Study On Substrate Specificity And Thermal Stability Of The ?-glucosidase From Hevea Brasiliensis Seeds By Molecular Docking

?-glucosidase is an enzyme that can hydrolyze the non-reducing P-D-glucosidic linkages which bound to the terminal and release the corresponding ligands and glucose,which has a wide range of application prospects in food flavoring,cellulose degradation,synthesis of glycosyl compounds,cyanogenic glycosides and other aspects.Hevea brasiliensis seed is a tropical crop with potential economic value,which is rich in unsaturated fatty acids,proteins,vitamins and other nutrients,but this resource cannot be reasonably used due to the presence of cyanide.In order to make full use of Hevea brasiliensis resources,the endogenous P-glucosidase of Hevea brasiliensis seeds was used as the research object to explore its thermostability and substrate specificity,in order to promote the research and utilization of ?-glucosidase from Hevea brasiliensis seeds in various fields.The mechanism of action of the ?-glucosidase from Hevea brasiliensis seeds and fluorescence quencher quercetin was investigated by fluorescence quenching combined with molecular docking;Determing the hydrolysis kinetic parameters of?-glucosidase hydrolyze different types of substrates,combined with molecular docking data,to explore the substrate specificity of the ?-glucosidase from Hevea brasiliensis seeds;pNPG was used as the substrate to determine the mechanical parameters of heat loss activity of ?-glucosidase after adding different concentrations of urea and carbohydrate,and to investigate the influence of urea and carbohydrate on the thermal stability of glycosidase.The primary findings are as follows:The ?-glucosidase structure template obtained by homologous modeling is used to connect docking with quercetin,the result in Ligplot showed that the hydrogen bonding is existed between quercetin and Gln45,His336,Glu467,and Asn468.At the same time,quercetin is located in the hydrophobic cavity of the protein,and there is a hydrophobic interaction between the hydrophobic group of?-glucosidase and quercetin.The contribution of binding energy was analyzed by AutoDock Tools.It was inferred that the interaction process between quercetin and enzyme proteins,van der Waals forces,hydrogen bonding and hydrophobic interactions were the most important forces.Quercetin and ?-glucosidase form complexes,causing static quenching,and ?S0>0 shows that there is hydrophobic interaction during fluorescence quenching.?G0<0,H0<0 illustrates hydrogen bonding and van der Waals forces are exist.The protein structure is complex,and multiple interactions between small molecules and proteins often present at the same time.The results of AutoDock Tools molecular docking and fluorescence quenching are different,but the results of interaction between quercetin and enzyme protein are consistent,reliable analysis of the interaction between enzyme and substrate by Software Simulation.The kinetic parameters of hydrolyzing different types of substrates with ?-glucosidase from Hevea brasiliensis seeds were determined by HPLC,and the Km value was compared.It was found that the affinity of the enzyme to cyanogenic glycosides and isoflavones was greater,followed by flavonoids and phenol glycosides.The ?-glucosidase is relatively weak in the hydrolysis of cello-oligosaccharides.The binding energies and inhibitory constants for molecular docking were obtained in AutoDock Tools,and the order of binding energy was linamarin,daidzin,pNPG,amygdalin,glycitin,genistin,salicin,cellobiose,sucrose,maltose,consistent with the results of Km,salicin is not included.In the low concentration urea,the activity of the ?-glucosidase from Hevea brasiliensis seeds on the substract pNPG be improved,the heat-inactivation half-life(t1/2)of the enzyme is also prolonged,the value of t1/2 extended by 47.62%at 72?.In high concentration urea ?-glucosidase activity decreased significantly,the value of t1/2 reduced by 30%at 72?.With the adding of low concentration urea,the activity and stability of?-glucosidase can be improved,which may be beneficial to the interaction between enzymes and substrate due to changes in protein structure,and the enzyme activity is improved.At the same time,the structure is stable and the stability of heat treatment is improved.By comparing the heat-inactivation half-life of ?-glucosidase after addition of monosaccharides and oligosaccharides,it was found that the inactivation half-life at the extreme temperatures(68,70 and 72?)were extended to varying degrees after the addition of sugars.The protective effect of the thermostability of the enzymes by the three oligosaccharides is clearly better than that of monosaccharides.The results of molecular docking and determination of ?-glucosidase hydrolysis of various substrates by HPLC showed that the affinity between ?-glucosidase and oligosaccharides was much lower than that of pNPG.When the two co-existed,the oligosaccharides formed hydrogen bonds with water in the reaction system.The binding energy of glucose to the enzyme was lower,and the ability of binding to the protein was stronger in the form of hydrogen bond,compared with the result of the docking of glucose,maltose and ?-glucosidase.The protective effect of glucose on ?-glucosidase activity was weaker than that of maltose.