Construction Of High-yield ?-glucosidase Engineered Strain And Research Of The Effect On Ethanol Production By Saccharomyces Cerevisiae WXY12

Lignocellulosic biomass resources are renewable,rich but low utilization resources in the world.Corn stalk is one of the important sources of lignocellulose,it consists of lignin,cellulose and hemicellulose.Cellulose and hemicellulose are composed of carbohydrate.So how to convert cellulose and hemicellulose efficiently becomes the key problem of utilizing stalk resource.Convert waste corn stalk into ethanol effectively,not only solve the energy crisis but also reduce environmental pollution.Endocellulase,exocellulase and?-glucosidase convert the cellulose into glucose during the hydrolysis mainly.However,the enzyme component of cellulase is unbalanced,?-glucoside enzyme activity was low generally,thus leading to the accumulation of cellobiose.Cellobiose is an inhibitor of other enzymes in the cellulase system,it inhibits the entire hydrolysis process of the cellulose.Increasing the activity and content of?-glucosidase in the hydrolysis system can degrade the cellobiose into glucose,so the inhibition from cellobiose is alleviated,and the hydrolysis of cellulose was accelerated.That promotes the saccharification and utilization of corn stalk.In this study,a strain of Aspergillus niger with rapid growth and high?-glucosidase production was obtained by screening and mutagenesis,its?-glucosidase gene sequence was cloned,constitutive high expression?-glucosidase multi-copy S.cerevisiae engineered strain was constructed by rDNA integration method,and the copy number of the engineered strains was identified by digital drop PCR?dd-PCR?technique.Fermentation of glucose and xylose in pretreated corn stover hydrolysate by high-efficiency ethanol-producing S.cerevisiae WXY12,which can utilize both glucose and xylose at the same time,then increase ethanol production by optimizing fermentation conditions.The main results are as follows:1.P-nitrophenol-glucoside?p-NPG?as the sole carbon source for screening strains with high?-glucosidase production,then a strain with high?-glucosidase production was isolated from nature.The strain was identified as Aspergillus niger by biology.In order to improve the?-glucosidase activity further,the Aspergillus niger was mutagenized by ARTP mutagenesis technology,then a mutant library of Aspergillus niger strain was successfully constructed and a good genetic stability mutant was screened from it.The mutant strain reached the log phase and the peak of enzyme production for 3 days earlier than the wild type strain.The wild type reached the peak of enzyme production on the 12th day,the enzyme activity was 77 IU/mL;the mutant reached the peak of enzyme production on the 9th day,the enzyme activity was 104.31 IU/mL;the mutant strain produced the enzyme more quickly and the enzyme activity increased 35.47%.2.The?-glucosidase gene sequences of the wild type strain and the mutant strain of Aspergillus niger were analyzed for the difference,6 base mutations were detected;and two amino acid mutations were detected by protein sequence difference analysis.The mutant?-glucosidase gene was combined with the promoter PGK,the secretion signal peptide?-factor,and the terminator CYC1 to obtain an expression cassette-P?BC,then the pYES2 plasmid and the rDNA core sequence were ligated to the expression cassette-P?BC to construct pYES2-P?BC-rDNA expression vector.Then transformed the pYES2-P?BC-rDNA expression vector into S.cerevisiae INVSc1,the multi-copy?-glucosidase S.cerevisiae engineering strain was constructed by rDNA integration method.It was hoped that the expression of?-glucosidase protein can be increased by the introduction of a promoter,a secreted signal peptide,and an increasing in gene dose.A total of 23positive transformants were obtained by preliminary screening of SC-U auxotrophic medium and secondary screening of bacterial liquid PCR.3.The copies number of positive transformants was determined by digital droplet PCR?dd-PCR?assay.The ATC1 gene was used as the internal reference gene.The detected copy number of?-glucosidase gene sequence were as follows:1,2,3,5,6,9,10,12,13 and 15.The relationship between the copy number and the expression level of?-glucosidase was analyzed:when the copy number was 10,the?-glucosidase activity was the highest?29.8 IU/mL?,when the copy number exceeded 10,the?-glucosidase activity of transformant activity decreased.The stability of the transformant?-glucosidase were verified,the result showed the transformants were stable in genetic.4.It was found that ammonium sulfate with 60%saturation had the best purification effect on?-glucosidase in the fermentation broth of S.cerevisiae,its apparent molecular weight was about 60kDa by SDS-PAGE.Characterization of the enzymatic properties of?-glucosidase showed that the optimum temperature was 55?,it was relatively stable at 40-55?;the optimum pH is 5.5,relatively stable in a slightly acidic environment at pH5.0-6.0;Mn²⁺,Fe²⁺,and Zn²⁺have significant synergistic effects on?-glucosidase activity,while Ag⁺and Cu²⁺have significant inhibitory effects on?-glucosidase activity.5.The corn stalk was pretreated,and the content of lignocellulose in the corn stalk was determined before and after pretreatment with the different pretreated method according to the National Energy Laboratory's standardized method?NREL?.Through the analysis of enzymatic saccharification experiments,we found that the addition of?-glucosidase to the enzymatic hydrolysis system significantly increased the glucose content?21.53%?in the pretreated corn stalk hydrolysate.By optimizing the pretreatment conditions,the optimum conditions to pretreated corn stalk with 2%Na2CO3+2%H2O2 were 70 min,130°C,and the ratio of material to liquid was 1:10.The results of scanning electron microscopy showed that the pretreatment made the surface of corn stalk from the original smooth and orderly structure rough and messy.The X-ray diffraction results showed that the crystallinity of corn stalk was decreased after pretreatment.The result of the Fourier infrared spectrum showed the absorption peak of lignin was attenuated,the absorption peaks of cellulose and hemicellulose were enhanced.The results showed that the pretreatment method could reserve the cellulose and hemicellulose components well and removed the lignin fraction effectively.6.Investigated the optimal addition ratio of endocellulase,exocellulase,?-glucosidase and xylanase during the process of pretreatment corn stalk with saccharification and enzymatic hydrolysis:the yield of monosaccharide was the best when the ratio was 3:2:3:4.It means that in the process of fermentation,when 15 IU of endocellulase,10 IU of exocellulase,15 IU of?-glucosidase were added per gram of cellulose raw material and 20 IU of xylanase was added per gram of hemicellulose raw material,the effect of enzymatic saccharification of pretreated corn stalk was the best.7.Selected high-efficiency ethanol producing Saccharomyces cerevisiae WXY12 that can utilize xylose and glucose simultaneously,the glucose and xylose in the pretreated corn stalk hydrolysate were fermentation by simultaneous saccharification and fermentation to improve the stalk utilization and the ethanol production.The ethanol concentration was determined by gas chromatography,single factor method and response surface analysis method were used to optimal conditions for ethanol production by fermentation:nitrogen source 15 g/L,metal ion 3 g/L,initial pH5.98,liquid volume 120 mL/250 mL,temperature 28°C,speed 139 r/min,the ratio of solid to liquid was 1:12,inoculation amount 1.49%.It showed the highest ethanol yield in this condition,the concentration was 46.87 g/L?...