The Study On Production Optimization,directed Evolution And Bioethanol Application Of Lignocellulase

The work is to turn agricultural waste into treasure and protect the environment,the solid state co-fermentation process of lignocellulase was optimized,and the optimal temperature of lignocellulase was modified in order to solve the production bottleneck of second generation bioethanol.The work provided a technical support for high-level production of lignocellulase and second generation bioethanol,and scientific evidence for molecular modification of lignocellase optimum temperature.The research content and results are as follows:1)Based on the respective enzyme-producing advantages of Penicillium oxalicum 16?Po16?and Trichoderma reesei Rut-C30,agricultural wastes containing rice straw and wheat bran were used as raw materials to ferment lignocellulase under solid state co-fermentation.The production process was optimize by single factor method and response surface method.The yields of FPase,xylanase,amylase,cellobiohydrolase and?-glucosidase?BGL?reached 38.0 IU/gds,352.9 IU/gds,713.2 IU/gds,15.7 IU/gds,and 188.6 IU/gds,respectively.Compared with pre-optimization,those hydrase yields increased by 4.2 times,2.9 times,2.03 times,1.08 times,and 1.96 times,respectively.2)The lignocellulase from 1)was utilized to hydrolyze untreated wheat bran?WB?,pretreated rice straw?pre-RS?,and the mixture of untreated wheat bran and pretreated rice straw?WB+pre-RS?.Second generation bioethanol was produced by isothermal semi-synchronized saccharification fermentation process.Specifically,the dosage of lignocellulase?FPase/g dry matter?was 20 IU/gds,solids loading was10%,and saccharification was carried out at pH 5,45? and 150 rpm for 48 h.The saccharification efficiency of WB was 94.26%,that of pre-RS obtained 42.04%,and that of WB+pre-RS reached 93.23%.Then,Saccharomyces cerevisiae UV-20 was added into the above broth at 35? for 48 h.The ethanol conversion efficiency of WB was 98.41%,that of pre-RS was 67%,and that of WB+pre-RS was 90.8%.3)BGL is a key rate-limiting enzyme for the hydrolysis of lignocellulose.Po16BGL can withstand any concentration of lignin derivatives and furan derivatives,and also has a good tolerance to ethanol,inorganic salts and organic acid salts,which indicates its potential application value in bioethanol production.The inhibitory effect of the inhibitors on Po16BGL is salicin-organic acids>organic acids>salicin-organic sodium salts>organic sodium salts>salicin>salicin-KCl>salicin-NaCl>salicin-ethanol=ethanol.4)To shorten the gap between the optimal fermentation temperature of S.cerevisiae and the optimal temperature of BGL,Po16BGL was the subject in the work and mutated by error-prone PCR method,and then the mutated fragment was integrated into pGAPZ?A-bgl vector to gain mutated BGLs with lower optimal temperature and higher activity according to high-throughout screening of 96-well plates.Compared with the primitive enzyme Po16BGL,the enzyme activity of the mutant Y-1-B₁ was increased to 1.8 times,the optimum temperature of Y-1-B₁ was reduced from 70? to 50?,the K_cat/K_m reached 76.521 mL/mg.min,and catalytic efficiency of Y-1-B₁ was 1.53 times higher.