Cloning and Characterization of a Sucrose Isomerase from Erwinia rhapontici NX-5 for Isomaltulose Hyperproduction

• DOI: 10.1007/s12010-010-9015-z
• Publication Date: 2010-06-29T12:52:19Z
• Authors: Sha Li; Heng Cai; Yujia Qing; Ben Ren; Hong Xu; Hongyang Zhu; Jun Yao
• Keywords: Sucrose; 0303 health sciences; Molecular Sequence Data; Gene Expression; Isomaltose; Kinetics; 03 medical and health sciences; Bacterial Proteins; Glucosyltransferases; Enzyme Stability; Escherichia coli; Erwinia; Amino Acid Sequence; Cloning, Molecular; Sequence Alignment; Soil Microbiology

The sucrose isomerase (SIase) gene from an efficient strain of Erwinia rhapontici NX-5 for isomaltulose hyperproduction was cloned and overexpressed in Escherichia coli. Protein sequence alignment revealed that SIase was a member of the glycoside hydrolase 13 family. The molecular mass of the purified recombinant protein was estimated at 66 kDa by SDS-PAGE. The SIase had an optimal pH and temperature of 5.0 and 30 °C, respectively, with a K (m) of 257 mmol/l and V (max) of 48.09 μmol/l/s for sucrose. To the best of our knowledge, the recombinant SIase has the most acidic optimum pH for isomaltulose synthesis. When the recombinant E. coli (pET22b- palI) cells were used for isomaltulose synthesis, almost complete conversion of sucrose (550 g/l solution) to isomaltulose was achieved in 1.5 h with high isomaltulose yields (87%). The immobilized E. coli cells remained stable for more than 30 days in a "batch"-type enzyme reactor. This indicated that the recombinant SIase could continuously and efficiently produce isomaltulose.