Hydrolases as Catalysts for Green Chemistry and Industrial Applications - Esterase, Lipase and Phytase
(2012)- Abstract
- The use of enzymes in industrial applications has been recognised for providing clean
processes with minimal impact on the environment. This thesis presents studies on
engineering of enzymes and enzyme-based processes in the light of green chemistry and
environmental sustainability, and focuses on three hydrolases: esterase, lipase and phytase.
The use of esterase has been investigated to provide an alternative clean route for the
synthesis of a chiral pharmaceutical compound, S-clopidogrel, by selective hydrolysis of
the racemic precursor. Current production of the pure S- clopidogrel isomer involves the
use of a resolving agent, L-camphorsulfonic acid, and organic solvents.... (More) - The use of enzymes in industrial applications has been recognised for providing clean
processes with minimal impact on the environment. This thesis presents studies on
engineering of enzymes and enzyme-based processes in the light of green chemistry and
environmental sustainability, and focuses on three hydrolases: esterase, lipase and phytase.
The use of esterase has been investigated to provide an alternative clean route for the
synthesis of a chiral pharmaceutical compound, S-clopidogrel, by selective hydrolysis of
the racemic precursor. Current production of the pure S- clopidogrel isomer involves the
use of a resolving agent, L-camphorsulfonic acid, and organic solvents. Screening of
different hydrolases revealed that crude pig liver esterase (PLE), a mixture of different
isoenzymes, selectively acts on the R isomer, with E = 8.3. Two PLE isoenzymes, PLE-1
and PLE-3 were tested individually as catalysts for the reaction. Molecular modeling
simulations indicated that a phenylalanine F407 residue is destabilizing for the R-isomer-
PLE-1 tetrahedral intermediate. PLE-1 and its mutants were expressed in Escherichia coli
with a chaperon system. Interestingly, mutations of F407 to alanine or leucine led to a
dramatic increase in activity but with reversed selectivity (E=3.3 and E>100 respectively
towards the S isomer). On the other hand, PLE-3 isoenzyme selectively hydrolysed the
correct isomer, R, with E=10.
Immobilized lipase B from Candida antarctica, was employed for the production of
biodegradable specialty chemicals from renewable resources. N-alkanoyl-Nmethylglucamide,
a bio-based surfactant, was produced in a solvent-free reaction.
Engineering the molar ratio of the substrates in the reaction and adding a step involving
hydrolysis of the by-product resulted in final yield of 99 %. Comparison with earlier
reports based on green metrics showed the method to have a greener profile. Another
product, trimethylolpropane-oleate, a biolubricant, was produced by lipase-catalysed
esterification with high yield, better product quality and a greener profile compared to the
process catalysed by other heterogeneous chemical catalysts. Reliable metrics of the
greenness of a process are essential for the progress of green chemistry. As an important
contribution in this area, a java-based software HPLC-EAT was developed to evaluate the
greenness of liquid chromatographic methods. The tool is freely available at
www.biotek.lu.se/hplc-eat.
The enzyme phytase hydrolyses phytate (myo-inositol 1,2,3,4,5,6-hexakisphosphate),
an anti-nutrient compound present in cereals and grains, and increases the bioavailability
of phosphorus and other nutrients. A recombinant thermostable Bacillus sp. MD2 alkaline
phytase, a metallo-enzyme, was characterized and the effect of various divalent metal ions
on its stability and catalytic properties was studied. The presence of calcium ions on both
the enzyme and the substrate was required for optimal activity and stability of the
enzyme. Furthermore, site-directed mutagenesis of the enzyme was done to improve its
activity and stability in the acidic environment. Mutation of a glutamate residue in the
enzyme active site to serine E227S led to a slight decrease of the optimum pH and higher
stability at low pH. (Less) - Abstract (Swedish)
- Popular Abstract in English
The chemical industry has greatly contributed to the advancement of the human
civilization. However, the side effects of this industry have become a real
environmental burden. The message in this thesis is that enzymes have a great
potential as catalysts for clean and sustainable processes. With three enzymes:
an esterase, a lipase and a phytase, the thesis demonstrated how to rationally
engineer the enzyme structure to improve its performance for a certain task,
how to engineer an enzyme-based reaction to run solvent-free processes, the
advantages of biocatalysts over conventional chemical catalysts, and the importance
of... (More) - Popular Abstract in English
The chemical industry has greatly contributed to the advancement of the human
civilization. However, the side effects of this industry have become a real
environmental burden. The message in this thesis is that enzymes have a great
potential as catalysts for clean and sustainable processes. With three enzymes:
an esterase, a lipase and a phytase, the thesis demonstrated how to rationally
engineer the enzyme structure to improve its performance for a certain task,
how to engineer an enzyme-based reaction to run solvent-free processes, the
advantages of biocatalysts over conventional chemical catalysts, and the importance
of green metrics in the evaluation of the developed processes. The integration
of computer science, bioinformatics, molecular biology, and chemistry is
traced throughout this thesis. Such an interdisciplinary approach is promising
and necessary for the future development of biocatalysis and green chemistry. (Less)
Please use this url to cite or link to this publication:
https://lup.lub.lu.se/record/2518358
- author
- Gaber, Yasser LU
- supervisor
- opponent
-
- Professor Sheldon, Roger, Professor Emeritus of Biocatalysis and Organic Chemistry at Delft University of Technology and Chief Executive Officer of CLEA Technologies B.V., Delft, The Netherlands.
