Expression and Purification of the Enzymes are involved in Isomalt production from Saccharose

Diabetes, a metabolic syndrome, is the most common endocrine disease and the third disease causing death in the world. More than 10 % of Iranian people have diabetes of type 1 and 2. Diabetes caused of metabolic disorders in patients with cardiovascular and cancer. Today, improving the nutritional regime is accepted as the most effective way for prophylaxis and control of diabetes. For this purpose, societies are encouraged to use sugar substitutes. Isomalt or Palatinose is one of the most important members of this group which can be used instead of sucrose. Isomalt consists of an equimolal mixture of α-D-Glucopyranosyl-(1, 6)-Sorbit (GPS) and α-D-Glucopyranosyl-(1, 1)-Mannit (GPM) and is produced as a result of enzymatic or semienzymatic methods. The former method consists of two distinct stages. In the first stage Saccharose is converted to Isomaltulose, being used as a substrate by mannitol dehydrogenase and Sorbitol dehydrogenase in second stage to produce 1, 6-GPS and 1, 1- GPM. In the present study, Sequences coding of β-galactosidase, sucrose isomerase, mannitol dehydrogenase and Sorbitol dehydrogenase, e.i. bgaB, PalI, mtlD and sdh from Geobacillus stearothermophilus, Erwinia rhapontici ، Gluconobacter Oxidants ، Pseudomonas fluorescence, Previously, inserted in expression vector. The accuracy of cloning was verified via PCR. The plasmids were used for transformation and expression in E. coli B121 (DE3) plysS. The purification of expressed enzymes was done by using affinity chromatography. For purification using FPLC, the column Ni-NTA and Gel filtration were used. SDS-PAGE of purified enzymes showed, that proteins had high purify. The best condition for protein stabilization and activities in vitro were detected. The HPLC analysis with RI detector showed that the purified enzymes were capable in using all these substances as its substrates. Furthermore, sucrose isomerase converted Saccharose to Isomaltulose, Mannitol dehydrogenase and Sorbitol dehydrogenase respectively 1, 1-GPM and 1, 6-GPS from Isomaltulose. In addition β-galactosidase is able to produce Galactooligosaccharide (GOS).