Activity And Enantioselectivity Of Modified Lipases In Organic Solvents

Lipases from Rhizomucor miehei and Candida antarctica component B (CalB), and cutinase from Fusarium solani pisi were chemically modified with tresyl activated polyethylene glycol monomethyl ether (MPEG). The enantioselectivity of the native and the modified lipases in the transesterification reaction of three different chiral alcohols was measured in o-xylene and tert-butyl methyl ether at the optimal water activities. Both activity and enantioselectivity changed upon modification of the enzymes. To study the effect of the hydrophobicity of the modifier and degree of modification on the activity and enantioselcctivity of CalB in more depth, this lipase was chemically modified with MPEG, polyethylene glycol monooctyl ether (OPEG) and n-octanol (OCT). The hydrophobicity of the modifier had a profound effect on the enantioselectivity. Whereas MPEG modification increased the cnantioselectivity of CalB, modification with OPEG and OCT decreased the enantioselectivity as compared to native CalB. With higher degrees of modification both activity and enantioselectivity increased. The enthalpic and en tropic contributions to the enantioselectivity, ΔΔH#R-S, and ΔΔS#R-S, were calculated from the E-values measured at various temperatures. No clear correlation was observed between ΔΔH#R-S or ΔΔS#R-S, and the degree of modification, structure or hydrophobicity of the modifier. Linear enthalpy-entropy compensation was observed for the different CalB spccies. The present results indicate that, in order to increase enzyme activity and enantioselectivity, a modifier should be used which positively affects the porosity of the enzyme aggregates or enzyme flexibility. Also higher degrees of modification are preferred.

[1]  M. Egmond,et al.  Effect of chemical modification on the activity of lipases in organic solvents , 1999 .

[2]  G. Carrea,et al.  Optimization of Pseudomonas cepacia lipase preparations for catalysis in organic solvents. , 1999, Biotechnology and bioengineering.

[3]  J. Ottosson,et al.  The Temperature Dependence of Enzymatic Kinetic Resolutions Reveals the Relative Contribution of Enthalpy and Entropy to Enzymatic Enantioselectivity , 1999 .

[4]  J. Jongejan,et al.  Enthalpic and entropic contributions to lipase enantioselectivity , 1998 .

[5]  P. Adlercreutz,et al.  Physical characterization of porous materials and correlation with the activity of immobilized enzyme in organic medium , 1998 .

[6]  K. Hult,et al.  CHIRAL RECOGNITION OF ALCOHOL ENANTIOMERS IN ACYL TRANSFER REACTIONS CATALYSED BY CANDIDA ANTARCTICA LIPASE B , 1998 .

[7]  A. Straathof,et al.  The enantiomeric ratio: origin, determination and prediction , 1997 .

[8]  G. Carrea,et al.  Activity, stability, and conformation of methoxypoly(ethylene glycol)-subtilisin at different concentrations of water in dioxane. , 1997, Biotechnology and bioengineering.

[9]  Y. Okahata,et al.  Lipid-coated enzymes as efficient catalysts in organic media , 1997 .

[10]  C. Lowe,et al.  Surfactant modified enzymes: Solubility and activity of surfactant-modified catalase in organic solvents , 1997 .

[11]  M. Reetz,et al.  Efficient immobilization of lipases by entrapment in hydrophobic sol‐gel materials , 2000, Biotechnology and bioengineering.

[12]  H. Anthonsen,et al.  Calculation of enantiomer ratio and equilibrium constants in biocatalytic ping-pong bi-bi resolutions , 1996 .

[13]  A. Margolin.  Novel crystalline catalysts , 1996 .

[14]  P. Wangikar,et al.  Active‐site titration of serine proteases in organic solvents , 1996, Biotechnology and bioengineering.

[15]  F. Plou,et al.  Effect of Surfactants on Activity and Stability of Native and Chemically Modified Lipases A and B from Candida Rugosa , 1996 .

[16]  D. Reinhoudt,et al.  Flexibility of Enzymes Suspended in Organic Solvents Probed by Time-Resolved Fluorescence Anisotropy. Evidence That Enzyme Activity and Enantioselectivity Are Directly Related to Enzyme Flexibility , 1995 .

[17]  P. Halling,et al.  Biocatalyst behaviour in low-water systems , 1995 .

[18]  K. Hult,et al.  Kinetics of acyl transfer reactions in organic media catalysed by Candida antarctica lipase B. , 1995, Biochimica et biophysica acta.

[19]  H. Gais,et al.  Polyethylene glycol monomethyl ether-modified pig liver esterase: Preparation, characterization and catalysis of enantioselective hydrolysis in water and acylation in organic solvents , 1995 .

[20]  Y. Inada,et al.  Chemical modification of proteins with polyethylene glycols , 1995 .

[21]  D. Reinhoudt,et al.  Large activation of serine proteases by pretreatment with crown ethers , 1995 .

