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Lipase engineering, production and application : Expanding the lipids tailoring toolbox with applied bioinformatics

Dong, Zehui LU (2024)
Abstract
Enzymes, as efficient and environmentally friendly catalysts, have gained
significant scientific and industrial attention for their mild operating conditions,
high substrate selectivity and reusability when immobilized. Among them, lipases
dominate the global industrial enzyme market, catalyzing various reactions with a
focus on substrate specificity and selectivity. This thesis investigates the
optimization of Rhizopus oryzae lipase (ROL) for lipid modification, emphasizing
its potential for industrial applications.
The research is structured into four key phases aimed at optimizing ROL and its
application: 1) Screening the binding interactions between different fatty acid
substrates and the ROL... (More)
Enzymes, as efficient and environmentally friendly catalysts, have gained
significant scientific and industrial attention for their mild operating conditions,
high substrate selectivity and reusability when immobilized. Among them, lipases
dominate the global industrial enzyme market, catalyzing various reactions with a
focus on substrate specificity and selectivity. This thesis investigates the
optimization of Rhizopus oryzae lipase (ROL) for lipid modification, emphasizing
its potential for industrial applications.
The research is structured into four key phases aimed at optimizing ROL and its
application: 1) Screening the binding interactions between different fatty acid
substrates and the ROL molecule, and investigating relevant structural features
(Paper Ⅰ); 2) Designing ROL mutants based on structural insights from phase 1,
with the objective of altering substrate selectivity (Paper Ⅲ); 3) Developing an
efficient and convenient production system for ROL in the oleaginous yeast
Yarrowia lipolytica (GRAS) (Paper Ⅱ); and 4) Evaluating ROL immobilization
using promising methods and carriers (Paper Ⅳ).
Through the integration of molecular docking, molecular dynamics (MD)
simulations and enzymatic experiments, this study explored the interactions
between various fatty acids and the ROL enzyme, correlating these interactions with
substrate selectivity. Structural insights guided the creation of ROL mutants with
targeted mutations in the lid region, leading to the development of a lid-swapped
chimera mutant that exhibited increased selectivity for long-chain fatty acids over
medium-chain fatty acids – a novel contribution to the field.
Additionally, a high-yield production platform for ROL was developed in
Y. lipolytica using minimal media, simplifying lipase purification and enabling the
evaluation of different expression factors.
Lipase immobilization is crucial for ensuring efficient applications, especially in
non-aqueous environments. This study investigated immobilization strategies
involving hydrophobic interactions and a combination of hydrophobic interaction
and covalent coupling. The results indicate that polyacrylic supports functionalized
with a short alkyl chain and a minimal number of epoxy groups are effective for
ROL immobilization. Furthermore, the study reveals that ROL immobilization
influences its fatty acid selectivity, regardless of whether the process involves pure
adsorption or a combination of adsorption and covalent coupling. (Less)
Please use this url to cite or link to this publication:
author
supervisor
opponent
  • Associate Prof. Pavlidis, Ioannis, University of Crete, Greece.
organization
publishing date
type
Thesis
publication status
published
subject
pages
65 pages
publisher
Biotechnology, Lund University
defense location
Lecture Hall KC:B, Kemicentrum, Naturvetarvägen 22, Faculty of Engineering LTH, Lund University, Lund.
defense date
2024-11-22 09:30:00
ISBN
978-91-8096-078-6
978-91-8096-079-3
language
English
LU publication?
yes
id
e53a0051-476f-491d-b529-05bac7d7079b
date added to LUP
2024-10-30 09:51:08
date last changed
2025-04-04 14:59:07
@phdthesis{e53a0051-476f-491d-b529-05bac7d7079b,
  abstract     = {{Enzymes, as efficient and environmentally friendly catalysts, have gained<br/>significant scientific and industrial attention for their mild operating conditions,<br/>high substrate selectivity and reusability when immobilized. Among them, lipases<br/>dominate the global industrial enzyme market, catalyzing various reactions with a<br/>focus on substrate specificity and selectivity. This thesis investigates the<br/>optimization of Rhizopus oryzae lipase (ROL) for lipid modification, emphasizing<br/>its potential for industrial applications.<br/>The research is structured into four key phases aimed at optimizing ROL and its<br/>application: 1) Screening the binding interactions between different fatty acid<br/>substrates and the ROL molecule, and investigating relevant structural features<br/>(Paper Ⅰ); 2) Designing ROL mutants based on structural insights from phase 1,<br/>with the objective of altering substrate selectivity (Paper Ⅲ); 3) Developing an<br/>efficient and convenient production system for ROL in the oleaginous yeast<br/>Yarrowia lipolytica (GRAS) (Paper Ⅱ); and 4) Evaluating ROL immobilization<br/>using promising methods and carriers (Paper Ⅳ).<br/>Through the integration of molecular docking, molecular dynamics (MD)<br/>simulations and enzymatic experiments, this study explored the interactions<br/>between various fatty acids and the ROL enzyme, correlating these interactions with<br/>substrate selectivity. Structural insights guided the creation of ROL mutants with<br/>targeted mutations in the lid region, leading to the development of a lid-swapped<br/>chimera mutant that exhibited increased selectivity for long-chain fatty acids over<br/>medium-chain fatty acids – a novel contribution to the field.<br/>Additionally, a high-yield production platform for ROL was developed in<br/>Y. lipolytica using minimal media, simplifying lipase purification and enabling the<br/>evaluation of different expression factors.<br/>Lipase immobilization is crucial for ensuring efficient applications, especially in<br/>non-aqueous environments. This study investigated immobilization strategies<br/>involving hydrophobic interactions and a combination of hydrophobic interaction<br/>and covalent coupling. The results indicate that polyacrylic supports functionalized<br/>with a short alkyl chain and a minimal number of epoxy groups are effective for<br/>ROL immobilization. Furthermore, the study reveals that ROL immobilization<br/>influences its fatty acid selectivity, regardless of whether the process involves pure<br/>adsorption or a combination of adsorption and covalent coupling.}},
  author       = {{Dong, Zehui}},
  isbn         = {{978-91-8096-078-6}},
  language     = {{eng}},
  month        = {{10}},
  publisher    = {{Biotechnology, Lund University}},
  school       = {{Lund University}},
  title        = {{Lipase engineering, production and application : Expanding the lipids tailoring toolbox with applied bioinformatics}},
  url          = {{https://lup.lub.lu.se/search/files/198610160/electronic_nailing.pdf}},
  year         = {{2024}},
}