Journal of Molecular Catalysis B: Enzymatic 22 (2003) 45–53 A cycloamylose-forming hyperthermostable 4--glucanotransferase of Aquifex aeolicus expressed in Escherichia coli Shakhawat Hossain Bhuiyan, Motomitsu Kitaoka ∗ , Kiyoshi Hayashi Enzyme Laboratory, National Food Research Institute, 2-1-12 Kannondai, Tsukuba, 305-8642 Ibaraki, Japan Received 8 November 2002; received in revised form 27 December 2002; accepted 27 December 2002 Abstract The gene (malM) encoding for 4--glucanotransferase (4GTase) from the hyperthermophilic bacterium Aquifex aeolicus was cloned and expressed in Escherichia coli. The recombinant enzyme was purified to homogeneity and its behavior towards various substrates was examined. The enzyme was hyperthermostable, exhibiting maximal activity at 90 ◦ C and it retained 70% of its original activity after incubation at 90 ◦ C for 30 min. A low affinity was observed for the enzyme towards maltose (K m = 71 mM) and the k cat /K m value for maltotriose was approximately 166 times greater than that observed for maltose but the values did not change significantly with larger maltooligosaccharides. The A. aeolicus 4GTase produced cycloamylose with a minimum degree of polymerization (DP) of 16, whereas the cycloamylose produced by the amylomaltase from the thermophilic bacterium Thermus aquaticus, which is also a Type II 4GTase, produced a cycloamylose with a minimum DP of 22. These findings indicate that the A. aeolicus 4GTase differs from the T. aquaticus amylomaltase and the glucanotransferases produced by other hyperthermophilic organisms. © 2003 Elsevier Science B.V. All rights reserved. Keywords: Aquifex aeolicus; 4--Glucanotransferase; Hyperthermostable; Cycloamylose 1. Introduction 4--Glucanotransferase (4GTase) (EC 2.4.1.25) are enzymes that catalyze glucan transfer from one -1,4-glucan molecule to another -1,4-glucan or to glucose. This enzyme is also known as the dis- proportionating enzyme (d-enzyme) in plants and it was first found in potato tubers by Peat et al. [1]. It has since been found in many hyperthermophilic organisms, with varying degrees of substrate speci- ficities [2–4]. 4--Glucanotransferase is also named ∗ Corresponding author. Tel: +81-298-38-8071; fax: +81-298-38-7321. E-mail address: mkitaoka@nfri.affrc.go.jp (M. Kitaoka). as amylomaltase in bacterial species [5,6], and the crystal structure of the Thermus aquaticus amylo- maltase has recently been reported [7]. A compar- ison of the d-enzyme and amylomaltase indicates that these enzymes display significant homology in their amino acid sequences and they display strong similarities in their catalytic properties [8]. These en- zymes catalyze an intermolecular transglycosylation reaction called a “disproportionating reaction” and they also catalyze intramolecular transglycosylation, which creates a cyclic glucan (cycloamylose) from a single linear glucan molecule. It has been shown that maltooligosaccharides are effective donors and maltooligosaccharides and glucose serve as accep- tors in vitro. Glucosyl or maltosyl groups have been 1381-1177/03/$ – see front matter © 2003 Elsevier Science B.V. All rights reserved. doi:10.1016/S1381-1177(03)00005-5