Lipases as Biocatalysts in Organic Media. Influence of Enzyme Preparation and Reaction Conditions on Activity and Selectivity
(2001)- Abstract
- The discovery that enzymes can function in organic solvents has broadened the scope of biocatalysis, making enzymes highly useful tools for organic chemists. The widespread use of enzymes as catalysts in organic solvents has, however, been limited by the low activity obtained for enzymes in organic solvents compared to aqueous solutions. The increasing demand for optically pure enantiomers in pharamaceuticals and in agrochemicals has further broadened the interest in using enzymes as catalysts for the production of enantiopure compounds. In the present study an evaluation of different methods of enzyme preparation for increasing the activity of lipases in organic solvent is performed. The influence of different reaction conditions on... (More)
- The discovery that enzymes can function in organic solvents has broadened the scope of biocatalysis, making enzymes highly useful tools for organic chemists. The widespread use of enzymes as catalysts in organic solvents has, however, been limited by the low activity obtained for enzymes in organic solvents compared to aqueous solutions. The increasing demand for optically pure enantiomers in pharamaceuticals and in agrochemicals has further broadened the interest in using enzymes as catalysts for the production of enantiopure compounds. In the present study an evaluation of different methods of enzyme preparation for increasing the activity of lipases in organic solvent is performed. The influence of different reaction conditions on enantioselectivity in lipase catalysed kinetic resolution reactions is also investigated. Furthermore the importance of controlling the water activity to obtain high catalytic activity is emphasised. By changing the solvent composition and the water activity the enantioselectivity can be altered but the effects upon a change of the reaction medium is difficult to rationalise and predict. The temperature was found to influence enantioselectivity in all of the lipase catalysed reactions investigated in the present work and a thermodynamic model describing the effects of temperature on enantioselectivity enabled such effects to be rationalised. Addition of different additives such as KCl, crown ether and phosphate buffer during the lyophilisation of Humicola lanuginosa lipase increases the esterification activity up to 46 fold compared to the crude powder. The increase in activity for preparations lyophilised in the presence of KCl or phoshate buffer is probably due to the additives serving as an immobilisation matrix for the enzyme. The higher activity obtained for the crown ether preparation is probably due to a molecular imprinting effect. The crown ether supposedly binds to the active site during the dehydration step, keeping the active site of the enzyme in a conformation that is catalytically active. Immobilisation by adsorption onto a porous polypropylene support (Accurel EP-100) proved to be the most efficient method for using lipases in organic solvents. The specific activity was 400 fold higher as compared with the crude powder when lipase from Humicola lanuginosa was immobilised by this adsorption technique. Active site titration revealed that part of the explanation for the high activity obtained when lipases are immobilised onto Accurel EP-100 is due to a higher accessibility of the enzyme molecules as compared with lyophilised preparations. At optimal enzyme loading 93 % of the enzyme molecules were titrated at a high rate indicating that this adsorption on a hydrophobic surface was a very efficent way for reducing mass transfer limitations and to immobilise the enzyme in active conformation for use in organic solvents. A further advantage with the immobilisation technique is that a rather high protein loading (100 mg/g carrier) can be combined with the high specific activity, making this enzyme preparation interesting for industrial applications. (Less)
Please use this url to cite or link to this publication:
https://lup.lub.lu.se/record/42271
- author
- Persson, Mattias LU
- supervisor
- opponent
-
- Dr Secundo, Fransesco, Instituto di Biocatalisis e Riconoscimento Moleculare, via Mario Branco 9,20131 Milano
- organization
- publishing date
- 2001
- type
- Thesis
- publication status
- published
- subject
- keywords
- Biochemical technology, Bioteknik, Biotechnology, water activity, temperature, enantioselectivity, immobilisation, organic solvents, lyophilisation, Lipases, Accurel EP-100, Biokemisk teknik, Proteins, enzymology, Proteiner, enzymologi
- pages
- 150 pages
- publisher
- Mattias Persson Sölvegatan 39, kemicentrum, Lunds universitet
- defense location
- Hörsal K:B på kemicentrum, Sölvegatan 39, Lund
- defense date
- 2001-01-25 10:15:00
- external identifiers
-
- other:ISRN: LUTKDH/TKBT--02/1057-SE
- ISBN
- 91-89627-05-9
- language
- English
- LU publication?
