| Traditional fuel ethanol is obtained from corn starch by fermentation with Saccharomyces cerevisiae,which has the problems of competing for food and high energy consumption.In recent years,the shortage of corn supply leads to the problem of raw materials,which seriously restricts the further development of the ethanol industry in our country.The development of industrial or agricultural processing wastes as raw materials for ethanol production will effectively solve these problems.Sweet potato residue(SPR),a by-product of sweet potato starch extraction,still contains considerable amounts of starch and polysaccharides such as cellulose and hemicellulose,and is widely available and inexpensive,with potential as a raw material for fuel ethanol production.In this study,SPR was used as raw material.Firstly,the effect of gelatinization on the yield of reducing sugar was compared when the raw material was pretreated,and it was found that gelatinization significantly improved the yield of reducing sugar.The effects of the proportion of α-amylase,glucoamylase,cellulase,hemicellulose,and pectinase in the mixedenzyme on the yield of reducing sugar were investigated.It was found that increasing the proportion of cellulase,hemicellulose,and pectinase(300 U:300 U:800 U)was beneficial to cellulose degradation.The effects of pH,solid-liquid ratio,mixed enzyme addition amount,temperature,and time of enzymatic hydrolysis on reducing sugar yield were tested.It was found that the optimum pH for one-step enzymatic hydrolysis of SPR was 4.5,the optimum solid-liquid ratio was 1:7,the optimum mixed enzyme addition amount was 0.3 g/10 g SPR,the optimum temperature of enzymatic hydrolysis was 50℃,and the optimum enzymatic hydrolysis time was 6 h.Response surface(P<0.01)optimization was used to study the factors of pH,solid-liquid ratio,and mixed-enzyme addition amount.The optimal enzymatic hydrolysis process of SPR was initial pH 4.56,solid-liquid ratio 1:7,the addition amount of mixed enzyme 0.32 g/10 g SPR,enzymatic hydrolysis temperature 50℃,and enzymatic hydrolysis time 6 h.Under this condition,the yield of reducing sugar reached 65.1%,which was close to the predicted value(64.83%).Compared with the best process combination in the single-factor experiment,the reducing sugar yield was increased by 2.27%.High-performance liquid chromatography was used to analyze the composition of reducing sugar produced by enzymatic hydrolysis.The content of glucose,xylose,and arabinose was 58.9%(w/w),0.09%(w/w),and 0.32%(w/w),respectively.According to scanning electron microscopy and saccharification residue composition analysis,it was found that starch in SPR was completely hydrolyzed,and cellulose was partially hydrolyzed(64.98%).The ethanol fermentation of this hydrolysate using industrial diploid Saccharomyces cerevisiae strain resulted in an ethanol yield of 46.9 g/L within 28 h,corresponding to an ethanol yield of 27.27%.Development of Saccharomyces cerevisiae with saccharification function to reduce enzyme consumption.The codon-optimized glucoamylase gene from Saccharomycopsis fibuligera was optimized and the α-amylase gene from Talaromyces emersonii were optimized.GAP and TEF1 strong promoters were used to drive expression and CYC1 and ADH1 terminators were used to stop transcription.The glucoamylase gene expression frame(GA),α-amylase gene expression frame(temA),and glucoamylase superimposed α-amylase gene expression frame(GA-temA)were integrated into Saccharomyces cerevisiae strain 1974,respectively.Saccharomyces cerevisiae strain 1974-GA integrating 8 copies of the GA gene,Saccharomyces cerevisiae strain 1974-temA integrating 8 copies of the temA gene,and Saccharomyces cerevisiae strain 1974-GA-temA integrating 8 copies of GA and 8 copies of the temA gene were obtained.Using the iodine-starch hydrolysis circle experiment to screen recombinants,it was found that the starch hydrolysis circle diameter of Saccharomyces cerevisiae strain 1974-GA-temA was the largest,indicating that Saccharomyces cerevisiae engineering strains co-expressing α-amylase and glucoamylase had a stronger ability to hydrolyze starch,followed by Saccharomyces cerevisiae strain 1974-temA and Saccharomyces cerevisiae strain 1974-GA was the least capable of hydrolyzing starch.The results of SDS-PAGE showed that α-amylase and glucoamylase expressed by Saccharomyces cerevisiae strain 1974-GA,1974-temA,and 1974-GA-temA recombinants were successfully expressed and secreted extracellularly.The genetic stability of the 1974-GA-temA strain was tested.When the strain was passaged 30 times in the YPD medium,the diameter of the starch hydrolysis circle of the 10 th,20th,and 30 th passages was almost the same,indicating that its starch hydrolysis ability was unchanged.The recombinant α-amylase enzyme activity was0.121 U/m L for Saccharomyces cerevisiae strain 1974-temA,1.36 U/m L for Saccharomyces cerevisiae strain 1974-GA,and 1.47 U/m L for Saccharomyces cerevisiae strain 1974-GAtemA.The enzymatic properties of the recombinant enzymes were examined and the optimum pH for α-amylase was about 4.0,the optimum pH of glucoamylase was about 5.0,and the optimum hydrolysis temperature of α-amylase and glucoamylase was 60℃ ～ 70℃.The ethanol fermentation of soluble starch substrates was performed using recombinant Saccharomyces cerevisiae strain 1974-GA-temA.The ethanol yield of the control Saccharomyces cerevisiae strain 1974-GA and 1974-temA was 28.4 g/L and 33.0 g/L,respectively.The Saccharomyces cerevisiae strain 1974-GA-temA strain produced 42.2 g/L ethanol after 7 days of fermentation,and the starch was completely hydrolyzed.Finally,the successfully constructed Saccharomyces cerevisiae strain 1974-GA-temA was applied to the raw fermentation process of SPR.The optimal process conditions for raw fermentation of SPR were determined by orthogonal optimization and supplementation with exogenous α-amylase or glucoamylase tests,which were solid-liquid ratio 1:6,initial pH 4.0,inoculum volume 10%(v/v),and addition of cellulase(300 U/g SPR),pectinase(800 U/g SPR)and 50% of exogenous glucoamylase(150 U/g SPR).Under this optimal condition,the starch of SPR was completely hydrolyzed after 7 ～ 8 days of fermentation.The ethanol yield reached51.32 g/L,and the corresponding ethanol yield was 30.79%.Finally,the optimal fermentation process of SPR was verified by the amplification test of the 2 L fermenting tank.The ethanol yield was 45.8 g/L after 8 days of fermentation,and the corresponding ethanol yield was 31.7%.In this study,the important raw material sugar production process in the fermented SPR alcohol production process was changed from a two-step enzymatic hydrolysis process to one-step enzymatic hydrolysis,and no corrosive auxiliary sugar reagents such as acid and alkali were added,which provided a convenient method for releasing fermentable sugar from SPR for bioethanol production.At the same time,the Saccharomyces cerevisiae strain 1974-GA-temA constructed in this study realizes the direct utilization of SPR starch without adding exogenous α-amylase and glucoamylase,omitting the liquefaction and saccharification steps in the traditional process,which is conducive to promoting the development of fermentation process and high-density fermentation process while saccharification,improving and optimizing the alcohol production process,and has certain industrial application value. |
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