Isomaltulose synthase from Klebsiella sp. strain LX3: gene cloning and characterization and engineering of thermostability.
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 alpha-amylase domain and (beta/alpha)(8)-barrel structures, suggesting that it belongs to the alpha-amylase family. Sequence alignment indicated that the five amino acid residues of catalytic importance in alpha-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 Km of 54.6 +/- 1.7 mM for sucrose, and maximum activity (approximately 328.0 +/- 2.5 U/mg) at pH 6.0 and 35 degrees 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 degrees 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 degrees C.
Zhang
Daohai
D
Laboratory of Biosignals and Bioengineering, Institute of Molecular Agrobiology, Singapore 117604.
Li
Xianzhen
X
Zhang
Lian-Hui
LH
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D003001:Cloning, Molecular; D004795:Enzyme Stability; D005656:Fungal Proteins / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D019751:Intramolecular Transferases / Q000235:genetics* / Q000378:metabolism; D007534:Isomaltose / Q000031:analogs & derivatives* / Q000378:metabolism; D007709:Klebsiella / Q000201:enzymology* / Q000235:genetics; D008565:Membrane Proteins; D008969:Molecular Sequence Data; D011392:Proline / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013696:Temperature; D000516:alpha-Amylases / Q000737:chemistry
D005656:Fungal Proteins; D008565:Membrane Proteins; C115834:PALI protein, Emericella nidulans; D007534:Isomaltose; D011392:Proline; D000516:alpha-Amylases; D019751:Intramolecular Transferases; C042002:isomaltulose synthase; C008189:isomaltulose