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

2002; American Society for Microbiology; Volume: 68; Issue: 6 Linguagem: Inglês

10.1128/aem.68.6.2676-2682.2002

1098-5336

Daohai Zhang, Xianzhen Li, Lian‐Hui Zhang,

Enzyme Structure and Function

ABSTRACT 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 α-amylase domain and (β/α) 8 -barrel structures, suggesting that it belongs to the α-amylase family. Sequence alignment indicated that the five amino acid residues of catalytic importance in α-amylases and glucosyltransferases (Asp 241 , Glu 295 , Asp 369 , His 145 , and His 368 ) 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 Fe 3+ and Hg 2+ and was enhanced by Mn 2+ and Mg 2+ . 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 Glu 498 and Arg 310 with proline resulted in an 11-fold increase in the half-life of PalI at 50°C.