TY - CHAP

T1 - Stability and selectivity of alkaline proteases in hydrophilic solvents

AU - Pedersen, Lars Haastrup

AU - Ritthitham, Sinthuwat

AU - Pleißner, Daniel

N1 - Conference code: 4

PY - 2008

Y1 - 2008

N2 - Hydrophilic, organic solvents can be used as co-solvents with water to produce one phase systems sustaining optimal mass transfer of substrates and products of mixed polarity in biocatalysed processes. At concentrations below 50 % hydrophilic solvents can even have a stabilising effect on alkaline proteases, but at higher concentrations and particularly in anhydrous systems most enzymes including alkaline proteases will denature and consequently loose activity [1]. However, partial denaturing and increased structural flexibility due to the interaction between hydrophilic solvents and alkaline proteases has been agued as the primary reasons for increasing activity, influencing regio-selectivity and improving the enantio-selectivity of these enzymes [2]. Alkaline proteases have been shown to be active not only on peptides, but on a wide range of renewable resources for synthesis of biologically active molecules and carriers, and in synthesis of carbohydrate derivatives with designed functional properties. When it comes to regio-selectivity of alkaline proateses on carbohydrates both the properties of the particular enzyme and the influence of the solvent is determining for the position of substitution. Some of the most abundant hexoses were all substituted at the primary hydroxyl group at the C-6 position in processes catalysed by different alkaline proteases [3,4,5]. However by adding DMSO to the reaction medium the regio-selectivity in a Streptomyces sp protease catalysed reaction was shifted from the primary hydroxyl to the secondary hydroxyl group at the C-2 position of galactose [3]. Subtilisins from B. subtilis showed regio-selectivity towards the primary hydroxyl group situated at the non reducing end of three important reducing disaccharides, respectively [4,6,7]. Sucrose monoesters were synthesised in anhydrous DMF and pyridine, respectively with different acyl donors and a number of different subtilisins as biocatalysts - in all cases the 1'-O-monoester was the major product [6,7,8,9]. But the alkaline protease AL89 revealed regio-selectivity towards the C-2 position of sucrose [10]. This way acylation of a secondary hydroxyl group situated on the glucose moiety of sucrose was obtained. The initial reaction rate of acylation was not effected by the fatty acid chain length of the acyl donor. The half life of the enzyme in de-ioniset water was 4 minutes whereas in 100% DMF it was 10 minutes. The activity was effected by the solvation of the enzyme in both DMSO and DMF [11].Literature[1] H. Ogino, H. Ishikawa, J. Biosci. Bioeng. 2001, 91, 109.[2] K. Watanabe, S. Ueji, Biotechnol. Lett. 2000, 22, 599.{3] M. Kitagawa, H. Fan, T. Raku, S. Shibatani, Y. Maekawa, Y. Hiraguri, R. Kurane, Y. Tokiwa, Biotechnol. Lett.1999, 21, 355.[4] S. Riva, J. Chopineau, A.P.G. Kieboom, A. Klibanov, J. Am. Chem. Soc.,1988, 110, 584.[5] T. Watanabe, R. Matsue, Y. Honda, M. Kuwahara, Carbohydr. Res., 1995, 275, 215.[6] P. Potier, A. Bouchu, G. Descotes, Y. Queneau, Tetrahedron: Lett. 2000, 41, 3597.[7] Q. Wu, N. Wang, Y.M. Xiao, D.S. Lu, X.F. Lin, Carbohydr. Res., 2004, 339, 2059.[8] H.G. Park, H.N. Chang, Biotechnol. Lett. 2000, 22, 39[9] S. Riva, M. Nonini, G. Ottolina, B. Danieli, Carbohydr. Res., 1998, 314, 259.[10] N.R. Pedersen, R. Wimmer, R. Matthiesen, L.H. Pedersen, A. Gessesse, Tetrahedron: Asymmetry 2003, 14, 667.[11] L. H. Pedersen, S. Ritthitham and M. Kristensen (2008) in Modern Biocatalysis Eds W. D. Fessner and T. Anthonsen, Wiley-VCH in press

