Study On The Mechanism Of Indigenous Saccharomyces Cerevisiae High Yielding ?-d-glucosidase

The?-D-glucosidase is a kind of hydrolase which can increase the aroma of wine,and is widely found in various plant cells and microorganisms.At present,the?-D-glucosidase produced by Aspergillus niger is widely used and studied,while the production of?-D-glucosidase of Saccharomyces cerevisiae is less.The addition of foreign?-D-glucosidase during wine-making can lead to instability of the wine,and Saccharomyces cerevisiae,which produces?-D-glucosidase,is used as a wine fermentative bacterium,not only to complete the fermentation process,but also to achieve the effect of increasing the aroma of wine.So more and more attention has been paid to the study of Saccharomyces cerevisiae producing?-D-glucosidase.In this study,the growth,enzyme production,enzymatic properties and wine-making properties of high-yielding?-D-glucosidase KDLYS9-16 were studied,which provided data support for the industrial application of this strain.First,the relationship between KDLYS9-16 cell growth and cellulase production is studied,and the optimal conditions of cell growth and cellulase production were optimized,and the transformation point of cell growth and enzyme production was found to provide the basis production of enzymes.The effects of cell growth and cellulase production conditions,cell growth and cellulase production and metal on the growth and cellulase production were optimized and studied by using orthogonal optimization method and mathematical modeling method.The results showed that KDLYS9-16 cell growth and cellulase production is semi-coupling relationship,and there is a coupling relationship between cell growth and enzyme production at a specific time with.The growth rate of Saccharomyces cerevisiae was the fastest was cultured in the medium that 1.0%yeast extract,2.0%peptone and 2.0%glucose were used as media components when the culture temperature was 30?,pH 5.0 and dissolved oxygen amount 9.365 mg/L.The enzyme production is the largest when the medium composition was 1.0%yeast powder,2.0%glucose,%MgCl₂,0.5%CaCl₂,and 0.5%BaCl₂,and the optimum conditions were culture temperature 30?,culture medium pH 5.0 and dissolved oxygen 9.365 mg/L.The Ca²⁺,Mg²⁺,Ba²⁺,Na⁺and K⁺could promote the production of the enzyme,in which Ca²⁺,Mg²⁺and Ba²⁺could increase the production of enzyme by 35.5%,27.2%and 38.3%.The other metal elements had inhibitory effects on the enzyme production,and the enzyme activity was not detected when the CO²⁺,Cu²⁺and Fe²⁺were added in the fermentation broth of KDLYS9-16 S.cerevisiae,indicating that these three metal ions completely inhibited the production of Enzyme.Vitamin C and tryptophan can promote the production of the enzyme,and the promotion was more significant when the amount of added 2.5%and2.0%,respectively.The 2.5%of vitamin C and 2.0%of tryptophan were added during the lag phase?start of fermentation?,and the 0.2%Na⁺,1.0%Mg²⁺,0.2%Mg²⁺,0.5%Ca²⁺and 0.5%Ba²⁺were added at the beginning of the logarithmic phase?8¹0 h?.The pH of medium was adjusted to 5.0,and the temperature was 30?,and the dissolved oxygen was 9.365 mg/L.The enzyme activity was increased to 0.046818 U/mL.The effects of cell growth,The kinetic relationship between the enzyme production and the enzyme production were described,and the dynamic relationship model was established.Secondly,the?-D-glucosidase produced by Saccharomyces cerevisiae KDLYS9-16was isolated and purified,and the physicochemical properties and catalytic properties of the enzyme were studied.The purified?-D-glucosidase was purified by Sephadex G-150gel column chromatography.The molecular weight of the protein was determined by SDS-polyacrylamide gel electrophoresis.K_m and V_maxax were determined by the reciprocal method?L-B plotting method?.The reaction substrate with different chemical bonds was selected to select the optimal hydrolysis chemical bond of the enzyme.The experimental design and data analysis were carried out using the Design Expert 7.0 statistical software,According to BoxBenhnken center combination experiment design principle,the reaction conditions of?-D-glucosidase produced by KDLYS9-16 Saccharomyces cerevisiae were optimized.The results showed that the purified?-D-glucosidase was 272.748 times more purified than the original fermentation broth,the specific activity of the enzyme was0.98462 U/mg,the enzyme activity was 0.06529 U/mL.After SDS-polyacrylamide Gel electrophoresis.The results showed that the purified enzyme had only one band,which indicated that the purified?