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

University dissertation from Lund University, Biotechnology Department

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.

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