Modification of β-galactosidase for use in organic mono-phase hexanol system

Daniela Nikolovska-Nedelkoska, Irina Mladenoska, Filimena Poposka, Eleonora Winkelhausen, Slobodanka Kuzmanova

Abstract


Different methods for preparation of the Aspergillus oryzae β-galactosidase were evaluated and the total enzyme activity was determined in hexanol mono-phase system using p-nitrophenyl-β-D-galactoside as a substrate. Several supports such as Acurell EP-100, Amberlite IRC (50), Celite, Dowex, Eupergit C and silica gel were tested in order to select the most suitable matrix for immobilization of the β-galactosidase for performing transgalactosylation reactions in hexanol. Celite and Amberlite IRC (50) were selected as the most appropriate carriers. Albumin, PEG 6000, starch and glycine, added prior to the immobilization procedure, acted as stabilizers of the galactosidase. By adding albumin on Celite, a 3.3-fold increase of enzyme activity was achieved. Sodium dodecyl sulphate (SDS), dioctyl sulfosuccinate (AOT), Tween 65 and crown ether were used directly in the reaction medium in order to increase its homogeneity. Addition of SDS to the medium resulted in a 3.65-fold increased activity of the β- galactosidase deposited on Celite. A micro-emulsion system created by addition of AOT resulted in an increased catalytic activity. The β-galactosidase showed enhanced total activity with increased water activity in the system having the highest value for the water activity close to the saturation level (0.92).


Keywords


β-galactosidase; immobilization; enzyme modification; Amberlite IRC (50); Celite; hexanol mono-phase system

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References


J. S. Dordick, Enzymatic catalysis in monophasic organic solvents. Enzyme Microb. Technol., 11, 194–211 (1989).

M. Woudenberg-van Oosterom, H.J.A. van Belle, F. van Rantwijk, R.A. Sheldon, Immobilized β-galactosidase and their use in galactoside synthesis. J. Mol. Catal., 134, 267–274 (1998).

A. Ducret, M. Trani, R. Lortie, Screeninig of various glycosidases for the synthesis of octyl glucoside. Biotechnol. Bioeng., 77 (7), 752–757(2002).

F. van Rantwijk, M. Woudenberg-van Oosterom, R. A. Sheldo, Glycoside-catalysed synthesis of alkyl glycosides. J. Mol. Catal. B: Enzymatic, 6 (6), 511–532 (1999).

O. S. Kouptosova, N. L. Klyachko, A. V. Levashov, Synthesis of alkyl glycosides catalyzed by β-glycosidases in a system of reverse micelles. Rus. J. Bioorg. Chem., 27 (6), 429–433 (2001).

M. Persson, I. Mladenoska, E. Wehtje, P. Adlercreutz, Preparation of lipases for use in organic solvents. Enzym. Microb. Techn., 31, 833–841 (2002).

M. Persson, E. Wehtje, P. Adlercreutz, Immobilization of lipases by adsorption and deposition: high protein loadings gives lower water optimum. Biotechnol. Lett., 22, 1571– 1575 (2000).

J.A. Arcos, L. Robledo, C. Otero, Stability of a Pseudomonas sp. lipase: comparison between solubilased enzyme in reverse micelles and suspended lipase in dry solvents. Biotechnol. Bioeng., 57(5), 505–509 (1997).

T. Hansson, P. Adlercreutz, Optimization of galactooligosaccharide production from lactose using β-glycosidases from hyperthermophiles. Food Biotechnol., 15: 79–97 (2001).

M. Reslow, P. Adlercreutz, B. Mattiasson, On the importance of the support material for bioorganic synthesis. Eur. J. Biochem., 172, 573–578 (1988).

R. Barros, E. Wehtje, P. Adlercreutz, Mass-transfer studies on immobilized α-chymotrypsin biocatalists prepared by deposition for use in organic medium. Biotechnol. Bioeng., 59(3), 364–373 (1998).

Y. Khmelnitsky, S. Welch, D. Clark, J. S. Dordick, Salts dramatically enhance activity of enzymes suspended in organic solvents. J. Am.Chem. Soc., 116, 2647–8 (1994).

E. Wehtje, P. Adlercreutz, B. Mattiasson, Improved activity retention of enzymes deposited on solid supports. Biotechnol. Bioeng., 41, 171–178 (1993).

R. Affleck, Z.-F. Xu, V. Suzawa, K. Focht, D.S. Clark, J.S. Dordick, Enzymatic catalysis and dynamics in lowwater environments. Proc. Natl. Acad. Sci. USA, 89, 1100–1104 (1992).

I. Mladenoska, D. Nikolovska Nedelkoska, E. Winkelhausen, S. Kuzmanova, Aspergillus oryzae-β-galactosidase – an efficient catalyst for alkyl-β-galactoside synthesis in organic mono-phase system. Maced. J. Chem. Chem. Eng., 26 (1), 17–24 (2007).

A. Zaks, A. M. Klibanov, Enzyme-catalyzed processes in organic solvents. Proc. Natl. Acad. Sci. USA., 82, 3192– 3196 (1985).

T. Hansson, E. Wehtje, P. Adlercreutz, Enzymatic synthesis of hexyl glycosides from lactose at low water activity and high temperature using hyperthermostable β- galactosidases. Biocatal. Biotrans., 20, 167–178 (2001).




DOI: http://dx.doi.org/10.20450/mjcce.2009.224

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