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Applied and Environmental Microbiology, June 2002, p. 2676-2682, Vol. 68, No. 6
0099-2240/02/$04.00+0     DOI: 10.1128/AEM.68.6.2676-2682.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Isomaltulose Synthase from Klebsiella sp. Strain LX3: Gene Cloning and Characterization and Engineering of Thermostability

Daohai Zhang, Xianzhen Li, and Lian-Hui Zhang^*

Laboratory of Biosignals and Bioengineering, Institute of Molecular Agrobiology, Singapore 117604

Received 26 November 2001/ Accepted 20 March 2002

The gene (palI) encoding isomaltulose synthase (PalI) from a soil bacterial isolate, Klebsiella sp. strain LX3, was cloned and characterized. PalI converts sucrose into isomaltulose, trehalulose, and trace amounts of glucose and fructose. Sequence domain analysis showed that PalI contains an -amylase domain and (ß/)₈-barrel structures, suggesting that it belongs to the -amylase family. Sequence alignment indicated that the five amino acid residues of catalytic importance in -amylases and glucosyltransferases (Asp²⁴¹, Glu²⁹⁵, Asp³⁶⁹, His¹⁴⁵, and His³⁶⁸) are conserved in PalI. Purified recombinant PalI displayed high catalytic efficiency, with a K_m of 54.6 ± 1.7 mM for sucrose, and maximum activity (approximately 328.0 ± 2.5 U/mg) at pH 6.0 and 35°C. PalI activity was strongly inhibited by Fe³⁺ and Hg²⁺ and was enhanced by Mn²⁺ and Mg²⁺. The half-life of PalI was 1.8 min at 50°C. Replacement of selected amino acid residues by proline significantly increased the thermostability of PalI. Simultaneous replacement of Glu⁴⁹⁸ and Arg³¹⁰ with proline resulted in an 11-fold increase in the half-life of PalI at 50°C.

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* Corresponding author. Mailing address: Institute of Molecular Agrobiology, 1 Research Link, National University of Singapore, Singapore 117604. Phone: 65-8727400. Fax: 65-8727012. E-mail: lianhui{at}ima.org.sg.

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Applied and Environmental Microbiology, June 2002, p. 2676-2682, Vol. 68, No. 6
0099-2240/02/$04.00+0     DOI: 10.1128/AEM.68.6.2676-2682.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

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