Cloning and characterization of a sucrose isomerase from Erwinia rhapontici NX-5 for isomaltulose hyperproduction.
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.
Li
Sha
S
State Key Laboratory of Materials-Oriented Chemical Engineering, College of Food Science and Light Industry, Nanjing University of Technology, No. 5 New Model Road, Nanjing, People's Republic of China.
Cai
Heng
H
Qing
Yujia
Y
Ren
Ben
B
Xu
Hong
H
Zhu
Hongyang
H
Yao
Jun
J
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D003001:Cloning, Molecular; D004795:Enzyme Stability; D004885:Erwinia / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics / Q000302:isolation & purification; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D005964:Glucosyltransferases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D007534:Isomaltose / Q000031:analogs & derivatives* / Q000096:biosynthesis; D007700:Kinetics; D008969:Molecular Sequence Data; D016415:Sequence Alignment; D012988:Soil Microbiology; D013395:Sucrose / Q000378:metabolism
D001426:Bacterial Proteins; D013395:Sucrose; D007534:Isomaltose; D005964:Glucosyltransferases; C116853:sucrose isomerase; C008189:isomaltulose