BIOCATALYSIS Engineered glucose isomerase from Streptomyces sp. SK is resistant to Ca 2+ inhibition and Co 2+ independent Hajer Ben Hlima • Nushin Aghajari • Mamdouh Ben Ali • Richard Haser • Samir Bejar Received: 9 September 2011 / Accepted: 8 November 2011 / Published online: 4 December 2011 Ó Society for Industrial Microbiology and Biotechnology 2011 Abstract The role of two amino acid residues linked to the two catalytic histidines His54 and His220 in kinetics and physicochemical properties of the Streptomyces sp. SK glucose isomerase (SKGI) was investigated by site-directed mutagenesis and molecular modeling. Two single muta- tions, F53L and G219D, and a double mutation F53L/ G219D was introduced into the xylA SKGI gene. The F53L mutation increases the thermostability and the catalytic efficiency and also slightly shifts the optimum pH from 6.5 to 7, but displays a profile being similar to that of the wild- type enzyme concerning the effect of various metal ions. The G219D mutant is resistant to calcium inhibition retaining about 80% of its residual activity in 10 mM Ca 2? instead of 10% for the wild-type. This variant is activated by Mn 2? ions, but not Co 2? , as seen for the wild-type enzyme. It does not require the latter for its thermostability, but has its half-life time displaced from 50 to 20 min at 85°C. The double mutation F53L/G219D restores the thermostability as seen for the wild-type enzyme while maintaining the resistance to the calcium inhibition. Molecular modeling suggests that the increase in thermo- stability is due to new hydrophobic interactions stabilizing a2 helix and that the resistance to calcium inhibition is a result of narrowing the binding site of catalytic ion. Keywords Streptomyces Á Glucose isomerase Á Site-directed mutagenesis Á Thermostability Á Calcium inhibition Introduction Xylose isomerase (XI) (EC 5.3.1.5) plays an essential role in the metabolism of sugars in microorganisms. The enzyme is found in a number of bacteria and catalyzes the isomerization of D-xylose to D-xylulose in vivo. The practical importance of the enzyme stems from its ability to isomerize D-glucose to D-fructose under certain conditions in vitro; therefore, the enzyme is often referred to as glu- cose isomerase (GI) and utilized in industry for the pro- duction of high-fructose corn syrup (HFCS) from corn starch. This process involves several separate enzymatic steps, including liquefaction of corn starch by a-amylase, saccharification by glucoamylase, and isomerization by glucose isomerase [17]. Typically, high temperatures have the advantage of a higher equilibrium concentration of fructose, a faster reaction rate, and a decreased viscosity of the substrate in the product stream [10]. The enzyme is active in dimeric or tetrameric forms and requires Mg 2? , Mn 2? or Co 2? for catalytic activity and thermostability, whereas Ca 2? is a strong competitive inhibitor even at low concentrations [4]. The latter ion has a practical importance in the starch bioconversion process, since a-amylases require Ca 2? ions for its activity. Thus, it is very important to identify a GI not being inhibited by this ion or an a-amylase, which acts at low calcium concentration. In this context, several studies have been developed concerning H. Ben Hlima Á M. Ben Ali Á S. Bejar (&) Laboratoire de Microorganismes et de Biomole ´cules, Centre de Biotechnologie de Sfax, Universite ´ de Sfax, Route de Sidi Mansour Km 6, B.P 1177, 3018 Sfax, Tunisia e-mail: samir.bejar@cbs.rnrt.tn N. Aghajari Á R. Haser Laboratoire de BioCristallographie et Biologie Structurale des Cibles The ´rapeutiques, Bases Mole ´culaires et Structurales des Syste `mes Infectieux, UMR 5086–CNRS/Universite ´ Lyon 1, Institut de Biologie et Chimie des Prote ´ines, FR3302, 7 Passage du Vercors, 69367 Lyon cedex 07, France 123 J Ind Microbiol Biotechnol (2012) 39:537–546 DOI 10.1007/s10295-011-1061-1 Downloaded from https://academic.oup.com/jimb/article/39/4/537/5994583 by guest on 05 January 2023