Study On Enzymatic Degradation Of Different Cellulosic Biomass To Prepare Feruloylated Oligosaccharides

Feruloyl oligosaccharides（FOs）are esterified products of xylooligosaccharides and ferulic acid,combining the physiological activities of both,and can be obtained by hydrolysis of ferulylated polysaccharides,are compounds with high-added value.As important by-products of food and agricultural processing,brewer’s spent grain,wheat bran and corncob are biomass with huge output.Because the main structures of hemicellulose of brewer’s spent grain,wheat bran and corncob are ferulylated arabinoxylans,they are important source for FOs preparation.Filamentous fungi can secrete large amounts of lignocellulosic enzymes,which play an important role in the production of various lignocellulosic degrading enzymes,including cellulase and hemicellulase,etc.These enzymes are mainly used to degrade cellulosic biomass for the production of biofuels and bio-based chemicals.By studying the enzymatic action of different cellulase systems and hemicellulase on three cellulosic biomass substrates,the hemicellulose in these cellulosic biomass can be efficiently converted into feruloyl oligosaccharides,which has important practical significance for converting various unused and underutilized agricultural by-products into products with high-added value.In this thesis,three kinds of cellulosic biomass including brewer’s spent grain,wheat bran and corn cob were selected.The cellulase systems of Trichoderma reecei and xylanases derived from Penicillium oxalicum were used for enzymolysis to produce FOs.The effects of different cellulase systems and different xylanases on FOs yield were studied,and the synergistic hydrolysis of cellulase systems and different xylanases was optimized to improve FOs yield.The main research contents and results are as follows:1.Cloning and expression of xylanases from Penicillium oxalicum in Trichoderma reecei and construction of ferulic acid esterase gene knockout strains of Penicillium oxalicumRecombinant strains expressing eight xylanases Xyn11A,Xyn11B,Xyn11C,Xyn11D,Xyn11E,Xyn1OB,Xyn10C and Xyn30B derived from P.oxalicum were constructed using T.reesei QMP strain as the expression host.The xylanase activity of recombinant strains at different times of fermentation broth under glucose conditions and it was found that the culture supernatants at 24 hours of fermentation had significant xylanase activity,and SDS-PAGE results of the extracellular proteins of these 8 recombinant strains showed that the target protein band present as single band and the secretory background of the protein was clean,which could be used for enzymatic hydrolysis of BSG to prepare FOs.The ferulic acid esterase genes fae1A,PDE₀9178,PDE 08709 and PDE 08238 were knocked out in high cellulase-producing Penicillum oxalicum strain RE-6,respectively,and ferulic acid esterase knockout strains Δfae1A-8,Δ09178-4,Δ08709-15 and Δ08238-2 were obtained.The FPsae activity and xylanase activity in the culture supernatants were determined,and the FOs could be prepared by enzymatic hydrolysis of BSG with cellulase solution at 96 hours of fermentation under cellulose conditions.2.Preparation of feruloyl oligosaccharides from brewer’s spent grain by enzymatic hydrolysisThe effect of crude enzyme solution of P.oxalicum strains RE-6,RE-7,RE-8,and T.reesei strain h61 in the enzymatic hydrolysis of BSG for the preparation of FOs was evaluated.It was found that the enzyme system of T.reesei strain h61 was much better than the enzyme system of P.oxalicum strains RE-6,RE-7,RE-8,and could be better used to degrade BSG to prepare FOs.The enzyme system of T.reesei h61 was used as the basic enzyme system for the enzymatic hydrolysis of deproteinized and undeproteinized BSG.It was found that the yield of FOs prepared with deproteinized BSG as substrate was about 25.62%higher than that prepared with undeproteinized BSG as substrate,and the yield of FOs reached up to 39.66%.The enzyme dosage was determined based on the total xylanase activity,and the optimal addition amount for enzymatic hydrolysis of BSG to prepare FOs was 120 U/g BSG.The effect of adding different xylanases to the enzyme solution of h61 as the basic enzyme system on the preparation of FOs was investigated,and it was found that adding Xyn10B,Xyn10C and Xyn30B could better promote the hydrolysis of BSG to prepare FOs,and the yield of FOs prepared by hydrolysis of BSG with only Xyn30B and Xyn10A single enzyme could reach more than 20%.The effect of complex enzyme of the enzyme solution of h61 and different xylanases in different ratios for the enzymatic hydrolysis of BSG on the preparation of FOs was investigated,and it was found that the yield of FOs reached its maximum when h61:Xyn10B:Xyn10C was 1.5:0.75:0.75,at which ratio the production was 8.80 μmol/g and the yield was able to reach 44.52%,which was 9.37%higher than the basic enzyme system added only with h61.The effect of α-L-arabinofuranosidase Abf51B and α-glucuronidase Agu67A and Agu115A on the preparation of FOs was investigated,and it was found that Agu67A could promote the hydrolysis of BSG and also synergized with Abf51B to promote the hydrolysis of BSG,which improved the yield of FOs.The effect of the enzyme solutions of P.oxalicum ferulic acid esterase gene knockout strains Δfae1A-8,Δ09178-4,Δ08709-15,and Δ08238-2 on the preparation of FOs was investigated,and it was found that the yield was only half of that with adding T.reesei h61.3.Preparation of feruloyl oligosaccharides from wheat bran and corncob by enzymatic drolysisThe optimal enzyme dosage for the preparation of FOs by hydrolysis of wheat bran and corncob insoluble dietary fiber with the enzyme solution of h61 was optimized.Based on the total xylanase activity,the optimal addition amount for enzymatic hydrolysis of wheat bran insoluble dietary fiber to prepare FOs was 15 U/g WB,and the optimal addition amount for enzymatic hydrolysis of corncob insoluble dietary fiber to prepare FOs was 300 U/g CC.The effect of adding different xylanases to the enzyme solution of h61 as the basic enzyme system on the preparation of FOs from wheat bran by enzymatic hydrolysis was investigated,and it was found that adding Xyn10A and Xyn11B could better promote the hydrolysis of wheat bran insoluble dietary fiber to prepare FOs,and the yield of FOs prepared by hydrolysis of wheat bran insoluble dietary fiber with only Xyn10A and Xyn30B single enzyme could reach more than 20%.The effect of adding different xylanases to the enzyme solution of h61 as the basic enzyme system on the preparation of FOs from corncob by enzymatic hydrolysis was investigated,and it was found that the enzyme solution of h61 was added first for enzymatic hydrolysis,and then the enzymatic hydrolysate was drained at the end of enzymatic hydrolysis,followed by the addition of different xylanases.Xyn30B and Xyn10A could better promote the hydrolysis of corncob insoluble dietary fiber to prepare FOs.The effect of complex enzyme of the enzyme solution of h61 and different xylanases in different ratios by two-step enzymatic hydrolysis on the preparation of FOs by enzymatic hydrolysis of corncob insoluble dietary fiber was investigated,and it was found that the yield of FOs reached its maximum when h61:Xyn10A（h61+Xyn10A=200 U+100 U）was 2:1,at which ratio the production was 23.22μmol/g and the yield was able to reach 39.65%,which was 9.05%higher than that of the enzyme solution with 300 U/g CC h61 added directly by the one-step enzymatic hydrolysis.The effect of secondary enzymatic hydrolysis with the enzyme solution of h61 on the preparation of FOs from deproteinized BSG,wheat bran insoluble dietary fiber and corncob insoluble dietary fiber was investigated,and it was found that the yield of FOs from corncob increased by 20.42%and that of FOs from BSG increased by 14.94%.