Chemical modification of lysine residues in Bacillus -amylases: effect on activity and stability Khosro Khajeh a , Hossein Naderi-Manesh b , Bijan Ranjbar b , Ali akbar Moosavi-Movahedi a , Mohsen Nemat-Gorgani a, * a Institute of Biochemistry and Biophysics, University of Tehran, P.O. Box 13145-1384, Tehran, Iran b Department of Biophysics, School of Sciences, Tarbiat Modarres University, P.O. Box 14115–175, Tehran, Iran Received 10 August 2000; received in revised form 6 December 2000; accepted 19 December 2000 Abstract Chemical modification of lysine residues in two bacterial -amylases, a mesophilic enzyme from Bacillus amyloliquefaciens (BAA) and a thermophilic enzyme from Bacillus licheniformis (BLA) was carried out using citraconic anhydride. 13  1 residues in BAA and 10  1 residues in BLA were found modified under defined experimental conditions. Modification brought about dramatic enhancement of thermal stability of BAA and catalytic activity of BLA. Such alterations were found dependent on the temperature and pH. Results obtained on Tm, the extent of deamidation, changes in the circular dichroism (CD) spectra and kinetic parameters before and after modification are discussed in terms of their contributions to the mechanism of irreversible thermoinactivation and activity enhancement. © 2001 Elsevier Science Inc. All rights reserved. Keywords: -amylase; Activation; Modification; Thermostability; Mesophilic & thermophilic enzymes; Deamidation 1. Introduction It is now generally believed that proteins are only mar- ginally stable and this property has been described as a biologic necessity related to function of these macromole- cules [1–3]. It is also an accepted fact that the protein sequence ultimately determines its assigned function. To this end, information on the three-dimensional structure of the macromolecule is necessary in order to unequivocally determine the specific contribution of each amino acid to its functional property as a whole [4,5]. Since bacterial -amy- lases are widely used in industry [6] and often at high temperatures, a clear understanding of the molecular mech- anisms of their inactivation would be very useful. A number of factors have been found important in relation to thermal stabilization of various proteins examined (see for example ref. 1 and references cited therein). However, no universal set of rules can be offered regarding improvement of ther- mostability of an enzyme. In a study related to elucidation of the mechanisms of irreversible thermal inactivation of Bacillus -amylases, it has been concluded that these en- zymes are inactivated due to formation of incorrect (scram- bled) structures which subsequently undergo aggregation and deamidation [7,8]. Chemical modification of proteins is widely used as a tool for studying localization of individual amino acids, their participation in the maintenance of the native confor- mation [9] and for their stabilization [10 –14]. In a number of cases, dramatic stabilization has been achieved [11–16]. This relatively simple procedure would complement other techniques such as site-directed mutagenesis used for struc- tural modification of a protein. In two recent communications, we described our at- tempts in elucidating structure-stability relationships of BAA and BLA [17,18]. In this connection, the thermody- namic and kinetic stability of these proteins were investi- gated in the presence of a number of stabilizing agents and organic solvents and resistance to proteolysis was com- pared. In the present report, a mild and selective procedure is described for modification of -amino groups of lysine residues in these proteins using citraconic anhydride. Ob- served changes in kinetic and thermodynamic stabilities and catalytic properties of these enzymes brought about by mod- ification are analyzed. It appears that this simple method * Corresponding author. Tel.: +98-21-6113377; fax: +98-21- 6404680. E-mail address: gorganim@ibb.ut.ac.ir (M. Nemat-Gorgani). www.elsevier.com/locate/enzmictec Enzyme and Microbial Technology 28 (2001) 543–549 0141-0229/01/$ – see front matter © 2001 Elsevier Science Inc. All rights reserved. PII: S0141-0229(01)00296-4