Polynucleotide for recombinant expression of isomaltulose synthase
The present invention is directed towards genetic modification of native gene encoding for sucrose isomerase and isomaltulose synthase to substantially increase the expression level of these enzymes and use of said enzymes in a process to produce rare disaccharides such as isomaltulose and trehalulose. Also disclosed in the present invention is expression constructs comprising the modified genes and a host cells to express the same.
1. A modified polynucleotide encoding an isomaltulose synthase, wherein the modified polynucleotide comprises the nucleotide sequence of SEQ ID NO: 2.
2. An expression construct comprising the modified polynucleotide according to claim 1 .
3. The expression construct according to claim 2 , wherein the modified polynucleotide is operably linked to a T7 promoter.
4. An isolated host cell comprising the expression construct of claim 2 .
5. The host cell of claim 4 , wherein the host cell is a prokaryotic host cell.
6. A process for production of an isomaltulose synthase, said process comprising the steps of:
1. culturing the host cell of claim 4 in a suitable medium in presence of IPTG or lactose for 2-3 hours to produce the isomaltulose synthase,
2. isolating the isomaltulose synthase from the host cell, and
3. purifying the isomaltulose synthase using chromatographic techniques.
7. A process for the production of isomaltulose from sucrose, said process comprising the steps of:
1. culturing the host cell of claim 4 in a suitable medium in presence of IPTG or lactose for 2-3 hours to produce the isomaltulose synthase,
2. isolating the isomaltulose synthase from the host cell, and purifying the isomaltulose synthase using chromatographic techniques,
3. immobilizing the purified isomaltulose synthase in a suitable matrix, and
4. contacting sucrose with the immobilized isomaltulose synthase for a period in the range of 4 to 14 hours to produce isomaltulose.
8. The process of claim 7 , wherein 91% of total sugar produced is isomaltulose.
9. The process according to claim 6 , wherein the host cell is a prokaryotic host cell.
10. The process according to claim 9 , wherein the host cell is an Escherichia coli cell.