Characterization of a thermostable alkaline phosphatase from a novel species Thermus yunnanensis sp. nov. and investigation of its cobalt activation at high temperature Ningping Gong a,1 , Chaoyin Chen c,1 , Liping Xie a,b , Hongtao Chen a , Xianzhi Lin c , Rongqing Zhang a,b, * a Institute of Marine Biotechnology, Department of Biological Sciences and Biotechnology, Tsinghua University, Beijing, 100084, China b Protein Science Laboratory of the Ministry of Education, Tsinghua University, Beijing, 100084, China c Biological and Chemical Engineering School, Kunming University of Science and Technology, Kunming, 650224, China Received 21 September 2004; received in revised form 10 April 2005; accepted 9 May 2005 Available online 23 May 2005 Abstract A thermostable alkaline phosphatase with high specific activity and thermal resistance was purified from a novel species of Thermus sp. named as Thermus yunnanensis sp. nov. The enzyme contains a single peptide with a molecular mass of about 52 kDa on SDS-PAGE analysis and appears to be a homodimer in solution with the molecular mass of 104 kDa. The optimal pH and temperature for its activities are pH 8.0 – 10.0 and 70–80 -C, respectively. The catalytic activities of the enzyme are metal ion dependent, and Mg 2+ , Zn 2+ and Co 2+ are the main activators. Among these, Co 2+ is the most active stimulator and has unique activation effect at high temperature. Metal binding analysis showed the binding of Mg 2+ at the metal binding site was easy to loss in the thermoinactivation, and Co 2+ was apt to bind at that site and kept the favorable configuration of catalysis, which would result high activation in the incubation with Co 2+ at high temperature. According to this study, a model was proposed for the explanation of the activation and the results of actual experiments demonstrated the validity of the model. D 2005 Elsevier B.V. All rights reserved. Keywords: Thermostable alkaline phosphatase; Thermus yunnanensis sp. nov.; Thermostability; Cobalt activation 1. Introduction Alkaline phosphatase (AP, EC 3.1.3.1) is a non-specific phosphomonoesterase which hydrolyzes a wide variety of phosphate esters and is classified as alkaline phosphatase according to its optimum pH [1]. In a very wide variety of organisms, AP plays a vital role in phosphate transportation and metabolism and is a most crucial enzyme for the survival of organisms under phosphate starvation [2,3]. In practical applications, the enzyme has a wide use in the diagnostics, immunology and molecular biology, such as served as biochemical markers in quantitative measurements of disease, linked enzymes in ELISA and used in non- radioactive detection techniques, probing, blotting and sequencing systems [4–8]. Because of the important principle and practical values of the enzyme, more and more research interests have been attracted in the field. So far, the most commonly used APs are Escherichia coli AP and calf intestine AP, but their inherently low thermal resistance and shelf lives have restricted their further applications under some special circumstances, for instance, under high temperature. Therefore, thermostable alkaline phosphatase (TAP) has attracted many attentions because it has the advantage of being used under some extreme conditions. Until now, TAPs have been purified and characterized from many thermophilic microorganisms, such as Thermus 1570-9639/$ - see front matter D 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.bbapap.2005.05.007 * Corresponding author. Tel.: +86 10 62772899; fax: +86 10 62772899. E-mail address: rqzhang@mail.tsinghua.edu.cn (R. Zhang). 1 Contributed equally to this work. Biochimica et Biophysica Acta 1750 (2005) 103 – 111 http://www.elsevier.com/locate/bba