-D-glucosidase had a molecular weight of 78 kD and the?-D-glucoside produced by KDLYS9-16 The kinetics parameters of the enzyme were K_m=8.808038627 mmol/L,V_max=1.304971943 U/?L·min?;In addition,the isoelectric point of?-D-glucosidase produced by KDLYS9-16 was in the range of 4.5⁴.8,The enzyme activity was maintained above 60%in the range of-20⁴0?,and remained above 40%after 120 min in the pH range of 3⁷.The activity of nitrobenzene?-D-glucoside?pNPG?for the best substrate,indicating that KDLYS9-16 produced by?-D-glucosidase of Saccharomyces cerevisiae hydrolysis?-1,4 glycosidic bond;when the reaction system buffer solution pH value of 5.10,The reaction temperature was 48?,the substrate concentration was 41.4 mmol/L,and the enzyme activity was 0.0170033 U/mL.Thirdly,using the commercial bacteria as the control,the fermentation characteristics of the enzyme producing bacteria were studied by simulating the grape juice and wine grape juice.The results showed that KDLYS9-16 could quickly adapt to the fermentation conditions when the fermentation medium was used as the fermentation substrate.The fermenting time was short after 144 h,while the control bacteria SC and F15 had 8 h adaptation time.The fermentation time was longer than that of the self-selected bacterium,168 hours,and the alcoholicity of the KDLYS9-16 fermentation simulated juice was more than 12°,while the SC of the control strain SC was between 11.5¹2.0°,The fermentation temperature of the strain F15 was lower than that of the commercial strain F15,which was lower than 11.5°.The fermented wine KDLYS9-16 still showed the advantage of quick fermentation,rapid fermentation and short fermenting period.And the control bacteria SC and F15 were almost in the adaptation stage from 0 to 24 h.After fermentation,the ethanol content of KDLYS9-16 fermented wine was significantly higher than that of the control bacteria,especially in Changli Mei The fermenting wine of Le grape was 13.2°,while that of the control bacteria was only 12¹2.5°.The alcohol conversion rate of the selected bacteria was 96%,while that of the control bacteria was only about 90%.In general,the curves of CO₂ generation,residual sugar curve and alcohol content were compared with those of sugar conversion rate,which indicated that KDLYS9-16 had the advantages of fast fermentation start,short fermenting period,high alcohol production and fast fermentation.Sugar conversion of alcohol high.Finally,the wine grape was used as raw material and commercial bacteria as control.The wine-producing experiment was carried out to evaluate the wine-producing and flavor-producing characteristics.The wine was sealed and stored in 10¹5?environment after fermentation.The contents of monoterpenes and total aroma components in the wine were detected by GC and GC-MS respectively after 30 days of fermentation.The results showed that a total of 13 monoterpenes were detected,and 11monoterpene substances were detected in the Cabernet Sauvignon and Cabernet Sauvignon wines with the concentration of 2.454 and 2.855 mg/L,while the content of monoterpene was 0.765¹.416 mg/L.The results showed that KDLYS9-16 could not only maintain the original monoterpenes in the grape,but also could produce new,While the control bacteria not only did not produce new monoterpenoids,but also difficult to retain the original monoterpenes of grape;the choice of bacteria KDLYS9-16 fermentation of the original wine is not only a variety of aroma,and the content should be obvious The aroma components of Changli Cabernet Sauvignon wine were the highest,reaching 98.94%,while the aroma substances in the control bacteria wine were generally lower than 30Species,the total content of less than 90%.In general,compared with the control bacteria,KDLYS9-16 fermented wine has more fragrant,monoterpene content is higher,more wine has a positive impact.In conclusion,the growth and enzyme production of KDLYS9-16 were favorable for cell growth and enzyme production.The?-D-glucosidase could be used in the low temperature and low pH of the wine-making process.The fermented experiment shows that the strain has the advantages of quick fermentation start,short fermentation cycle,short fermentation cycle,high alcohol production rate and high alcohol conversion rate.The aroma substances in the fermented wine have the advantages of good heat and acid-base stability.And monoterpene substances content and type are far higher than the control bacteria,has obvious advantages of producing fragrance.In summary,the choice of Saccharomyces cerevisiae KDLYS9-16 can be used for industrial promotion.