Study On The Technology Of Trehalose Synthase On The Surface Of Pichia Pastoris

Trehalose synthase is a kind of enzyme conversing maltose into trehalose by one step,its substrate specificity is high,the process of using the enzyme for trehalose production is simple and not affected by the substrate concentration of maltose,it is the first choice for industrial producting trehalose.In order to obtain pichia yeast surface display vector with production of trehalose synthase ability,laboratory screening Pseudomonas putida bacterium Pseudomonas putide P06 and based trehalose synthetic enzyme as a template,PCR amplification by trehalose synthase gene(tres,2064 bp),then connected to the pPICZ?A plasmid and obtain recombinant plasmid pPICZ?A-tres.We enhancing green fluorescent protein gene(egfp,720 bp)from pEGFP-N1 plasmid as the presentation protein,and enhancing Saccharomyces cerevisiae covalently linked cell wall of PIR series protein grains mature Pir1 p peptides as pichia yeast surface displayed anchor protein,pir1p(847 bp)was connected to the recombinant plasmid pPICZ?A-tres and construct pPICZ?A-tresegfp-pir1 p recombinant plasmid.The recombinant plasmid was transferred into Pichia pastoris GS115 by electroporation,and the ?-factor signal peptide was used to direct the foreign protein secretion and anchored to the cell wall,which was displayed on the surface of Pichia pastoris.The positive clones were screened by Zeocin.The fluorescence intensity was detected by flow cytometry,and the expression of exogenous protein was demonstrated to Pichia pastoris,and we basing on the optimization of the fermentation conditions,the expression of exogenous protein was optimized.Pir1p(847 bp)was continued to connected to the recombinant plasmid pPICZ?A-tres,reconstructing to the recombinant plasmid pPICZ?A-tres-pir1 p,and transforming to Pichia pastoris GS115,screening positive clones for fermentation test.Centrifugate fermentation products,crushing,and use the tangle polysaccharide hydrolysis,elution,SDS polyacrylamide gel electrophoresis analysis of visible fusion protein band,indicating that the trehalose synthase has been successfully anchored on the surface of the yeast Pichia pastoris.The recombinant Pichia yeast was washed and suspensioned by pH 7.5 buffer,mixed with the concentration of 300 g / L maltose in 30?to 60? water bath conditions roling 2 h,we can detected enzymatic activity 300.65 U / g by HPLC.In the optimized conditions of enzymatic reaction after pH 7.5,50?,the surface of trehalose synthase activity was 600.65 U/g.The enzyme activity was stable between 40? and 50?,and the relative activity of the residual enzyme activity was up to 75% or more after heat preservation 60 min,and the optimum reaction pH was 7.5,and it was stable in alkaline environment.