Research paper More stable structure of wheat germ lipase at low pH than its native state Ejaz Ahmad 1 , Sadaf Fatima 1 , Mohd Moin Khan, Rizwan Hasan Khan * Interdisciplinary Biotechnology Unit, Aligarh Muslim University, Aligarh 202002, India article info Article history: Received 7 November 2009 Accepted 26 March 2010 Available online 2 April 2010 Keywords: Acid-induced state Enzyme activity Molten globule pH-induced denaturation Wheat germ lipase abstract Wheat germ lipase is a cereal lipase which is a monomeric protein. In the present study we sought to structurally characterize this protein along with equilibrium unfolding in solution. Conformational changes occurring in the protein with varying pH, were monitored by circular dichroism (CD) spec- troscopy, fluorescence emission spectroscopy, binding of hydrophobic dye, 1-anilino 8-naph- thalenesulfonic acid (ANS) and dynamic light scattering (DLS). Our study showed that acid denaturation of lipase lead to characterization of multiple monomeric intermediates. Native protein at pH 7.0 showed far-UV spectrum indicating mixed structure with both alpha and beta-type of characteristics. Activity of lipase was found to fall on either sides of pH 7.0e8.0. Acid-unfolded state was characterized at pH 4.0 with residual secondary structure, disrupted tertiary spectrum and red-shifted fluorescence spectrum with decreased intensity. Further decrease in pH lead to formation of secondary structure and acid- induced molten globule state was found to be stabilized at pH 1.4, with exposed tryptophan residues and hydrophobic patches. Notably, interesting finding of this study was characterization of acid-induced state at pH 0.8 with higher secondary structure content than native lipase, regain in tertiary spectrum and induction of compact conformation. Although enzymatically inactive, acid-induced state at pH 0.8 was found to be structurally more stable than native lipase, as shown by chemical and thermal denaturation profiles. Ó 2010 Elsevier Masson SAS. All rights reserved. 1. Introduction Lipids constitute large part of earth biomass and hence lipases are indispensable for bioconversion of lipids from one organism to another and within the organism [1]. Lipases catalyze wide range of reactions such as hydrolysis and interesterification specifically, in addition alcoholysis, acidolysis and aminolysis. Multi-faceted lipases have an unsurpassed role in swiftly growing modern biotechnology [2]. This is primarily due to their ability to act under mild conditions, to utilize wide spectrum of substrates and show high stability in organic solvents. Lipases find their widespread application in food technology, detergents and dairy industry, biomedical sciences and chemical synthesis [3]. Enantioselective and regiospecific nature of lipases have been utilized for resolution of chiral drugs, ester and amino acid derivatives, fat modifications; synthesis of agrochemicals, biofuels, biosensors, biosurfactants, bioremediations, cosmetics and perfumery, flavor enhancement etc. [4]. Nowadays, lipases are the enzymes of choice for organic chemists, pharmacists, biophysicists, biochemical and process engineers, biotechnologists, microbiologists and biochemists. Lipases belong to the class of serine hydrolases (triacylglycerol acylhydrolases EC 3.1.1.3) having Ser-His-Asp triad in the active site. It has been directly observed in the three-dimensional structure of two fungal lipases [5,6] that are unrelated in sequence yet contain structurally analogous catalytic triad. A model of interfacial acti- vation has been proposed as mechanism of action of lipases, in which lipases become catalytically competent at lipidewater interface. Mammalian, microbial and plant lipases from different sources have been purified and characterized. Wheat germ lipase was first isolated by Singer and Hofstee [7] which has molecular weight of 42,000 and intrinsic viscosity is 4.72 ml/g indicating that it is elongated in shape [8]. The protein consists of approximately 20% a-helix and 40% b-sheet. Effect of acidic and alkaline pH on structural integrity and activity of wheat germ lipase has also been investigated [8,9]. This enzyme was found to be stabilized by sugars (glucose, sucrose etc.), polyhydric alcohols (glycerol) and DMSO [10] out of which DMSO was found to be most stabilizing. Guani- dine hydrochloride induced denaturation of lipase was found to be non-cooperative process [11]. In this communication, we sought to characterize partially folded intermediates stabilized during pH-induced denaturation of wheat germ lipase. We have characterized multiple * Corresponding author. Tel.: þ91 571 2720388; fax: þ91 571 2721776. E-mail address: rizwanhkhan@hotmail.com (R.H. Khan). 1 These authors contributed equally to this work. Contents lists available at ScienceDirect Biochimie journal homepage: www.elsevier.com/locate/biochi 0300-9084/$ e see front matter Ó 2010 Elsevier Masson SAS. All rights reserved. doi:10.1016/j.biochi.2010.03.023 Biochimie 92 (2010) 885e893