Rational Desigh Of ?-galactosidase Based On Computer Dynamic

?-galactosidase can be used to hydrolyze lactose during milk processing,solving the lactose intolerance;P-galactosidase also can be taken use of producing low poly galactose,which was important for functional food and food additives.Thennostable enzymes not only greatly reduced the risk of microbial contamination,but also catalyzed reaction ?very quickly,with high initial reaction rate and substrate concentration.Good thermostability was an improtant characteristics for enzymes.Now,studies on P-galactosidase with high thermostability and catalytic activity were rare.This study was on the basis of the amino acid sequence,providing theoretical proof for the reasonable design by computer simulation.Firstly,Several kinds of bioinformatic online servers,including Protparam,ProtScal,SignalP,TMHMM,SOPMA,were used to carry out bioinformatic analysis of the thermostable P-galactosidase from Bacillus coagulans T-242.Then,three amino acids side chains with high scores as templates through the searching of procedure BLAST.The sequence of enzymatic amino acides chain and templates were contrast with software CLUSTALX and then the alignment result was submitted to SWISS-MODEL to predict the spatial structure of ?-galactosidase by homology-modelling.At last the quality of that three models were evaluated by online server.Results wed that the thermostable?-galactosidase was composed of 665 amino acids residues.The molecular weight was 76.09 KDa,and its formula was C345-H5221N907O988S27.total number of atoms was 10594,the theory of isoelectric point was 6.06 without signal peptide and transmembrane region.The secondary structure was mainly composed of ?-Helix and Random coli.The spatial structure of the ?-galactosidase was homo-trimer.The best quality could be obtained when the template of model was 3TTS and reliability was higher.This model could be a foundation of the rational molecular modification of the P-galactosidase.Pymol could be used to compare and find the active region of that T-242 thermostable ?-galactosidase,which could help with the molecular docking in further.At last,the molecular weight of the single chain was proved to be 78 kDa,which was close to the value of predicted by online server.According to Native-PAGE,the molecular weight of enzyme with catalytic activity could be known,which proving this lactase was homo-trimer.Then,software AutoDock was used to carry out molecular docking between thermostable ?-galactosidase and ONPG.The docking result was used to find out all the amino acids less than 5A away the substrate,excluding the conservative ones,mutating the hydrophilic amino acid into the hydrophbic one of the same property.In that way,the binding pocket can be enlarged,affinity of the lactase can be promoted.Mutating Gln 314 to Gly,Cys,Ser separately,and naming them as mutating enzyme Lac 1,Lac 2,Lac 3.The result showed that three mutants' affinity with substrate was increased.Among them,mutated enzyme Lac3 was the best,and the combination energy was reduce from-5.49kJ to-6.76kJ.At last,software GROMACS was used for molecular dynamic simulation,simulating temperature was 350K(77 ?).Mutating amino acids of high RMSF to Pro,analyzing thermostability before and after mutation according to the RMSD and gyrate value.Mutating Lys 3 to Pro 3,naming as Lac a;mutating Lys 3,Asp 101,Glu 6 to Pro,naming as Lac b.Molecular dynamic simulation was carried out then.The result showed that both mutated enzymes,thermostability were promoted,Lac b was better,RMSD was reduced from 0.267 nm to 0.221nm;And gyrate was reduced from 3.85 nm to 3.0nm.