Molecular Modification Of Trehalose Synthase And Secretory Expression In Pichia Pastoris

Trehalose is a disaccharide linked by two glucose molecules through ?-1,1 glycosidic bonds,it have none-reducing ends and it has strong biological activity.And it has widely used in food and medicine.Trehalose synthase can convert maltose to trehalose in only one step.The reaction can be carried out at low temperature,and the cost is low.In this study,Trehalose synthase(TreS),which delived from Pseudomonas putida P06,was transformed and the covertion rate was improved,the enzyme was expressed it in Pichia pastoris.The results were as follows:(1)The three dimensional structure of TreS was predicted by I-TASSER online server,and AUTODOCK was uesd for the enzyme-substrate flexible docking.The flexible was analyzed by PyMOL,and the amino acids ranging from 4-8 ? in the substrate binding region were selected.We construct a sequence alignment library which contain 500 differents TreS by BLAST.The amino acids in the range of 4-8 ? aera was analyzed and the highly conserved residues were excluded.Eventually,the Asp232 located above the catalytic pocket,the Val407 at the substrate entrance channel and the Lys490 at the bottom of the pocket were selected for mutation.Saturation mutant primers were designed and the mutants library was constructed by using pET-15b-tres plasmid as a template.Three mutants(V407M,K490 L,V407M/K490L)with improved conversion rate were obtained by screening.Its conversion rate were increased by 7.2%,9% and 11.9%,respectively.The enzymatic properties analysis showed that the thermal stability and acid resistance of V407M/K490 L were all enhanced.The reaction kinetic parameters proved that the mutation had a positive influence on the substrate binding capacity and catalytic efficiency.(2)The recombinant expression plasmids pGAP-Z?A-tres3 was constructed by molecular biological method,then it was transferred to Pichia pastoris GS115 by electricity.The recombinant Pichia pastoris GS115/pGAP-Z?A-tres3 was obtained by positive verification and high copy screening.Fermentation results showed that the recombinant intracellular enzyme activity was 120U/g highestly.(3)We have optimized the fermentation medium and fermentation conditions for recombinant Pichia pastoris GS115/ pGAP-Z?A-tres3,in the shake flask level,the intracellular enzyme activity of recombinant yeast was increased by 35%.The optimized medium was used for high density fermentation of recombinant yeast,and we control the growth and metabolism rate by glucose flowing.Fermentation results showed that the highest intracellular enzyme activity was 695 U/g,and the highest extracellular enzyme activity was 29.3 U/mL.