Preparation of new lipases derivatives with high activity- stability in anhydrous media: adsorption on hydrophobic supports plus hydrophilization with polyethylenimine

J.M. Guisan, P. Sabuquillo, R. Fernandez-Lafuente, G. Fernandez-Lorente, C. Mateo, P.J. Halling, D. Kennedy, E. Miyata, D. Re

Research output: Contribution to journalArticle

53 Citations (Scopus)

Abstract

A novel method to prepare immobilized lipases derivatives is hereby proposed. Lipases are firstly adsorbed on supports having large internal surfaces covered by hydrophobic groups (e.g. polyacrylic resins covered by C18 moieties). Then, immobilized lipases are incubated in the presence of polyethyleneimine (PEI) at a pH value over the isoelectric point of the enzyme in order to cover the lipase surface with this polymer. In this way, we try to minimize all possible direct interactions between immobilized lipase and organic solvents when using these derivatives in anhydrous media. Lipases from Rhizomucor miehie (RML) and Candida rugosa (CRL) were immobilized according to the proposed protocol. These derivatives were very active and very stable when catalyzing esterifications and transesterifications in anhydrous media. For example, RML derivatives exhibited a very high synthetic activity (more than 1000 Units/g immobilized biocatalyst) even when catalyzing the esterification of lauric acid with octanol at water activity values very close to zero. On the contrary, covalently immobilized derivatives exhibited a much lower synthetic activity under similar conditions (less than 10 Units/g of immobilized biocatalyst), Moreover, these new RML derivatives preserve 100% activity after incubation for 3 days in anhydrous butanone in the presence of molecular sieves. Under the same conditions, commercial immobilized RML lost more than 90% of activity in less than 10 min.
LanguageEnglish
Pages817-824
Number of pages7
JournalJournal of Molecular Catalysis B: Enzymatic
Volume11
Issue number4-6
DOIs
Publication statusPublished - 22 Jan 2001

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Polyethyleneimine
Lipases
Lipase
Adsorption
Derivatives
lauric acid
Biocatalysts
Esterification
Enzymes
Rhizomucor
Butanones
Octanols
Candida
Transesterification
Molecular sieves
Isoelectric Point
Organic solvents
Polymers
Resins
Acids

Keywords

  • lipases
  • interfacial adsorption
  • enzymes in organic medium
  • artificial enzyme microenvironment
  • protein surface hydrophilization
  • sepabeads Selective adsorption
  • organic-solvents
  • immobilization
  • enantioselectivity

Cite this

Guisan, J.M. ; Sabuquillo, P. ; Fernandez-Lafuente, R. ; Fernandez-Lorente, G. ; Mateo, C. ; Halling, P.J. ; Kennedy, D. ; Miyata, E. ; Re, D. / Preparation of new lipases derivatives with high activity- stability in anhydrous media: adsorption on hydrophobic supports plus hydrophilization with polyethylenimine. In: Journal of Molecular Catalysis B: Enzymatic. 2001 ; Vol. 11, No. 4-6. pp. 817-824.
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abstract = "A novel method to prepare immobilized lipases derivatives is hereby proposed. Lipases are firstly adsorbed on supports having large internal surfaces covered by hydrophobic groups (e.g. polyacrylic resins covered by C18 moieties). Then, immobilized lipases are incubated in the presence of polyethyleneimine (PEI) at a pH value over the isoelectric point of the enzyme in order to cover the lipase surface with this polymer. In this way, we try to minimize all possible direct interactions between immobilized lipase and organic solvents when using these derivatives in anhydrous media. Lipases from Rhizomucor miehie (RML) and Candida rugosa (CRL) were immobilized according to the proposed protocol. These derivatives were very active and very stable when catalyzing esterifications and transesterifications in anhydrous media. For example, RML derivatives exhibited a very high synthetic activity (more than 1000 Units/g immobilized biocatalyst) even when catalyzing the esterification of lauric acid with octanol at water activity values very close to zero. On the contrary, covalently immobilized derivatives exhibited a much lower synthetic activity under similar conditions (less than 10 Units/g of immobilized biocatalyst), Moreover, these new RML derivatives preserve 100{\%} activity after incubation for 3 days in anhydrous butanone in the presence of molecular sieves. Under the same conditions, commercial immobilized RML lost more than 90{\%} of activity in less than 10 min.",
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Preparation of new lipases derivatives with high activity- stability in anhydrous media: adsorption on hydrophobic supports plus hydrophilization with polyethylenimine. / Guisan, J.M.; Sabuquillo, P.; Fernandez-Lafuente, R.; Fernandez-Lorente, G.; Mateo, C.; Halling, P.J.; Kennedy, D.; Miyata, E.; Re, D.

