Abstract The effects of the water-miscible or-
ganic solvents (methanol, ethanol, 1-propanol,
2-propanol, acetonitrile, N,N¢-dimethylforma-
mide and tetrahydrofuran) on the stability and
catalytic activity of a-chymotrypsin (CT) immo-
bilized on Eupergit CM were studied. Enhanced
stabilities and activities were observed both as a
consequence of immobilization and the presence
of organic solvent, which in combination provide
long term (at least 24 h) retention of activity, and
up to 50-fold increase in 50% (v/v) methanol in
buffer. Low quantities (20%, v/v) of acetonitrile
not only prevented CT inactivation by autolysis at
20°C but also induced a significant increase in the
activity of both free (six-fold) and immobilized
(two-fold) CT.
Keywords a-Chymotrypsin Æ Eupergit Æ
Immobilization Æ Organic solvent
Introduction
The immobilization of enzymes is of major
importance for many processes of biotechnological
significance (Cao 2005). The covalent attachment
to, physical adsorption on, or entrapment in,
various solid phase matrices has been used to
improve the tolerance of these biomolecular
structures towards physical and chemical stress
(Wang et al. 2001; Kreiner and Parker 2005) and
can afford improved conditions for substrate and
product mass transfer, and even provide
enhanced stabilities (Kudryashova et al. 2003).
Other approaches, such as use of lyoprotectants
(Dabulis and Klibanov 1993), additives (Laszlo
et al. 2001), chemical modification (Davis 2003),
imprinting (Rich et al. 2002), and protein engi-
neering (Eijsink et al. 2004), have also proven
useful to this end.
A wide variety of processes utilise enzymes in
the presence of organic solvents, either water-
miscible or immiscible, in order to facilitate the
solubility of reaction substrates or products, or
even to alter the specificity of an enzyme (Drauz
and Waldmann 2002). Nonetheless, the activity of
enzymes is often compromised by the greater
hydrophobicity of organic solvents, or organic
solvent containing media (Klibanov 1997).
Accordingly, the development of approaches
which provide stabilization of the protein in a
functional form under such conditions is most
desirable. Immobilization on or in a matrix pro-
vides one possible method for achieving this goal.
In the present study, the influence of a range of
water miscible organic solvents on the activity of
Linus Olofsson and Pernilla So
¨
derberg authors have
contributed equally to the work.
L. Olofsson Æ P. So
¨
derberg Æ I. A. Nicholls (&)
Department of Chemistry & Biomedical Sciences,
University of Kalmar, SE-391 82 Kalmar, Sweden
e-mail: ian.nicholls@hik.se
Biotechnol Lett (2006) 28:929–935
DOI 10.1007/s10529-006-9025-7
123
ORIGINAL PAPER
Influence of water miscible organic solvents
on a-chymotrypsin in solution and immobilized
on Eupergit CM
Linus Olofsson Æ Pernilla So
¨
derberg Æ
Ian A. Nicholls
Received: 10 January 2006 / Accepted: 28 February 2006 / Published online: 15 June 2006
Ó
Springer Science+Business Media B.V. 2006