The Study Of The Relationship Between The Structure And Function, And On The Directed Evolution Of Trehalose Synthase From Meiothermus Ruber CBS-01

Trehalsoe is a non-reducing disaccharide widely used in pharmaceutical industry,cosmetic industry, food, agriculture, and many other fields. Because of its ability ofprotection, trehalose attracts lots of interest. Nowadays, trehalose is mainlymanufactured through enzymatic pathways. Among the enzymes which couldproduce trehalose, trehalose synthase (TreS) could convert maltose into trehalose inone step reaction, and the raw material is cheap, which makes it get more and moreconcerning. However, the relationship between the structure and function of TreS hasnot been investigated deeply. Moreover, the efficiency of some native TreS is not veryhigh. So it is important to study about the relationship between the structure andfunction and construct the method for directed evolution of TreS.In our previous study, the treS gene had been cloned from a thermophilicMeiothermus ruber strain CBS-01and expressed in Escherichia coli to characterizeits properties. Because of its thermophilicity and thermostability, TreS from M. ruber(TSM) was fit to produce trehalose in industry. However, the activity of TSM was nothigh enough to be applied in industry. It was necessary to improve the efficiency ofTSM for application and investigate the thermoadapatation mechanism of theenzyme.Firstly, the N-and C-terminal domains of TSM were constructed and expressedin E. coli. The C-termianl domain and wild-type TSM shared the similar secondarystructure, both of which were α/β type，a typical structure of α-amylase super family.While the N-terminal domain without C-terminal region underwent the change insecondary structure. It implied that the C-terminal domain was important for themaintenance of the structure of protein.Moreover, a series of proteins with different deletion at C terminus of TSM wereconstructed to find the region which affected the function of TSM. In a result, thetruncated protein ΔC44, which was deleted44amino acid residues from C terminus,changed the affinity to the substrates. It preferred maltose to trehalose as its substrate. Meanwhile, the truncated protein ΔC68and more amino acid residues missed from Cterminus could not fold correctly in solution form. The result showed that the44amino acid residues from C terminus may affect the linkage between the protein andsubstrate, while the region from the residue68from C terminus played a key role inthe protein folding.Secondly, the N-terminal and C-terminal domains of TSM and trehalosesynthase from Thermus thermophilus (TST) were switched to ascertain which of themplayed the important role in the characteristics and function of TreS. Two fusionproteins TSTtMr (N-terminal domain of TST fused with C-terminal domain of TSM)and TSMrTt (N-terminal domain of TSM and C-terminal domain of TST) wereconstructed. The enzymes with the same N-terminal domains shared the similarkinetics parameters and optimum temperature, indicating that the N-terminal domainsthemselves also play a major role in determining the thermostability and activity ofenzymes. Additionally, the fusion protein TSTtMr displayed the higher kcat/Kmvaluethan that of TSM, indicating that it could convert maltose to trehalose moreefficiently from a kinetic point of view.Thirdly, the three-dimensional structure of the N-terminal domain (3-543residues) of TSM was predicted at the tertiary level as a GH13domain of α-amylasesuper family. There was a putative catalytic cleft in the N-terminal domain. Fourconserved region and key sites of α-amylase super family were located in the cleft.Through site-directed mutagenesis, H104, D200, and the third conserved region werefound to play important roles in the activity of TSM. Besides, Y135, R388, and R392,which were also located in the cleft, could affect the function of TSM as well. Themutagenesis at these sites led to the complete loss or sharp decrease in the activity.The mutant R392A was inactive at50℃and kept a little activity at30℃, suggestingthat R392or the region near R392could influence the activity and thermophilicity ofTSM.Fourthly, the method for the directed evolution of TSM was constructed. Aproper process to prepare the crude enzyme was achieved with cell suspension treatedby2%toluene to get permeabilized cells. After DNS analysis, the absorbance at570nm was used to compare the activity of the mutants. Moreover, error-prone PCR and DNA shuffling for the directed evolution of TSM were performed to construct thelibrary of mutants. After screening the library, a mutant with6site substitution wasselected. The activity of the mutant was1.6fold of that of wild-type TSM. The Kmvalue of the mutant was a half of TSM, implying the affinity of mutant was2-foldhigher. And the catalytic efficiency was2-fold of wild-type TSM.