- organization
- publishing date
- 2012
- type
- Thesis
- publication status
- published
- subject
- keywords
- HPLC-EAT, sustainable environment, mutation, metallo-enzyme, phytate, b-propeller, biodegraqdable, metrics, green chemistry, Novozym 435, CalB, solvent-free, kinetic resolution, Biocatalysis, rational design, PLE, esterase, clopidogrel, enantioselectivity
- pages
- 142 pages
- publisher
- Lund University, Biotechnology Department
- defense location
- Lecture Hall A, Center for Chemistry and Chemical Engineering, Getingevägen 60, Lund University Faculty of Engineering
- defense date
- 2012-05-24 10:30:00
- ISBN
- 978-91-89627-81-9
- language
- English
- LU publication?
- yes
- additional info
- Author alternative e-mail: yassergaber2007@yahoo.com
- id
- c2e14c3f-938b-4a70-abe4-bcae226163e1 (old id 2518358)
- date added to LUP
- 2016-04-04 12:01:25
- date last changed
- 2018-11-21 21:08:33
@phdthesis{c2e14c3f-938b-4a70-abe4-bcae226163e1, abstract = {{The use of enzymes in industrial applications has been recognised for providing clean<br/><br> processes with minimal impact on the environment. This thesis presents studies on<br/><br> engineering of enzymes and enzyme-based processes in the light of green chemistry and<br/><br> environmental sustainability, and focuses on three hydrolases: esterase, lipase and phytase.<br/><br> The use of esterase has been investigated to provide an alternative clean route for the<br/><br> synthesis of a chiral pharmaceutical compound, S-clopidogrel, by selective hydrolysis of<br/><br> the racemic precursor. Current production of the pure S- clopidogrel isomer involves the<br/><br> use of a resolving agent, L-camphorsulfonic acid, and organic solvents. Screening of<br/><br> different hydrolases revealed that crude pig liver esterase (PLE), a mixture of different<br/><br> isoenzymes, selectively acts on the R isomer, with E = 8.3. Two PLE isoenzymes, PLE-1<br/><br> and PLE-3 were tested individually as catalysts for the reaction. Molecular modeling<br/><br> simulations indicated that a phenylalanine F407 residue is destabilizing for the R-isomer-<br/><br> PLE-1 tetrahedral intermediate. PLE-1 and its mutants were expressed in Escherichia coli<br/><br> with a chaperon system. Interestingly, mutations of F407 to alanine or leucine led to a<br/><br> dramatic increase in activity but with reversed selectivity (E=3.3 and E>100 respectively<br/><br> towards the S isomer). On the other hand, PLE-3 isoenzyme selectively hydrolysed the<br/><br> correct isomer, R, with E=10.<br/><br> Immobilized lipase B from Candida antarctica, was employed for the production of<br/><br> biodegradable specialty chemicals from renewable resources. N-alkanoyl-Nmethylglucamide,<br/><br> a bio-based surfactant, was produced in a solvent-free reaction.<br/><br> Engineering the molar ratio of the substrates in the reaction and adding a step involving<br/><br> hydrolysis of the by-product resulted in final yield of 99 %. Comparison with earlier<br/><br> reports based on green metrics showed the method to have a greener profile. Another<br/><br> product, trimethylolpropane-oleate, a biolubricant, was produced by lipase-catalysed<br/><br> esterification with high yield, better product quality and a greener profile compared to the<br/><br> process catalysed by other heterogeneous chemical catalysts. Reliable metrics of the<br/><br> greenness of a process are essential for the progress of green chemistry. As an important<br/><br> contribution in this area, a java-based software HPLC-EAT was developed to evaluate the<br/><br> greenness of liquid chromatographic methods. The tool is freely available at<br/><br> www.biotek.lu.se/hplc-eat.<br/><br> The enzyme phytase hydrolyses phytate (myo-inositol 1,2,3,4,5,6-hexakisphosphate),<br/><br> an anti-nutrient compound present in cereals and grains, and increases the bioavailability<br/><br> of phosphorus and other nutrients. A recombinant thermostable Bacillus sp. MD2 alkaline<br/><br> phytase, a metallo-enzyme, was characterized and the effect of various divalent metal ions<br/><br> on its stability and catalytic properties was studied. The presence of calcium ions on both<br/><br> the enzyme and the substrate was required for optimal activity and stability of the<br/><br> enzyme. Furthermore, site-directed mutagenesis of the enzyme was done to improve its<br/><br> activity and stability in the acidic environment. Mutation of a glutamate residue in the<br/><br> enzyme active site to serine E227S led to a slight decrease of the optimum pH and higher<br/><br> stability at low pH.}}, author = {{Gaber, Yasser}}, isbn = {{978-91-89627-81-9}}, keywords = {{HPLC-EAT; sustainable environment; mutation; metallo-enzyme; phytate; b-propeller; biodegraqdable; metrics; green chemistry; Novozym 435; CalB; solvent-free; kinetic resolution; Biocatalysis; rational design; PLE; esterase; clopidogrel; enantioselectivity}}, language = {{eng}}, publisher = {{Lund University, Biotechnology Department}}, school = {{Lund University}}, title = {{Hydrolases as Catalysts for Green Chemistry and Industrial Applications - Esterase, Lipase and Phytase}}, url = {{https://lup.lub.lu.se/search/files/5909285/2520402.pdf}}, year = {{2012}}, }