Cloning, Expression And Characterization Of Maltooligosyltrehalose Synthase From Nostoc Flagelliforme

The study on trehalose metabolic pathway and its related enzymes has been the focus and hotspots in bio-engineering research. Both its theoretical research or applications have shown significant value and meaning. Nostoc flagelliforme has a strong resistance to withstand extremely adverse environment. In 2007, our laboratory found the presence of trehalose in Nostoc flagelliforme under dehydration. This study did some further research on the relationship between the trehalose metabolism and resistance to adverse environment.A genomic DNA fragment encoding a putative maltooligosyltrehalose synthase (NfMTS) for trehalose biosynthesis was cloned by the degenerate primer-PCR from cyanobacterium Nostoc flagelliforme. The ORF of NfMTS is 2,799 bp in length and encodes 933 amino acid residues constituting a 106.6 kDa protein. The deduced amino acid sequence of NfMTS contains 4 regions highly conserved for MTSs. By expression of NfMTS in E. coli, the function of this protein was demonstrated that the recombinant protein catalyzed the conversion of maltohexaose to maltooligosyl trehalose. The Km of the recombinant enzyme for maltohexaose was 1.87 mM and the optimal temperature and pH of the recombinant enzyme was at 50℃and 7.0 respectively. The expression of MTS of N. flagelliforme was upregulated and both trehalose and sucrose contents increased significantly in N. flagelliforme during drought stress. However, trehalose accumulated in small quantities, about 0.36 mg·g-1 DW whereas sucrose accumulated in high quantities, about 0.90 mg·g-1 DW, indicating both trehalose and sucrose involved in dehydration stress response in N. flagelliforme and sucrose might act as a chemical chaperone rather than trehalose did during dehydration stress.This study examined the key enzymes in trehalose metabolic pathway in N. flagelliforme with molecular methods. Results obtained not only provides a certain foundation to study the metabolic pathway of trehalose in N. flagelliforme, but also helps us understand metabolism of trehalose and its function.