Heat-resistant And Catalytic Mechanism And Catalytic Activity Modification Of A New Kind Of Thermostable Trehalose Synthase From Thermobaculum Terrenum

Trehalose composed of two glucose molecules with an?-1,1-glycosidic bond is an important disaccharide in organisms.It plays many important roles in the organisms.Besides serving as the carbon and energy source,trehalose can protect the organism from extreme conditions such as hypothermia,dehydration,hypertonic,ethanol toxicity and oxidation.Based on Thermobaculum terrenum,the relationship between protein structure and heat-resistant mechanism,enzymatic properties,catalytic mechanism and molecular modification of trehalose synthase were studied by experiments.The main research results include the following aspects:?1?Determination of enzymatic properties and thermal resistance of trehalose synthase:The enzyme showed maximum activity at 45°C and the pH-dependency activities of TtTS were assayed,and the enzyme showed maximum activity at pH 7.5.Approximately 68.95%of the maltose substrate?150 mM?was converted to trehalose after a 4 h reaction.The effects of metal ions and reagents were analyzed at 1 mM and10 mM concentrations of a variety of substances.When treated with 1 mM SDS,EDTA,Cu²⁺,or Ni²⁺,TreS activity was clearly inhibited.All other metals and reagents showed no obvious effects at this concentration.At 10 mM,inhibitory effects of these four reagents were more obvious.However,Zn²⁺,Fe²⁺,Ca²⁺,Mn²⁺,Mg²⁺,Ba²⁺,and K⁺can improve the effects of TtTS.By analyzing the sequence and structure of TtTS and other trehalose synthases,it was found that the heat resistance of TtTS was mainly related to the following aspects:?1?it was found that the TtTS has four metal ion binding sites,the heat resistance of the mutant trehalose synthase decreased significantly.?2?TtTS has thermophilic structural characteristics.TtTS has more polar?charged?amino acids than mesophilic TreSs,which may lead to the formation of more hydrogen and ionic bonds.The hydrophobic interactions and aromatic-aromatic interactions may in turn lend the protein interior greater stability.?3?TtTS is more rigid and there was a close interaction between regions Asp4-Val129 and Tyr373-Gln450 of TtTS,which suggests that these two regions may significantly correlate with its thermostability.?2?Studies on the catalytic mechanism of trehalose synthase:Amino acids that may play a key role in the catalytic process were obtained by structural and molecular dynamics analysis.The mutation of polar amino acids near the active center by site mutation proved that in the catalytic process,when the?-1,4-glycoside bond was broken,the amino acids+1 position were reversed to form new?-1,1-glycoside bonds,thus the wheat was transformed into a new?-1,1-glycoside bond.Maltose is isomerized to trehalose.Computer calculation combined with enzymatic analysis of the key binding amino acids,which elucidated the glucose rotation after the?-1,4 glycoside bond broken up may be a key factor in determining the reaction direction.N246 is important to maintain the+1 subsite glucose stability during in its rotation stage.These results furtherly strength our understanding of the reaction mechanism of isomerase.?3?Site-directed modification of key amino acids in trehalose synthase substrate-binding channel:We furtherly probed the effect of the key aminoamides in the substrate gateway domain to trehalose synathase bioconversion.The result showed the most amino acids nearby substrate gateway entrance are very conserved.These amino acids have important function in maintaining a closed conformation of catalytic pocket.We established saturated mutation pointed at the non-conserved?Frequent<0.95?amino acids.The result showed that the conversation of K136T,Y137D,K138N and D139S mutations have increased the conversation slightly and were over 70%.Analysis of flexibility of mutations with wild TtTS demonstrated that flexible loop?residues60-80?around the active site were rigidified.This result furtherly demonstrated the importance of gateway amino acid for regulation the activity trehalose synthase.