Hydrolases as Catalysts for Green Chemistry and Industrial Applications - Esterase, Lipase and Phytase

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. 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.

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 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.

• author: Gaber, Yasser
• supervisor: Rajni Hatti-Kaul
• 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: Biotechnology
• publishing date: 2012
• type: Thesis
• publication status: published
• subject: Industrial Biotechnology
• 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
• 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: [e-mail]
• 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