- yes
- additional info
- Article: Mutants provide evidence of the importance of glycosidic chains in the activation of lipase 1 from Candida rugosa. Brocca S, Persson M, Wehtje E, Adlercreutz P, Alberghina L, Lotti M. (2000) Protein Science 9:985-990. Article: Effects of solvent, water activity and temperature on lipase and hydroxynitrile lyase enantioselectivity. Persson M, Costes D, Wehtje E, Adlercreutz P. Submitted.Immobilisation of lipases by adsorption and deposition. High protein loading gives lower water activity optimum. Persson M, Wehtje E, Adlercreutz P.Biotechnol. Lett. (2000) 22:1571-1575. Article: Preparation of lipases for use in organic solvents.Persson M, Mladenoska I, Wehtje E, Adlercreutz P. Submitted. Article: Factors governing the activity of lyophilised and immobilised lipase preparations in organic solvents. Persson M, Wehtje E, Adlercreutz P. Submitted.Enzymatic fatty acid exchange in digalactosyldiacylglycerol. Persson M, Svensson I, Adlercreutz P. (2000) Chem. Phys. Lipids. 104:13-21.
- id
- c3f4e558-ab4f-482c-a7f4-51584691d8f5 (old id 42271)
- date added to LUP
- 2016-04-04 11:35:35
- date last changed
- 2018-11-21 21:05:52
@phdthesis{c3f4e558-ab4f-482c-a7f4-51584691d8f5,
abstract = {{The discovery that enzymes can function in organic solvents has broadened the scope of biocatalysis, making enzymes highly useful tools for organic chemists. The widespread use of enzymes as catalysts in organic solvents has, however, been limited by the low activity obtained for enzymes in organic solvents compared to aqueous solutions. The increasing demand for optically pure enantiomers in pharamaceuticals and in agrochemicals has further broadened the interest in using enzymes as catalysts for the production of enantiopure compounds. In the present study an evaluation of different methods of enzyme preparation for increasing the activity of lipases in organic solvent is performed. The influence of different reaction conditions on enantioselectivity in lipase catalysed kinetic resolution reactions is also investigated. Furthermore the importance of controlling the water activity to obtain high catalytic activity is emphasised. By changing the solvent composition and the water activity the enantioselectivity can be altered but the effects upon a change of the reaction medium is difficult to rationalise and predict. The temperature was found to influence enantioselectivity in all of the lipase catalysed reactions investigated in the present work and a thermodynamic model describing the effects of temperature on enantioselectivity enabled such effects to be rationalised. Addition of different additives such as KCl, crown ether and phosphate buffer during the lyophilisation of Humicola lanuginosa lipase increases the esterification activity up to 46 fold compared to the crude powder. The increase in activity for preparations lyophilised in the presence of KCl or phoshate buffer is probably due to the additives serving as an immobilisation matrix for the enzyme. The higher activity obtained for the crown ether preparation is probably due to a molecular imprinting effect. The crown ether supposedly binds to the active site during the dehydration step, keeping the active site of the enzyme in a conformation that is catalytically active. Immobilisation by adsorption onto a porous polypropylene support (Accurel EP-100) proved to be the most efficient method for using lipases in organic solvents. The specific activity was 400 fold higher as compared with the crude powder when lipase from Humicola lanuginosa was immobilised by this adsorption technique. Active site titration revealed that part of the explanation for the high activity obtained when lipases are immobilised onto Accurel EP-100 is due to a higher accessibility of the enzyme molecules as compared with lyophilised preparations. At optimal enzyme loading 93 % of the enzyme molecules were titrated at a high rate indicating that this adsorption on a hydrophobic surface was a very efficent way for reducing mass transfer limitations and to immobilise the enzyme in active conformation for use in organic solvents. A further advantage with the immobilisation technique is that a rather high protein loading (100 mg/g carrier) can be combined with the high specific activity, making this enzyme preparation interesting for industrial applications.}},
author = {{Persson, Mattias}},
isbn = {{91-89627-05-9}},
keywords = {{Biochemical technology; Bioteknik; Biotechnology; water activity; temperature; enantioselectivity; immobilisation; organic solvents; lyophilisation; Lipases; Accurel EP-100; Biokemisk teknik; Proteins; enzymology; Proteiner; enzymologi}},
language = {{eng}},
publisher = {{Mattias Persson Sölvegatan 39, kemicentrum, Lunds universitet}},
school = {{Lund University}},
title = {{Lipases as Biocatalysts in Organic Media. Influence of Enzyme Preparation and Reaction Conditions on Activity and Selectivity}},
year = {{2001}},
}