A bioconjugate of Pseudomonas cepacia lipase with alginate with enhanced catalytic efficiency Kalyani Mondal a , Payal Mehta b , Bodh Raj Mehta c , Deepak Varandani c , Munishwar Nath Gupta a, ⁎ a Chemistry Department, Indian Institute of Technology, Delhi, Hauz Khas, New Delhi 110 016, India b Biochemistry Department, Ohio State University, 100 W, 8th Avenue, Columbus, OH 43210, USA c Physics Department, Indian Institute of Technology, Delhi, Hauz Khas, New Delhi 110 016, India Received 26 December 2005; received in revised form 14 March 2006; accepted 4 April 2006 Available online 5 May 2006 Abstract A bioconjugate of Pseudomonas cepacia lipase with alginate was prepared by simple adsorption. Atomic force microscope (AFM) images showed that this bioconjugate resulted from adsorption rather than entrapment of the enzyme as enzyme molecules were visible on the gel surface. The soluble bioconjugate exhibited increased enzyme activity in terms of high effectiveness factor (effectiveness factor was 3 for the immobilized preparation) and greater V max /K m value (V max /K m increased 25 times upon immobilization). This constitutes one of the less frequently observed instances of lipase activation by lid opening as a result of binding to a predominantly hydrophilic molecule. The bioconjugate was also more stable at 55 °C as compared to the free enzyme and could be reused for oil hydrolysis up to 4 cycles without any loss in activity. Fluorescence emission spectroscopy showed that the immobilized enzyme had undergone definite conformational changes. © 2006 Elsevier B.V. All rights reserved. Keywords: Pseudomonas cepacia lipase; Alginate; Noncovalent immobilization; Atomic force microscopy; Smart biocatalysts; Lipase bioconjugate 1. Introduction Immobilization of enzymes is an established technique for obtaining reusable enzyme derivatives [1,2]. Often, immobiliza- tion also results in a derivative more stable in aqueous and/or nonaqueous media [3,4]. Conventionally, enzymes are generally immobilized on solid matrices. However, use of reversibly sol- uble–insoluble polymers as matrices offer some distinct advan- tages. Such polymeric materials are also called smart polymers since their solubility can be altered in a dramatic fashion by applying a small stimulus like change in pH, temperature or ad- dition of a chemical species [5,6]. In such systems, the soluble immobilized enzyme functions as a homogenous catalyst. For recovery purposes, the application of appropriate stimulus results in the precipitation of the bioconjugate [7]. Alginate is one such smart polymer. It is a water-soluble polysaccharide (made up of guluronic acid and mannuronic acid units), which can be pre- cipitated by the addition of Ca 2+ ions [8]. An established approach is the use of Ca–alginate gels for entrapment of whole cells [9,10]. More recently, bioconjugates of alginate with enzymes have also been described [7,11]. It has earlier been shown that lipases (from wheat germ, porcine pancreas and Chromobacterium viscosum) bind to algi- nate and this phenomenon could be exploited for purification of lipases [12]. In such cases, lipases selectively picked up by alginate (from a crude extract) could be eluted off the latter because of weak binding. In the present work, it is shown that the lipase from Pseudomonas cepacia bound (noncovalently) to alginate rather firmly, hence the resultant bioconjugate could be used as an immobilized lipase preparation. Atomic force mi- croscopy (AFM) of the bioconjugate (in solid phase) provided a visual picture of the immobilized enzyme. Adsorption, as com- pared to covalent coupling, offers certain advantages [13].A somewhat unexpected and useful property of the bioconjugate was that the lipase molecules displayed enhanced catalytic effi- ciency when bound to the polymer. Lipases are one of the most useful enzymes in biotechnology and have been used for a variety of applications [14,15]. A recent Biochimica et Biophysica Acta 1764 (2006) 1080 – 1086 http://www.elsevier.com/locate/bba ⁎ Corresponding author. Tel.: +91 11 2659 1503; fax: +91 11 2658 1073. E-mail address: munishwar48@yahoo.co.uk (M.N. Gupta). 1570-9639/$ - see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.bbapap.2006.04.008