The Study Of Character And Activity Modification Of Trehalose Synthase From Pseudomonas Stutzeri CJ38

Trehalose is a non-reducing disaccharide that contains two glucose moieties linked by an ?,?-1,1-glycosidic linkage.It is present in a variety of organisms and has a non-specific protective effect.In this study,tres was cloned from Pseudomonas stutzeri Qlu3 genomic DNA.Following its overexpression in Escherichia coli BL21?DE3?and purification.Our experimental nature of its activity,protein structure and the transformation of trehalose synthase molecules were studied.The main findings include the following aspects:?1?The trehalose synthase enzymology properties:The conversion rate of maltose to trehalose,catalyzed by the recombinant TreS,at an initial maltose concentration of 150 mM was greater than 72%,with low byproduct formation?3.6%?in less than 2h,which is considerably shorter than the time previously reported.Trehalose conversion by recombinant TreS had an optimal pH of 8.0 and an optimal temperature of 35°C.The effect of metals on TreS activity was tested,and conversion was inhibited by the presence of 10 mM Zn²⁺,Cu²⁺,Fe²⁺,Ca²⁺,Co²⁺,Mn²⁺,Ni²⁺,and SDS.?2?Trehalose synthase crystal structure research: Through trehalose synthase crystal screening and optimization,we got the trehalose synthase crystals and collectted a native diffraction data.Since the trehalose synthase sequence similarity with the existing structure of the reported less than 30%,so the structural analysis is required to determine the phase.Subsequently,we had to extract Se-Met-TreS proteins and crystals was screened.Due to the low resolution of the crystal,it is insufficient for resolution of protein structure.Crystals need further optimization.?3?Trehalose synthase homology modeling and structural transformation: we predicted the structural characteristics of recombinant TreS bound to its substrate by using homology modeling and flexible docking studies of the enzyme-substrate system.The result was verified by biochemical experiments.In addition,an enclosed active site tunnel was revealed that controls substrate movement during intramolecular isomerization.The size of the channel and the channel switching state for trehalose synthase reaction rate plays a key role.According to biochemistry experiment,disruption of the tunnel by removing two loops led to total loss of isomerization activity.After amino acid mutations on the channel,it will significantly change the trehalose synthase activity.The A309 E mutant showed increased isomerase activity and decreased hydrolase activity.In contrast,the Q219 R,T308E,and L341 Q mutants showed decreased isomerase activity and increased hydrolase activity.