Study On Biological Degradation Of Corn Stalk And Improving Thermostability Of The Feruloyl Esterase

Feruloyl esterase can interrupt the ester linkages among cellulose,hemicellulose and lignin and break the complex network structure of plant cell walls,so that other fiber-degrading enzymes can be easier act on substrate.It is an important hydrolytic enzyme in industrial production.In this thesis,we first carried out degradation of corn stalk by microbial method,and then directional modification of feruloyl esterase A to improve its heat resistance.1.The fermentation temperature,pH,the ratio of solid to liquid and the total inoculation amount were optimized by single factor experiment,and the orthogonal experiment was then carried out based on the single factor experiment.The optimum pattern validated by orthogonal experimental design for corn straw fermentation was A2B1C2D3,with fermentation temperature 30 ?,pH 6.0,solid-liquid ratio 1: 9,inoculation amount 30.0%,and the final reducing sugar production was 14.509 mg/m L.The lignin,hemicellulose and cellulose contents of the treated corn straw by mixed bacteria liquid were 11.0%,22.1% and 32.2%;degradation rates were 31.2%,12.2% and 21.9%,respectively.2.Fermentation temperature,pH,solid to liquid ratio and the amount of enzymes were optimized by single factor experiment,and the orthogonal experiment was carried out based on the single factor experiment.It is proved that the optimum pattern for enzyme synergistic fermentation of corn straw was A2B3C1D2,with fermentation temperature 50?,pH 6.0,solid-liquid ratio 1:7 and the dosage of feruloyl esterase A 5g,and the final reducing sugar production was 30.768 mg/m L.Based on range analysis,it can be seen that the effect on reducing sugar production from large to small in turn was temperature,solid-liquid ratio,pH and the amount of feruloyl esterase A.The lignin,hemicellulose and cellulose contents of the corn straw after multi-enzyme treatment were 7.0%,19.5% and 21.2%;degradation rates were 56.3%,22.0% and 48.3%,respectively.Treatment of corn straw with multi-enzymes showed better effect on reducing sugar concentration and degradation of cellulose,hemicellulose and lignin than those of mixed bacteria.3.The gene of feruloyl esterase A was cloned from the recombinantional Pichia pastoris GS115 by PCR technique.The 161 th amino acid of the feruloyl esterase A was mutanted by the overlap extension PCR technique.Subsequently the pPIC9 K expression vector was linked with the mutated gene,and the constructed pPIC9K-FAE expression vector was linearized by restriction endonuclease Sac I.The linearized plasmid pPIC9K-FAE was then transformed into Pichia pastoris GS115.Through the induction by methanol,the final enzyme activity was 22.31 U/m L,which was 9.7% lower than that of the original recombinant feruloyl esterase A.After determination,the optimum temperature of 46? for the mutated enzymes,lower than the original recombinant feruloyl esterase A was 4?,thermal stability was also not as good as the original esterase.