Journal of Molecular Catalysis B: Enzymatic 15 (2001) 65–70 Enhancement of activity of sol–gel immobilized lipase in organic media by pretreatment with substrate analogues Shin-ya Furukawa ∗ , Tsutomu Ono, Hiroyuki Ijima, Koei Kawakami Department of Materials Process Engineering, Graduate School of Engineering, Kyushu University, Hakozaki, Higashi-ku, Fukuoka 812-8581, Japan Received 2 November 2000; received in revised form 12 January 2001; accepted 12 January 2001 Abstract By performing pretreatment with substrate analogues, an activation of lipase immobilized into organic–inorganic hybrid silicate was attempted in esterification of menthol with butyric acid in organic media. With decreasing water content of immobilized lipase preparations, the activity of immobilized lipases by (-)-menthol pretreatment was enhanced, and the highest activity was obtained at water activity, a w = 0.11. However, the pretreatment of immobilized lipases with (-)-menthol could not improve the enantioselectivity. In addition, the activity of pretreated immobilized lipases increased with increasing the chain length of alcohol as a substrate analogue. Consequently, it was found that hydrophobic and bulky alcohol was an effective substrate analogue to activate immobilized lipases. © 2001 Elsevier Science B.V. All rights reserved. Keywords: Sol–gel method; Lipase; Esterification; Substrate analogue; Organic–inorganic hybrid silicate 1. Introduction For the last decade, it has been reported that the sol–gel method is so useful for the immobilization of biomolecules in analytical and commercial applica- tions [1,2]. Recently, many biological materials such as enzymes [3,4], proteins [5], microbes [6], and mam- malian and plant cells [7–9] have been immobilized by the sol–gel method. The advantage of the sol–gel method is to form rigid and elastic silica matrices which bring about the high durability against the phys- ical damages [2]. The other advantage is possible in- sertion of any substituents, which give the efficient ∗ Corresponding author. Tel.: +81-92-642-4109; fax: +81-92-642-4109. E-mail address: furukawa@chem-eng.kyushu-u.ac.jp (S.-y. Furukawa). environment to the biomolecules entrapped into silica matrices [10]. In previous reports, we demonstrated that the en- trapment of lipase into organic–inorganic hybrid silicate formed on Celite 545 resulted in an en- hancement of thermal stability [10]. Moreover, we reported that an increase in the chain length or the number of alkyl groups of organic silanes, which were precursors to form a silica matrix, led to an enhancement of the catalytic activity and the enan- tioselectivity at high temperature of lipase entrapped into the hybrid gel [11]. Consequently, the hybrid gel-entrapped lipase on Celite derived from a mixture of dimethyldimethoxysilane (DMDMOS) and tetram- ethoxysilane (TMOS) showed the highest activity. Fur- thermore, it was found that the hybrid gel-immobilized lipase prepared at r(= [DMDMOS]/[TMOS]) = 4 showed the highest activity and enantioselectivity at 75 ◦ C. 1381-1177/01/$ – see front matter © 2001 Elsevier Science B.V. All rights reserved. PII:S1381-1177(01)00007-8