[22]  A. Klibanov,et al.  The solvent dependence of enzyme specificity. , 1994, Biochimica et biophysica acta.

[23]  M. Arroyo,et al.  Stereoselectivity of chemically modified α-chymotrypsin and immobilized lipases , 1994 .

[24]  B. Testa,et al.  The temperature dependence of steady-state kinetics: what can be learned about pig liver esterase stereospecificity? , 1994, Chirality.

[25]  A. Straathof,et al.  A simple method to determine the enantiomeric ratio in enantioselective biocatalysis. , 1993, Enzyme and microbial technology.

[26]  T. Mcnelley,et al.  Temperature dependence of , 1993, Metallurgical and Materials Transactions A.

[27]  B. Mattiasson,et al.  Improved activity retention of enzymes deposited on solid supports , 1993, Biotechnology and bioengineering.

[28]  B. Mattiasson,et al.  Reactions Catalyzed by Peg-Modified α-Chymotrypsin in Organic Solvents. Influence of Water Content and Degree of Modification , 1993 .

[29]  A. Russell,et al.  Determination of equilibrium and individual rate constants for subtilisin‐catalyzed transesterification in anhydrous environments , 1992, Biotechnology and bioengineering.

[30]  P. Halling,et al.  Lipases from different sources vary widely in dependence of catalytic activity on water activity. , 1992, Biochimica et biophysica acta.

[31]  J. Dordick.  Designing Enzymes for Use in Organic Solvents , 1992, Biotechnology progress.

[32]  C. Haynes,et al.  Solvent dielectric effects on protein dynamics. , 1992, Proceedings of the National Academy of Sciences of the United States of America.

[33]  A. Russell,et al.  Role of diffusion in nonaqueous enzymology. 1. Theory. , 1992, Enzyme and microbial technology.

[34]  P. Halling,et al.  Polyethylene glycol-modified subtilisin forms microparticulate suspensions in organic solvents , 1992 .

[35]  D. Clark,et al.  Enzymatic catalysis and dynamics in low-water environments. , 1992, Proceedings of the National Academy of Sciences of the United States of America.

[36]  G. Carrea,et al.  Effects of medium and of reaction conditions on the enantioselectivity of lipases in organic solvents and possible rationales , 1992 .

[37]  C. Sih,et al.  Improving the Enantioselectivity of the Candida Cylindracea Lipase Via Chemical Modification , 1992 .

[38]  Y. Khmelnitsky,et al.  The effect of water content and nature of organic solvent on enzyme activity in low-water media. A quantitative description. , 1991, European journal of biochemistry.

[39]  P. Adlercreutz.  On the importance of the support material for enzymatic synthesis in organic media. Support effects at controlled water activity. , 1991, European journal of biochemistry.

[40]  D. Clark,et al.  Activity and flexibility of alcohol dehydrogenase in organic solvents , 1991, Biotechnology and bioengineering.

[41]  A. Klibanov,et al.  The effect of water on enzyme action in organic media. , 1988, The Journal of biological chemistry.

[42]  A. Klibanov,et al.  Enzymatic catalysis in nonaqueous solvents. , 1988, The Journal of biological chemistry.

[43]  Shih-Hsiung Wu,et al.  Quantitative analyses of biochemical kinetic resolution of enantiomers. 2. Enzyme-catalyzed esterifications in water-organic solvent biphasic systems , 1987 .

[44]  A. Klibanov,et al.  Substrate Specificity of Enzymes in Organic Solvents vs. Water Is Reversed. , 1986 .

[45]  A. Klibanov,et al.  Enzymatic catalysis in organic media at 100 degrees C. , 1984, Science.

[46]  C. Sih,et al.  Quantitative analyses of biochemical kinetic resolutions of enantiomers , 1982 .

[47]  K. Tanizawa,et al.  The application of insolubilized alpha-chymotrypsin to kinetic studies on the effect of aprotic dipolar organic solvents. , 1974, The Journal of biological chemistry.

[48]  A. Glazer.  Specific chemical modification of proteins. , 1970, Annual review of biochemistry.

[49]  J. M. Thomas,et al.  Introduction to the principles of heterogeneous catalysis , 1967 .

[50]  W. Günther.  Hypophosphorous Acid, a Novel Reagent for the Reduction of Diselenides and the Selenol-Catalyzed Reduction of Disulfides1,2 , 1966 .

[51]  M. L. Bender,et al.  THE EFFECT OF APROTIC DIPOLAR ORGANIC SOLVENTS ON THE KINETICS OF ALPHA-CHYMOTRYPSIN-CATALYZED HYDROLYSES. , 1963, Biochemistry.

[52]  John E. Leffler,et al.  THE ENTHALPY-ENTROPY RELATIONSHIP AND ITS IMPLICATIONS FOR ORGANIC CHEMISTRY , 1955 .