Study The Effects Of Electrostatic Interaction On The Thermostability Of 1,4-?-glucan Branching Enzyme

The 1,4-?-glucan branching enzymes?GBEs;EC 2.4.1.18?,have been classified among the glycoside hydrolase?GH?families 13,catalyze the formation of branch points in natural starch.These branch points are formed through the transglycosylation reaction that cleaves?-?16?,4glucosidic linkages and attaches the released oligosaccharide chains to different glucose residues by synthesis of new?-1,6-glucosidic bonds.Through the transglycosylation reaction,GBEs are able to control the frequency and position of the branch points,increasing slow digestion of starch,by which they are considered to have potential industrial applications.Till now,most of GBEs generally have relatively low thermostability,which severely limits their industrial applications.Therefore,thermostabilization of GBEs is the key point and research hotspot of their industrial application.In this study,we investigated the biochemical properties and its thermostabilization of GBE from Geobacillus thermoglucosidans STB02 in terms of electrostatic interactions from outside and inside of protein.The results showed that strengthening electrostatic interactions on the surface of protein could enhance the thermostability of GBE.Based on these phenomena,sequence and structure alignment,as well as gene truncation,were applied to construct mutations which had higher thermostability.The results also demonstrated that there were strong electrostatic interactions between metal ions in solvent and the surface of GBE.In addition,mutants of GBE with salt bridges,which were constructed inside the protein,were determined to enhance their thermostability according to bioinformatics of salt bridges.Simultaneously,the corresponding mechanisms of thermostabilization involved in electrostatic interactions from the inside and outside of GBE were explored.The main results are summarized bleow:?1?First,the properties of G.thermoglucosidans STB02 GBE were investigated.The results showed that the half-time of G.thermoglucosidans STB02 GBE had low thermostability,whose half-time was approximately 6.9 min under 65?C.Additionally,the performances of G.thermoglucosidans STB02 GBE were affected by in the presence or absence of EDTA and metal ions.K⁺and Na⁺could enhance the activity of GBE,while Ca²⁺,Co²⁺,Cu²⁺and Fe²⁺inhibited the activity of GBE.Particularly,0.5 mM K⁺or 1.0 mM Na⁺enhanced thermostability of GBE.The improvements in thermostability of GBE may rise from electrostatic interactions introduced by K⁺and Na⁺in solvent.Previous reports showed that metal ions affected performance of enzymes via two mechanisms.One is metal ions bind to metal ion binding sites of enzymes,the other is metal ions gather around the surface of enzymes.Our survey showed that there were no metal ion binding sites in GBE from G.thermoglucosidans STB02.The electrostatic interactions between metal ions and GBE may rise from metal ions randomly bind to the surface of enzymes.Glycerol could expel the water around the enzymes,which is able to strengthen the electrostatic interactions between metal ions and GBE.The results revealed that adding glycerol further increased the thermostability of GBE in the presence of metal ions.?2?The electrostatic interactions between metal ions and GBE may rise from the metal ions in the solvent and charged residues on the surface of GBE.Comparison of the amino acid sequences and three-dimensional structures of mesophilic?Escherichia coli?and thermophilic?G.thermoglucosidans STB02?GBEs showed that the last 26 residues at the C-terminal end of the GBE from G.thermoglucosidans STB02 were not conserved,and that their 3-dimensional structure was flexible.Then,a truncated mutant of GBE_Gt made by removing the last 26 residues from its C-terminal end was constructed and named GBE_Gt?C.The results showed that GBE_Gt and GBE_Gt?C had similar catalytic properties.However,the GBE_Gt?C presented higher thermostability than GBE_Gt,of which the T_m value was 5?C higher than GBE_Gt.Besides,GBE_Gt?C could re-dissolve more aggregates than GBE_Gt after incubation at their individual T_m.The activities of GBE_Gt and GBE_Gt?C differently responded to EDTA and metal ions.GBE_Gt?C had stronger EDTA tolerance than GBE_Gt.Additionally,truncation of a portion of the C-terminus resulted in increase in stability of GBE_Gt?C in the absence of metal ions,while most of GBE_Gt tended into aggregate under the same condition.The metal ions response and thermostability of GBE_Gt and GBE_Gt?C were found to be different,which may result from different aggregate state introduced by deletion of flexible residues at the C-terminal end of GBEGt.This change may result in alternation of thermostabilization mechanisms between GBE_Gt and GBE_Gt?C.The results also confirmed that there were electrostatic interactions between full length GBE and metal ions in solvent.?3?Strengthening electrostatic interactions between GBE and metal ions increased stability of GBE,the electrostatic interactions inside GBE may also have effects on stability of GBE.The salt bridges in proteins were investigated via bioinformatics and statistical methods,then summarized basic rules that are universally applied in constructing salt bridges.Based on our bioinformatics studies,from the evolutionary point of view,we found that when one part of salt bridges was conserved,the other part of salt bridges would have about 80%chance to be conserved;On the contrary,when one part of salt bridges was unconserved,the other part of salt bridges would have about 10-20%chance to be unconserved.In addition,our research revealed that the evolutionary stability of salt bridges was important for regionally evolutionary stability.Particularly,one pair of salt bridges were conservative,the evolutionary stability of their region would be 61-100%,while the evolutionary stability of their region dropped to less than 45%.More importantly,the salt bridges contributing more to stability appeared on the low ET value?high conservation?of regions.?4?Accoding to the characterized properites of salt bridges in proteins,several residues were screened and mutanted in order to enhance the thermostability of GBE.Several mutants aiming at increasing thermostability of GBE,H224E,H224D,Q231R,Q231K,T339E and T339D,were constructed in?-helical structures in GBE.The results showed that they prolonged half time of GBE at 60?C approximately 40%,38%,21%,26%16%and 21%,compared to their original type of GBE.Some mutants,V37E,V37D,G98E,G98D and I571D,were constructed in?-sheet structures in GBE,whose half time at 60?C prolonged 80%,88%,95%,109%and 52%,compared to their original type.Nevertheless,the mutagenesis at 266 site in coil structure in GBE showed little effects on thermostability of GBE,demonstrating that strengthen stability of coil structure in GBE would have limited effects on thermostability of GBE.?5?Based on previouos work,mutants that involved in three charged residues were constructed,and their thermostability was investigated.The triple electrostatic interaction salt bridges,Q231R-D227-D131H,Q231K-D227-D131H,T339E-K335-I291H and T339D-K335-I291H,were constructed in?-helical structures in GBE,which strengthened correspondingly regional stability of GBE.The I571D-R569-R617H built in?-sheet structures in GBE enhanced the thermostability of GBE.Compared with wild type GBE,the mutants with higher thermostability were more suitable for industrial applications when they had similar effects on starch.The modifications on GBE enhanced usability of GBE.The mutants with higher thermostability could be applied in starch modification industry earlier than wild type GBE,reducing the energy consumption.Additionally,the strategies of truncation at C-terminal and construction of salt bridges enriched the thermostabilization strategies of amylases.