thesis, although a deeper knowledge for the reaction settings and conditions were acquired, resulting in promising opportunities for future development.}},
  author       = {{Bogojevic, Oliver}},
  language     = {{eng}},
  note         = {{Student Paper}},
  title        = {{Enzyme immobilization strategies for the synthesis of perdeuterated molecules}},
  year         = {{2018}},
}