| Hemicellulose is a heterogeneous polymer composed of several types of monosaccharides,including xylan and mannan.Hemicellulases can hydrolyze polysaccharides except for cellulose and pectin in plant cell walls.Hemicellulases can degrade hemicellulose into monosaccharides or oligosaccharides,unblock its effect on nutritional factors,and improve the availability of nutrients.Lignocellulose degradation can meet the needs of raw materials in different industries,such as prebiotic oligosaccharides,chemicals,proteins,biohydrogen,and biogas.Therefore,hemicellulase has excellent research value for the degradation of lignocellulosic biomass.Feruloyl esterase is a kind of hemicelluloses degrading enzyme common in the microorganism.Ferulic acid is produced when treated with feruloyl esterase,which has the functions of anti-radiation,anti-oxidation,antibacterial and antiviral.At the same time,it can play the role of skin protection in the human body.Feruloyl esterase has excellent potential in industrial production because of its biotechnological functions.However,in the existing research,feruloyl esterase production is mainly isolated and purified from various microorganisms.The yield is still maintained at a low level,more suitable for industrial production of enzyme activity,and higher activity of feruloyl esterase still needs to be developed.Escherichia coli is a classical prokaryotic gene expression host with the advantages of a short culture period and convenient operation and is the first choice for recombinant protein research and application.However,recombinant proteins produced by E.coli are usually located in the cytoplasmic or periplasmic space.Extracellular production of recombinant proteins has significant advantages,such as simplifying the purification of recombinant proteins or facilitating the direct function of recombinant proteins outside the cell.In this study,based on the phenomenon of soluble secretion of feruloyl esterase in E.coli,the yield,and activity of feruloyl esterase were optimized,and the N-terminal sequence of feruloyl esterase was used to guide the secretion of other enzymes,and multi-enzyme co-expression vector was constructed to realize the production of xylooligosaccharide,xylose and ferulic acid,which was mainly reflected in the following aspects:(1)Multiple strategies to improve the extracellular secretion of feruloyl esterase in E.coli.First,the DNA sequence of feruloyl esterase FaeLam was optimized for the codon preference of E.coli,and E.coli BL21/pET22b-Opt-Fae was constructed.The extracellular enzyme activity of E.coli BL21/pET22b-Opt-Fae was 41.97% higher than that of E.coli BL21/pET22b-Faelam,the yield of extracellular enzyme can be achieved 2858.85 U/L.We constructed the recombinant strains with different numbers of T7 promoters and 5’UTRs.The FaeLam secretion was significantly increased when4×T7 promoters were cascaded and A-5’UTR was replaced,which was 36.46% and31.25% higher than E.coli BL21/pET22b-Opt-Fae,respectively.Then,the random mutation kit performed random mutations on the N-terminal sequence.Based on the soluble secretion of the feruloyl esterase FaeLam,an efficient and accurate screening method was constructed to screen the mutant E.coli BL21/pET22b-Opt-Fae C66.Compared with E.coli BL21/pET22b-Opt-Fae,extracellular feruloyl esterase secretion was increased by 20.66%,the yield of extracellular enzyme can be achieved 3449.49U/L.In order to further improve the secretion of FaeLam,114 intracellular binding proteins of feruloyl esterase were identified by co-immunoprecipitation,and 11 secretory cofactors were screened out for FaeLam co-expression,among which SurA,DanK,and TolC had the most apparent secretory promoting effect.The extracellular yield of feruloyl esterase was increased by 20.09%,23.17%,and 25.75%,respectively.These results laid a theoretical foundation for increasing the yield of FaeLam and provided the possibility for further application in industrial production.