AB - Hydrophilic, organic solvents can be used as co-solvents with water to produce one phase systems sustaining optimal mass transfer of substrates and products of mixed polarity in biocatalysed processes. At concentrations below 50 % hydrophilic solvents can even have a stabilising effect on alkaline proteases, but at higher concentrations and particularly in anhydrous systems most enzymes including alkaline proteases will denature and consequently loose activity [1]. However, partial denaturing and increased structural flexibility due to the interaction between hydrophilic solvents and alkaline proteases has been agued as the primary reasons for increasing activity, influencing regio-selectivity and improving the enantio-selectivity of these enzymes [2]. Alkaline proteases have been shown to be active not only on peptides, but on a wide range of renewable resources for synthesis of biologically active molecules and carriers, and in synthesis of carbohydrate derivatives with designed functional properties. When it comes to regio-selectivity of alkaline proateses on carbohydrates both the properties of the particular enzyme and the influence of the solvent is determining for the position of substitution. Some of the most abundant hexoses were all substituted at the primary hydroxyl group at the C-6 position in processes catalysed by different alkaline proteases [3,4,5]. However by adding DMSO to the reaction medium the regio-selectivity in a Streptomyces sp protease catalysed reaction was shifted from the primary hydroxyl to the secondary hydroxyl group at the C-2 position of galactose [3]. Subtilisins from B. subtilis showed regio-selectivity towards the primary hydroxyl group situated at the non reducing end of three important reducing disaccharides, respectively [4,6,7]. Sucrose monoesters were synthesised in anhydrous DMF and pyridine, respectively with different acyl donors and a number of different subtilisins as biocatalysts - in all cases the 1'-O-monoester was the major product [6,7,8,9]. But the alkaline protease AL89 revealed regio-selectivity towards the C-2 position of sucrose [10]. This way acylation of a secondary hydroxyl group situated on the glucose moiety of sucrose was obtained. The initial reaction rate of acylation was not effected by the fatty acid chain length of the acyl donor. The half life of the enzyme in de-ioniset water was 4 minutes whereas in 100% DMF it was 10 minutes. The activity was effected by the solvation of the enzyme in both DMSO and DMF [11].Literature[1] H. Ogino, H. Ishikawa, J. Biosci. Bioeng. 2001, 91, 109.[2] K. Watanabe, S. Ueji, Biotechnol. Lett. 2000, 22, 599.{3] M. Kitagawa, H. Fan, T. Raku, S. Shibatani, Y. Maekawa, Y. Hiraguri, R. Kurane, Y. Tokiwa, Biotechnol. Lett.1999, 21, 355.[4] S. Riva, J. Chopineau, A.P.G. Kieboom, A. Klibanov, J. Am. Chem. Soc.,1988, 110, 584.[5] T. Watanabe, R. Matsue, Y. Honda, M. Kuwahara, Carbohydr. Res., 1995, 275, 215.[6] P. Potier, A. Bouchu, G. Descotes, Y. Queneau, Tetrahedron: Lett. 2000, 41, 3597.[7] Q. Wu, N. Wang, Y.M. Xiao, D.S. Lu, X.F. Lin, Carbohydr. Res., 2004, 339, 2059.[8] H.G. Park, H.N. Chang, Biotechnol. Lett. 2000, 22, 39[9] S. Riva, M. Nonini, G. Ottolina, B. Danieli, Carbohydr. Res., 1998, 314, 259.[10] N.R. Pedersen, R. Wimmer, R. Matthiesen, L.H. Pedersen, A. Gessesse, Tetrahedron: Asymmetry 2003, 14, 667.[11] L. H. Pedersen, S. Ritthitham and M. Kristensen (2008) in Modern Biocatalysis Eds W. D. Fessner and T. Anthonsen, Wiley-VCH in press

KW - Chemistry

UR - http://d-nb.info/99015677X

UR - http://vbn.aau.dk/en/publications/stability-and-selectivity-of-alkaline-proteases-in-hydrophilic-solvents(792422b0-8bac-11dd-93c5-000ea68e967b).html

M3 - Published abstract in conference proceedings

SN - 978-3-930400-74-4

BT - Fourth International Congress on Biocatalysis: Biocat 2008

A2 - Grote, Ralf

PB - TuTech Verlag

CY - Harburg

T2 - Fourth International Congress on Biocatalysis : Biocat 2008

Y2 - 31 August 2008 through 4 September 2008

ER -