Efficient Display Of ?-Glucosidase On Saccharomyces Cerevisiae Cell Wall For Aroma Enhancement In Wine

It is well known that most of the terpenoids in wine exist in the form of bound glycosides,which are not easily released.?-glucosidase(BGL)is essential in the hydrolysis of glycosides,while its activity is always inhibited by the factors such as high glucose,ethanol and low pH during the winemaking process.Moreover,the disadvantages of poor stability of commercial enzyme preparations and high production costs of immobilized enzymes limit the use of BGL in the winemaking process.In recent years,cell surface-display technique of Saccharomyces cerevisiae,as as a whole-cell biocatalyst,has been considered as one of the most promising methods.By displaying the enzyme on the cell surface,the activity and stability of enzyme could be significantly enhanced,and it does not need to consume the immobilized material,which makes it reusable,providing a new solution for the application of BGL in improving the wine aroma.In our study,BGL derived from Aspergillus niger was displayed on the surface of Saccharomyces cerevisiae cells,and different vectors,promoters,anchoring proteins and translational fusion partners(TFPs)were used to obtain an efficient and stable display of BGL.In addition,the enzymatic properties and the capacity of surface-displayed BGL in hydrolyzing aroma glycosides during fermentation were investigated.The main research contents and results are as follows:(1)The green fluorescent protein(GFP)was used as the target protein,and was fused with 3 anchoring proteins,including Sag1 p,Sed1p and Cwp2 p,which were inserted into the vector pYES2/CT/?-Factor containing a inducible promoter Gal1 using In-fusion technology.Then,the location of GFP was observed using laser confocal microscopy.The results showed that GFP expressed by the positive control strain PBy-eG was present in the cytoplasm,while in the experimental strains like PBy-eGSA?PBy-eGSE?PBy-eGCW,GFP had a tendency to be secreted to the outside of the cell,meanwhile no free GFP was detected,confirming that the Saccharomyces cerevisiae surface display platform has been successfully constructed.(2)The promoter GAL1 in the three GFP-anchoring vectors constructed in chapter 2 was replaced with the promoters of GPD and SED1,respectively,and the gfp gene was replaced by ?-glucosidase gene(Bgl1),thus the high-copy plasmids displaying BGL on the surface were successfully constructed and then electroporated into the host strain BY4741 for expression.Results showed that the enzyme activities displayed by the six recombinant strains all decreased with the prolongation of the culture time,among which the enzyme activities of the positive recombinant strain driven by the GPD promoter were higher than that of the SED1 promoter.As for the three anchoring proteins(Sag1p,Sed1 p,Cwp2p),Sag1 p performed best.Therefore,the recombinant strain PBy-GBSa carrying the plasmid GPD-HQM-Sag1 was able to display BGL more efficiently.When it was cultured for 24 h,the enzyme activity reached a maximum of 18.29±1.05 U/g.(3)The yeast integrative plasmids Yip-loxp-URA3 with HIS3 as the recombination arm and URA3 as the selection marker was constructed,and then the six surface-display BGL cassettes in chapter 3 were inserted into the integrative plasmids,thus the surface-displayed BGL integrative plasmids were successfully constructed.qRT-PCR and enzyme activity assay were performed to monitor the BGL expression at both transcription and protein levels during the fermentation process.It was found that the transcription level of the SED1 promoter was higher than that of the GPD promoter,while at the protein expression level,the enzyme activities displayed by the six recombinant strains all gradually increased with the prolongation of the culture time,which reached the maximum at 84 h.The enzyme activities of the positive recombinant strains driven by GPD promoter were higher than that of the SED1 promoter,which reached the maximum(25.22±0.81 U/g)when sed1 p was used as anchoring protein.(4)The MFalpha1 TFP in the plasmid Yip-GPD-BGL-Sed1 of the positive recombinant strain BY-GSE in chapter 4 was replaced with 23 different TFPs derived from Saccharomyces cerevisiae,and effects of TFPs on the BGL activities were investigated.The results showed that all the strains with different TFPs could successfully display BGL on the yeast cell surface,among which strains 13-HSP150?17-SED1(195 aa)?23-CCW14 and 24-SED1(170 aa)had higher BGL activities than the original strain BY-GSE,and activity of BGL in the strain with 17-SED1(195 aa)(74.58±5.88 U/g)was 3 times higher than that of the control.(5)The stability of surface-displayed BGL under different conditions(different concentrations of glucose,ethanol and pH)and its capacity of hydrolyzing glycosidic bonds during the fermentation process were investigated,using commercial enzyme preparation AR2000 as control.It was found that,the surface-display BGL was less inhibited by glucose less than 5%,and still had 12% residual activity under 5% glucose,while the free enzyme was completely inhibited.At pH 3.0,the residual activity of surface-displayed BGL was about 50%,which was more than three times higher than that of free enzyme(only about 15%).Ethanol had an inhibitory effect on the surface-displayed BGL and the commercial enzyme preparation AR2000,and the inhibition trend was similar.At 20%(v/v)ethanol concentration,more than 50% residual activity was retained.The concentration of terpenols released by the surface-displayed BGL was 2.1 times that of the commercial enzyme preparation AR2000(37.58±1.16 ?g/L).