In: Journal of Molecular Catalysis B: Enzymatic, Vol. 11, No. 4-6, 22.01.2001, p. 817-824.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Preparation of new lipases derivatives with high activity- stability in anhydrous media: adsorption on hydrophobic supports plus hydrophilization with polyethylenimine

AU - Guisan, J.M.

AU - Sabuquillo, P.

AU - Fernandez-Lafuente, R.

AU - Fernandez-Lorente, G.

AU - Mateo, C.

AU - Halling, P.J.

AU - Kennedy, D.

AU - Miyata, E.

AU - Re, D.

PY - 2001/1/22

Y1 - 2001/1/22

N2 - A novel method to prepare immobilized lipases derivatives is hereby proposed. Lipases are firstly adsorbed on supports having large internal surfaces covered by hydrophobic groups (e.g. polyacrylic resins covered by C18 moieties). Then, immobilized lipases are incubated in the presence of polyethyleneimine (PEI) at a pH value over the isoelectric point of the enzyme in order to cover the lipase surface with this polymer. In this way, we try to minimize all possible direct interactions between immobilized lipase and organic solvents when using these derivatives in anhydrous media. Lipases from Rhizomucor miehie (RML) and Candida rugosa (CRL) were immobilized according to the proposed protocol. These derivatives were very active and very stable when catalyzing esterifications and transesterifications in anhydrous media. For example, RML derivatives exhibited a very high synthetic activity (more than 1000 Units/g immobilized biocatalyst) even when catalyzing the esterification of lauric acid with octanol at water activity values very close to zero. On the contrary, covalently immobilized derivatives exhibited a much lower synthetic activity under similar conditions (less than 10 Units/g of immobilized biocatalyst), Moreover, these new RML derivatives preserve 100% activity after incubation for 3 days in anhydrous butanone in the presence of molecular sieves. Under the same conditions, commercial immobilized RML lost more than 90% of activity in less than 10 min.

AB - A novel method to prepare immobilized lipases derivatives is hereby proposed. Lipases are firstly adsorbed on supports having large internal surfaces covered by hydrophobic groups (e.g. polyacrylic resins covered by C18 moieties). Then, immobilized lipases are incubated in the presence of polyethyleneimine (PEI) at a pH value over the isoelectric point of the enzyme in order to cover the lipase surface with this polymer. In this way, we try to minimize all possible direct interactions between immobilized lipase and organic solvents when using these derivatives in anhydrous media. Lipases from Rhizomucor miehie (RML) and Candida rugosa (CRL) were immobilized according to the proposed protocol. These derivatives were very active and very stable when catalyzing esterifications and transesterifications in anhydrous media. For example, RML derivatives exhibited a very high synthetic activity (more than 1000 Units/g immobilized biocatalyst) even when catalyzing the esterification of lauric acid with octanol at water activity values very close to zero. On the contrary, covalently immobilized derivatives exhibited a much lower synthetic activity under similar conditions (less than 10 Units/g of immobilized biocatalyst), Moreover, these new RML derivatives preserve 100% activity after incubation for 3 days in anhydrous butanone in the presence of molecular sieves. Under the same conditions, commercial immobilized RML lost more than 90% of activity in less than 10 min.

KW - lipases

KW - interfacial adsorption

KW - enzymes in organic medium

KW - artificial enzyme microenvironment

KW - protein surface hydrophilization

KW - sepabeads Selective adsorption

KW - organic-solvents

KW - immobilization

KW - enantioselectivity

UR - http://dx.doi.org/10.1016/S1381-1177(00)00011-4

U2 - 10.1016/S1381-1177(00)00011-4

DO - 10.1016/S1381-1177(00)00011-4

M3 - Article

VL - 11

SP - 817

EP - 824

JO - Journal of Molecular Catalysis B: Enzymatic

T2 - Journal of Molecular Catalysis B: Enzymatic

JF - Journal of Molecular Catalysis B: Enzymatic

SN - 1381-1177

IS - 4-6

ER -