Obtaining high transesterification activity for subtilisin in ionic liquids Shweta Shah, Munishwar N. Gupta ⁎ Department of Chemistry, Indian Institute of Technology Delhi, Hauz Khas, New Delhi 110016, India Received 19 April 2006; received in revised form 6 October 2006; accepted 6 October 2006 Available online 13 October 2006 Abstract It is known that subtilisin shows poor transesterification activity in ionic liquids (ILs). The present work, taking subtilisin as the system, explores approaches for biocatalyst preparations, which are capable of yielding higher/adequate transesterification activity in these solvents. Of all the approaches tried, enzyme precipitated and rinsed with n-propanol (EPRP) gave the best results (about 10,000 times increase in initial rates in 1-butyl-3-methylimidazolium hexafluorophosphate ([Bmim][PF 6 ]) over what is obtained with pH tuned lyophilized powders). In case of water soluble ionic liquid 1-butyl-3-methylimidazolium tetrafluoroborate ([Bmim][BF 4 ]), pH tuned lyophilized subtilisin did not show any transesterification activity. EPRP, however, gave an initial rate (for transesterification) of 2.78 mmol mg - 1 h - 1 . © 2006 Elsevier B.V. All rights reserved. Keywords: Cryoprotectant; Ionic liquids; Lyoprotectant; Propanol rinsed enzyme preparation; Subtilisin; Three phase partitioning 1. Introduction Enzymatic catalysis in non-aqueous media finds wide applications in organic chemistry and biotechnology [1–6]. Recently, room temperature ionic liquids (ILs) have emerged as an exciting option while choosing a non-aqueous medium [7–17]. Some extensive reviews on properties of ILs and their advantages as ‘green solvents’ are available [9–11,18–20].A clear road map for process optimization for biocatalysis in ILs is slowly emerging. There is, already, a general agreement on few features. An enzyme is inactivated if it dissolves in the IL (the activity can generally be regained by dilution with water) [9– 11,15,16]. Anions play an important role: enzymes are active in ILs with BF 4 - , PF 6 - and NTf 2 - as anions and inactive in ILs with NO 3 - , CF 3 SO 3 - and CH 3 CO 2 - as anions [9–11,15,16]. With many enzymes, initial rates comparable (and occasionally even better) to those obtained in nearly anhydrous organic solvents have been observed (provided the IL is judiciously chosen). In some cases, initial rates have been very low [9–11,15,16,21]. The protease subtilisin (in free form), for example, fails to show any significant transesterification activity in any IL [22]. An application with even modified subtilisin catalyzed reaction needed 60 h as the time period [22]. The objective of the present work is to show that pretreatment of the enzyme before using it for catalysis in ionic liquids is a crucial step. The way enzyme preparation is dried, influencing the subsequent rate of catalyst reaction in a drastic manner. Based upon this understanding, it has been possible to obtain reasonable rates for transesterifica- tion activity of subtilisin. A water immiscible IL, 1-butyl-3-methylimidazolium hexa- fluorophosphate ([Bmim][PF 6 ]) and a water miscible ionic liquid 1-butyl-3-methylimidazolium tetrafluoroborate ([Bmim] [BF 4 ]) have been chosen as the reaction media. 2. Materials and method 2.1. Materials Subtilisin Carlsberg, Type VIII was obtained from Sigma, St. Louis, USA. Ionic liquids, were procured from Acros Organics USA. This is the vendor from which ionic liquid has been purchased by most of the other workers [10]. The purity of ionic liquids as specified by the vendor was: [Bmin][PF 6 ] was 99.6% pure by HPLC, water content 0.05% by Karl-Fischer and [Bmim] Biochimica et Biophysica Acta 1770 (2007) 94 – 98 www.elsevier.com/locate/bbagen Abbreviations: ILs, ionic liquids; EPRP, Enzyme precipitated and rinsed with propanol; [Bmim][PF 6 ], 1-butyl-3-methylimidazolium hexafluorophos- phate; [Bmim][BF 4 ], 1-butyl-3-methylimidazolium tetrafluoroborate; PEG, Poly (ethylene glycol); TPP, Three phase partitioning; Cal-B, Candida antarctica lipase B; [Omim][PF 6 ], 1-octyl-3-methylimidazolium hexafluoro- phosphate; PREP, propanol rinsed enzyme preparations ⁎ Corresponding author. Tel.: +91 11 26591503, +91 11 26596568; fax: +91 11 2658 1073. E-mail address: munishwar48@yahoo.co.uk (M.N. Gupta). 0304-4165/$ - see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.bbagen.2006.10.004