Construction Of Recombinant Industrial S. Cerevisiae Strain With β-glucanase Activity And PrA Low Expression By Homologous Recombination

Beta-glucans could reduce the extract yields in the brewhouse,affect filtration performance and participate in haze formation in wort and beer.Commercialβ-glucanase was added into the wort and beer during the fermentation to eliminate the impact ofβ-glucan.Yeast proteinase A (PrA),a proteolytic enzyme found in the vacuole of Saccharomyces cerevisiae,can be excreted into beer under stress or by yeast autolysis and is detrimental to beer foam stability.PrA inhibitor was used in brewhouse to reduce the negative impact of PrA on foam active-protein.To resolve the problems caused byβ-glucan and PrA in beer production,we constructed the recombinant S.cerevisiae strain with one of the PEP4 gene was replaced by bg1S gene from B.subtilis.Activelyβ-glucanase secreting by the recombinant strains was detected and the PrA was lower than the control strain.The fermentation property of the recombinants was test and show near to the control.Some of the process and results showed as follow:The bg1S gene encoding endo-1,3-1,4-β-glucanase from Bacillus subtilis mutant ZJF-1A5 was cloned.The bg1S expression cassette including PGK1 promoter,bg1S gene fused to MFα1 signal peptide sequence,and ADH1 terminator,with G418-resistence as the selection marker,was constructed in the shuttle vector Yeplac-181.A notableβ-glucanase activity was detected in the culture with DNS method when the recombinants,obtained after the expression vector Yeplac-181 was transformed into the industrial S.cerevisiae strain WZ65,were incubated.The maximum activity was detected after 60h of incubation in the YEPD yeast culture.With the modified Congo-red method the enzyme Properties of theβ-glucanase secreted by the recombinant yeast was tested and showed a variation contrast to the initial enzyme of the B.subtilis.There is an obvious change in the optimal temperature and heat-stability of the recombinant enzyme contrast to the native enzyme.Changing in temperature lead to a significant reduction of the active enzyme in a relatively low temperature,below 40℃.There was a small impact to the reduction of the activity by raising the temperature when it over 40℃,The optimal pH of the recombinant enzyme was lower to 5.0 contrasts to 6.0-7.0 of the native.To enhance the expression stability of theβ-glucanase by the recombinants,the bg1S expression cassette was integrated on the S.cerevisiae chromosome after replacing one of the PEP4 gene allele and resulting recombinant SC-βG1 and SC-βG2 with the gene type,PEP4/pep4∷KanMX-bg1S.The maximumβ-glucanase activity of SC-βG1 and SC-βG2 was between 56-64h of the incubation course,almost the same time point mentioned above.The recombinant strains, SC-βG1 and SC-βG2,exibit an unobvious feeble in growth capability but had the same ultimate cell concentration to the control.To make a considering safely yeast strain of the recombinant we have constructed,the KanMX,working as the resistance marker,was deleted under the action of Cre/loxP system constructed in the plasmid pSH-Z.Two strains with the bglS cassette preserved and the plasmids pSH-Z missing were screened during the subculture,were named SC-βG1A and SC-βG1B respectively.Experiment result showed that the recombinant strains SC-βG1A and SC-βG1B remained theβ-glucanase activity.The physiological characteristics of SC-βG1A and SC-βG1B were tested including genetic stability,growing performance,fermenting ability,flocculation ability,heat-resistance temperature, etc.The activity of proteinase A andβ-glucanase was also measured.It is shown there is a lower proteinase A activity,contract to the very initial strain WZ65,and notableβ-glucanase activity in the ultimate recombinant strains SC-βG1A and SC-βG1B.The genetic performance was stable and the fermenting ability,flocculation ability of these recombinants was similar to the control strain except for an unobvious slowly starting growth and a slight lower heat-resistance temperature.Saccharomyces cerevisiae SC-βG1A was chosed to brewing beer in a 250L fermentation tank. The groth performance and fermenting property were tested and physic-chemical indexes were measured including concentration,turbidness,colourity,bitterness value,pH,α-N,total acidity, alcohol by volume and by weight,attenuation degree,apparent sugar concentration,bi-acetyl, head retention,CO₂ and flavor substances were also detected.The results indicated that the flavour of the beer brewing with the recombinant sacchramosasice cerevisiae strain similar to the current dry beer.Every of the physic-chemical indexe was reached superior grade according to the national standard,especially the head retention time was enhangced from 202s to 274s.Further more investigation was needed to test the brewing performance of the next generations of the recombinant Saccharomyces cerevisiae SC-βG1A.