Role of the C-terminal domain of Thermus thermophilus trehalose synthase in the thermophilicity, thermostability, and efficient production of trehalose.
Trehalose synthase (TS) from Thermus thermophilus (TtTS) is a thermostable enzyme that catalyzes the conversion of maltose into trehalose by intramolecular transglucosylation. It has a relatively higher thermophilicity and thermostability and a better conversion ratio for trehalose production than other known TSs from different sources at present. By amino acid sequences and the schematic motif alignment of trehalose synthase-related enzymes, it was found that TtTS (965 amino acid residues) contains a particular C-terminal fragment that is not found in most other TSs. To verify the function of this fragment, C-terminal deletion and enzyme fusion were respectively performed to explain the important role this fragment plays in the formation of trehalose. First, the C terminus (TtTSDeltaN, 415 amino acid residues) of TtTS is deleted to construct a TtTSDeltaC containing 550 amino acids. Furthermore, a novel cold-active TS was cloned and purified from Deinococcus radiodurans (DrTS, 552 amino acid residues) and then a fusion protein was created with TtTSDeltaN at the C terminus of DrTS (DrTS-TtTSDeltaN). It was found that the recombinant TtTStriangle upC enzyme had a lower thermostability and a higher byproduct than TtTS in catalyzing the conversion of maltose into trehalose. On the other hand, the recombinant DrTS-TtTSDeltaN enzyme had a higher thermostability and a lower byproduct than DrTS in their reactions. The above-mentioned results allowed the inference that the C terminus of TtTS plays a key role in maintaining its thermostability and hence in modulating the side reaction to reduce glucose production at a high temperature. A new, simple, and fast method to improve thermophilicity by fusing this fragment with particular conformation to a thermolabile enzyme is offered.
Wang
Jia-Hung
JH
Institute of Bioscience and Biotechnology, National Taiwan Ocean University, Keelung, 202, Taiwan.
Tsai
Meng-Yin
MY
Chen
Jen-Jye
JJ
Lee
Guan-Chiun
GC
Shaw
Jei-Fu
JF
D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D004795:Enzyme Stability; D005964:Glucosyltransferases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006358:Hot Temperature; D008969:Molecular Sequence Data; D011994:Recombinant Proteins; D013329:Structure-Activity Relationship; D016963:Thermus thermophilus / Q000201:enzymology*; D014199:Trehalose / Q000096:biosynthesis* / Q000737:chemistry
D011994:Recombinant Proteins; D014199:Trehalose; D005964:Glucosyltransferases; C099692:trehalose synthase