Optimization Of A Secretory Expressed Feruloyl Esterase In Bacillus Subtilis

Feruloyl esterase belongs toα/βA subclass of hydrolase can hydrolyze the ester bond between ferulic acid and lignocellulose components in plant cell wall.Its synergistic effect with cellulase can improve the hydrolysis efficiency of lignocellulose,so it is widely used in papermaking,feed,fuel ethanol and other industries.In addition,ferulic acid produced by ferulic acid esterase hydrolysis can stabilize the free radicals produced by metabolic activities or chemical reactions,and has a strong inhibitory effect on a variety of microorganisms.It has been approved by many countries as a food or pharmaceutical additive,with high application value and broad market prospects.This study used molecular biology,protein structure analysis,and response surface optimization methods to heterologous express the codon optimized feruloyl esterase gene in Bacillus subtilis.After screening of signal peptides,rational structural design and solid-state fermentation significantly improved the secretion of feruloyl esterase.The specific research methods and contents are as follows:1.Heterologous expression of feruloyl esterase.After literature search,a prokaryotic feruloyl enzyme with high enzyme activity was selected,and a codon optimization gene(FAE_bac)for Bacillus subtilis was obtained by gene synthesis based on its protein sequence.Using restriction endonucleases HindⅢand NdeⅠ,they were digested with E.coli expression plasmids p ET28a and B.subtilis secretory expression plasmid p BE-s was linked.Using chemical transformation methods,p ET28a-FAE_bac and p BEs-FAE_bac were transferred into corresponding host cells and expressed in E.coli BL21 and B.subtilis RIK1285.After protein extraction and purification,the enzymatic parameters of wild type feruloyl esterase were determined.The optimal p H and temperature for wild-type FAE_bac are 7.0 and 45℃,respectively.At 60℃and 70℃,about 35%and 17%of the remaining activity can be retained respectively;After incubating for one hour at p H 4.0 and 9.0,the remaining activity was retained at approximately 42%and 55%,respectively.The initial activity of feruloyl esterase in the fermentation broth of B.subtilis RIK1285 was 235 m U/m L.2.Screening of the best signal peptide of feruloyl esterase.In order to improve FAE_bac’s performance in B.subtilis,restriction endonucleases Mlu I and Eag I were used to double cleave the vector plasmid p BEs-FAE_bac to obtain a plasmid fragment that did not contain SPapr E.After the above plasmid fragments were recovered,173DNA fragments encoding different signal peptides were replaced into the signal peptide coding region by In-Fusion cloning to prepare plasmid libraries containing different signal peptides.The signal peptide plasmid library was transformed into E.coli DH5αreceptive cells.After antibiotic screening,the transformant colonies on the plate were washed with sterile water,and the extracted plasmid was electrical transformed to B.subtilis.Pick the 1000 B.subtilis transformants were cultured in 96-well plates,and five strains with relatively high enzyme activity were obtained through screening.The enzyme activity ranged from 1430 m U/m L to 2260 m U/m L,with the highest being 10.2 times of the initial enzyme activity.The five plasmids were extracted and sequenced.After comparison,it was determined that the signal peptides that had a good effect on the secretion and expression of ferulic acid esterase were SP_{Cit H},SP_{Asp B},SP_{Rpm G},SP_{Ykw D},and SP_{Lyt B}.3.Design and enzymatic detection of feruloyl esterase mutants.The first 100protein amino acid sequences with high similarity to the target feruloyl esterase were extracted from the NCBI non-redundant database with 10-4 as the threshold.After comparison,the above 100 protein sequences were obtained.Taking the wild type feruloyl esterase gene as a reference,the changes of amino acid residues in the consensus sequence that promote protein folding were predicted.Two ferulic acid esterase mutants,FAE_bac1A85 and FAE_bacD2,were designed on the basis that the substrate catalysis,binding region and amino acid residues at the protein dimerization interface remained unchanged.After the two were induced and expressed in E.coli BL21,the mutant protein was extracted and purified,and the enzymatic properties of the two were determined.Compared with wild-type FAE_bac,their optimal catalytic p H remained stable,meanwhile their tolerance to acid and alkali was improved.After pretreatment at p H 9.0 for 1 hour,the residual enzyme activities of mutant FAE_bac1A85 and FAE_bacD2 increased 1.18 and 1.16 times respectively;The residual enzyme activity of FAE_bacD2 increased by 1.24 times at p H 4.0.In addition,the tolerance of the two mutants to temperature was also improved.After pretreatment at60℃for 1 hour,the residual enzyme activities of the mutants FAE_bac1A85 and FAE_bacD2 increased by 1.14 and 1.23 times respectively;At 70℃,the residual enzyme activity of the two increased 1.35 times and 1.47 times respectively.4.Optimization of solid state fermentation conditions of feruloyl esterase in Bacillus.The best carbon source and nitrogen source for the production of ferulic acid esterase by Bacillus in solid state fermentation were determined by single factor experiment.With the basic formula（5 g/L yeast extract,10 g/L tryptone and 10 g/L Na Cl）as the control,five carbon sources were selected:corn starch（10.2 g/L）,potato starch（10 g/L）,glucose（11 g/L）,sucrose（9.5 g/L）,mannitol（7.610 g/L）;Five nitrogen sources:industrial yeast extract（7.7 g/L）,peptone（3.15 g/L）,NH₄H₂PO₄（1.58 g/L）,（NH₄）₂HPO₄（2.76 g/L）,NH₄NO₃（1.92 g/L）,extract ferulic acid from fermentation products,and compare the hydrolysis amount of each group of ferulic acid by liquid phase detection.The results show that in the single-factor test,when the C source and N source are sucrose and industrial yeast extract respectively,the hydrolysis of ferulic acid is the most affected.The response surface method was used to set up a 3-factor and 3-level experiment to determine the optimal culture conditions for the production of ferulic acid esterase by solid fermentation of Bacillus.The results showed that the highest concentration of ferulic acid-3.52 mg/g was measured under the conditions of 50%water content,9.5 g/L sucrose concentration and 7.7 g/L industrial yeast extract concentration.