Expression And Characterization Of Feruloyl Esterase In Pichia Pastoris GS115 From Aspergillus Niger

Feruloyl esterase(FAE) is one type of hemicellulose degrading enzymes, which hydrolyzes the ester bonds between the arabinose substitutions and ferulic acid, resulting in releasing of ferulic acid and other functional materia from plant cell walls. It can coordinate with xylanase and ligninase to destroy the dense network structure of the wood fiber, making the cell wall structure becomes loose, thus promoting the degradation of lignocellulose completely. If FAE was used in animal feed, the utilization rate of feed ingredients would be improved and bioactive substances would be produced. The expression and characterization of feruloyl esterase from Aspergillus niger in Pichia pastoris GS115 were studied in this work,and the main research results were as follows:Firstly, serching of feruloyl esterase O42807 from the Uni Prot database was performed. The amino acid sequence of FAE O42807 was converted into fae gene sequence according to the codon bias preference expression of P. pastoris. The gene was synthetized and ligated into p AO815, and then intracellular expression vector p AO815-fae was successfully constructed. The expression plasmid was transformed into P. pastoris GS115 and then verified by PCR. Finally, genetically engineered bacteria GS1115/p AO815-fae was obtained. The expression product of GS115/ p AO815-fae were analyzed by SDS-PAGE. The highest enzyme activity was 2.8 U/L.Secondly, the fae was cloned from the intracellular expression vector p AO815-fae and connected to the extracellular expression vector p PIC9 K. The expression vector p PIC9K-fae was successfully constructed, and transformed into P. pastoris GS115, resulting in obtaining 5 positive transformants. The SDS-PAGE analysis revealed a single band of fermentation supernatant after induced for 4 d, and the apparent molecular weight was 42 k Da. The enzyme generated by one positive transformants exhibited highest activity of 4.71 U/m L with the specific activity of 31.44 U/mg.Thirdly, the single-factor experiment was conducted to optimize the fermentation conditions of recombinant bacteria GS1115/p PIC9K-fae. The results showed that the optimal parameters for inoculum, methanol concentration and induction time were 7.5%, 1% and 3 d, respectively.Fourthly, the enzymatic properties of recombinant FAE(ex-re FAE) were determined and the results showed that: the optimum p H value and temperature was 5.0 and 50? respectively. ex-re FAE was quite stable in the temperature range of 40 ~ 45? and at the p H 6.0. The enzymatic activity was slightly enhanced by K+, Ca2+ and Na+, whereas it was slightly inhibited by Fe2+, Zn2+ and strongly inhibited by Mn2+, Cu2+.Finally, through single-factor and orthogonal experiment, the optimal stabilizer combination consisted of 40% of wheat bran, 3% of PEG400 and 3% of cellulase. At 50?and the presence of the optimal composite stabilizer, residual enzyme activity was highest, it was of 97.3% and increased nearly 20%. At p H 5.5, the highest residual rate of enzymatic activity of enzyme solution in the experimental group was 81.75%, while that was 8.76% more than the control group. The enzyme residual rate of ex-re FAE enzyme liquid added with composite stabilizer was 86.34% after stored at 4? for 20 days, while that of the control group was 67.1%.This work provided the theoretical basis for feruloyl estrase in the development and application of feed and also laid the foundation of subsequent modification for the enzyme.