Enzyme and Microbial Technology 37 (2005) 735–738 Construction of new stable strain over-expressing the glucose isomerase of the Streptomyces sp. SK strain Monia Mezghani, Mohamed Ali Borgi, Radhouane Kammoun, Hedi Aouissaoui, Samir Bejar ∗ Laboratoire d’Enzymes et de M´ etabolites des Procaryotes, Centre de Biotechnologie de Sfax, BP “K” 3038 Sfax, Tunisia Received 6 October 2004; received in revised form 11 April 2005; accepted 28 April 2005 Abstract In order to over express the xylA gene of Streptomyces sp. SK strain, it was cloned under the control of the constitutive ermE-up promoter. This construct was integrated through site-specific recombination process into the chromosome of a Streptomyces violaceoniger glucose isomerase deficient strain using the non-replicative vector pTS55. The resulting CBS4 strain shows a perfect stability in the absence of selection pressure. Its glucose isomerase activity was about four and nine-fold greater, than that obtained from Streptomyces sp. SK, respectively fully induced or not by xylose. © 2005 Elsevier Inc. All rights reserved. Keywords: Glucose isomerase; Over-expression; CBS4 strain; ermE-up promoter 1. Introduction Xylose isomerase (d-xylose ketol isomerase EC 5.3.1.5) catalyses the reversible isomerization of d-xylose into d-xylulose. It is also referred to as glucose isomerase (GI) because of its ability to convert d-glucose to d-fructose. This property is widely exploited industrially for the production of high-fructose syrup from starch [1]. Isomerization at high temperature increases the reaction rate and allows the shift of equilibrium between glucose and fructose toward the later [2]. This is useful in developing a one step process for the conversion of starch to HFCS and avoided the multiple enzymatic steps at varying temperatures and reaction conditions [3]. Hence, many thermoactive GIs were isolated and studied including those from Thermotoga maritima [4], Thermus caldophilus [5] and Thermus ther- mophilus [6]. Certainly, these interesting features must be coupled to the enzyme’s tolerance to acidic pH because of the formation of bitter (browning) sub-products (mannose, psicose, and other acidic compounds) under the condition of higher temperature and alkaline pH [7]. Moreover, glucose ∗ Corresponding author. Tel.: +216 74 440451; fax: +216 74 440818. E-mail address: samir.bejar@cbs.rnrt.tn (S. Bejar). syrup production process from starch involves enzymes acting at acidic pH range (-amylase, Glucoamylase). Hence the pH of the resulting starch hydrolysis should be adjusted before making use of glucose isomerase enzyme. For a better industrial application, the properties of some described enzymes were ameliorated by genetic engineering. For example, it was demonstrated that the change of Asp56 to Asn and Glu221 to Ala, in the GI of Streptomyces ru- biginosus, decreased the optimum pH of this enzyme and enhanced its catalytic efficiency [8]. In spite of the existence of several works describing the improvement of the GI performances, no recent studies have been reported concerning the enhancement of the strains pro- ductivity. Such approach is certainly of importance due to the potential use of improved strains in biotechnological process, especially those producing glucose isomerases. We have previously reported the cloning and the prelim- inary characterization of a thermostable glucose isomerase (SKGI), isolated from a new thermophilic Streptomyces sp. SK strain, which has an optimum temperature of about 90 ◦ C and a wide pH range with an optimum of 6 and 6.5 at 60 and 90 ◦ C, respectively [9]. These properties coupled to some aa sequence originalities make it attractive for an industrial application [10]. Furthermore, the enzyme purification as 0141-0229/$ – see front matter © 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.enzmictec.2005.04.012