(2)Site-directed mutagenesis improves the catalytic efficiency of feruloyl esterase.In order to improve the unit activity of FaeLam,a more suitable feruloyl esterase was obtained.Six mutants of FaeLam,Fae-T40 A,Fae-Q134 T,Fae-F160 A,Fae-G161 A,Fae-Q198 A and Fae-5×Site,were constructed.The mutants and original strain E.coli BL21/pET22b-Opt-Fae were induced and purified by His-Trap affinity chromatography to obtain the pure enzyme solution.Enzymatic properties were determined,including optimal reaction temperature,pH,temperature stability,and pH stability.The enzyme activities of Fae-Q134 T and Fae-Q198 A were 39.21% and48.88% higher than FaeLam,and the enzyme activities of Fae-Q134 T and Fae-Q198 A reached 397.99±4.77 U/mg and 425.64±5.55 U/mg,respectively.It was found that the unit point mutation had little effect on the enzymatic properties of a more suitable feruloyl esterase was obtained.However,the enzyme activity of the Fae-5×Site mutant decreased sharply,which may be due to the destruction of the spatial structure of FaeLam by the multi-site mutation.The kinetic parameters of different enzyme mutants were determined,substrate affinity and substrate turnover were compared,and the hydrolysis efficiency of FaeLam was improved.The catalytic efficiency of Fae-Q134 T and Fae-Q198 A was increased to 4.62 times and 5.42 times,respectively,compared with the original enzyme.These results lay a theoretical foundation for reforming FaeLam with higher catalytic efficiency and have potential application value.(3)The N20 sequence guides the secretory expression of xylanase in E.coli.Adding the 20 amino acid sequences(N20)of the N-terminal of FaeLam fromLactobacillus amylovorus to the N-terminal of xylanase from Bacillus paralicheniformis as a signal peptide,it was successfully overexpressed in E.coli and its enzymatic properties were analyzed.The enzymatic properties showed that the optimum reaction temperature of the enzyme was 50 ℃,and the optimum pH was 7.0.However,the temperature stability of the enzyme was poor at the optimum temperature.the temperature stability was better at 37 ℃,which made it possible to use the recombinant strain E.coli BL21/pET22b-N20 Xylfor simultaneous growth and fermentation of xylan.The extracellular yield of xylanase N20 Xylwas increased to504.12±10.66 U/mL by optimizing fermentation conditions,including medium,the concentration of inducer,induction time,glycine concentration,and fermentation time.Then the strain was used for the simultaneous growth and fermentation of xylan.The reducing sugar content in the fermentation liquid was 8.79±0.10 g/L.TLC and HPLC results showed that the main end products were xylobiose(4.35±0.05 g/L),xylotriose(2.34±0.01 g/L),and xylotetraose(0.57±0.01 g/L).Subsequently,20 different strains of LAB were used to determine the growth-promoting effect of XOS.The study showed that L.brevis,Enterococcus faecium,Pediococcus acidilactici,and Weissella confusa could effectively use XOS for growth.These results indicate that ferulate esterase is an effective signal peptide with N20,which can realize extracellular production of recombinant proteins and simultaneous fermentation of substrates and further broaden the types of signal peptides.(4)Construction and application of single vector multi-enzyme co-expressing strain.First,the xylose utilization gene(xylose isomerase XylA and xylose kinase XylB)of E.coli BL21 was knocked out by Red/ET recombinant technology.Growth experiments in the M9 medium with xylose showed that E.coli BL21/ Δ xylAB completely lost the xylose utilization ability.Subsequently,constructed multi-enzyme expression strains of xylosidase Xys,feruloyl esterase FaeLam,and xylanase N20 Xyland screened the sequence of the three enzymes.After induction by IPTG(final concentration 0.5 mM),comprehensive enzyme activity was determined.The optimal sequence of recombinant strain E.coli BL21/ Δ xylAB/pDⅢ-2 was selected.Xylan was added to the medium at 50 g/L concentration for simultaneous growth and fermentation under optimized fermentation conditions,and the final extracellular reducing sugar content was 33.70±0.46 g/L.The antioxidant capacities of the obtained fermentation broth were determined and prepared into special MRS Medium for probiotic culture,in which the hydroxylradical scavenging capacity could reach 80.0%,and the total antioxidant capacity could reach 2.289±0.55.The special MRS Medium also promoted the growth of LAB better than xylose MRS Medium.These results provided the basis for constructing single vector multi-enzyme co-expression strains,using multi-enzyme co-expression strains to produce xylose and ferulic acid,which has potential application value. |
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