Study On The Structure Of Hemicellulose And Efficient Enzymatic Degradation Of Corn Fiber

As lignocellulose has the characteristics of being widely distributed on earth,abundant and renewable,converting lignocellulose into liquid fuels and bulk chemicals is one of the effective ways to solve the current challenges faced by human society,such as shortage of fossil resources and energy,environmental challenges,etc.Corn fiber mainly refers to the seed coat part of corn kernels,which is a by-product of the corn ethanol industry and corn starch processing industry,and is one of rich lignocellulosic biomass resources.The annual production of corn fiber in China was estimated to reach 10.9 million tons.it was estimated that the total ethanol production can be increased by 13%from the original basis if the carbohydrates in corn fiber can be effectively converted to ethanol,as while as the quality of DDGS(Distillers Dried Grains with Solubles)can also be significantly improved.Bioconversion of lignocellulose was mainly divided into three steps:pretreatment,enzymatic hydrolysis,and fermentation,of which the production of fermentable monosaccharides by enzymatic hydrolysis with cellulase was a key step.Corn fiber contains a large amount of hemicellulose(about 40%),and its complex sugar compositions and structure make corn fiber stubbornly resistant to enzymatic degradation.Therefore,to achieve efficient degradation of corn fiber,on the one hand,it is necessary to break the degradation barriers by suitable pretreatment for improving the enzymatic digestibility of corn fiber,on the other hand,corn fiber degrading enzyme system is still optimized to improve its hydrolysis ability.Up to now,however,the pretreatment processes of corn fiber reported in literature were not suitable for actual production because the low enzymatic digestibility of pretreated corn fiber,or more inhibitors produced by acid pretreatment,which need to be removed by detoxification treatment.increasing the process cost.On the other hand,as the lack of understanding of corn fiber hemicellulose structure,it is very difficult to screen suitable enzymes required for enzymatic degradation of corn fiber hemicelluloses,which result in the slow research progress in the optimization of the corn fiber degrading enzyme system.Based on the research backgrounds,in this thesis,the dilute acid pretreatment of corn fiber was optimized for maximizing sugars production and minimizing inhibitors formation.The structures of hemicellulose in corn fiber and solid residues from enzymatic hydrolysis of corn fiber with cellulase system produced from Penicillium oxalicum MCAX were characterized,and enzyme components required for the degradation of corn fiber were screened based on the structural characteristics of the hemicelluloses.Then,the cellulase system capable of effective degrading corn fiber was constructed by studying the effects of the screened enzymes additions on enzymatic hydrolysis of corn fiber with starting cellulase system from P.oxalicum MCAX using RSM(Response surface method),and hydrolysis performance of the optimized cellulase system was also validated.The main research contents and results of this thesis were as follows:1.The optimization of dilute acid pretreatment of corn fiber using RSM and establishment of ethanol production process by enzymatic hydrolysis and fermentation without detoxificationBy comparing the effects of different pretreatment methods,including mechanical pretreatment,hydrothermal pretreatment,steam pretreatment,and dilute acid pretreatment,on the enzymatic hydrolysis performance of corn fiber,it was found that dilute acid pretreatment was a more suitable pretreatment method for corn fiber.The dilute acid pretreatment conditions(temperature,time,and acid concentration)were optimized by RSM,and based on the sugar yield and inhibitors production,the optimal pretreatment conditions were that corn fiber was pretreated with 0.5%(w/v)H2SO4 at 105℃ for 43 min.Under these conditions,the pretreated corn fiber was hydrolyzed with P.oxalicum MCAX cellulase(5 mg protein/g DM)for 72 h,and the yield of glucose,xylose,and arabinose reached 354.133,and 114 mg/g corn fiber,respectively,which was equivalent to 81.8%of the total content of three sugars in corn fiber.Further,domestication of Saccharomyces cerevisiae strain was carried out to improve its tolerance to inhibitors.By combing pretreatment with anti-toxic yeast,an "one-pot" process of dilute acid pretreatment,enzymatic hydrolysis,and fermentation of ethanol production without detoxification was established.By using this process,after dilute acid pretreatment under optimal conditions,the pretreated corn fiber could be directly hydrolyzed with cellulase and fermented to produce ethanol.The ethanol concentration in fermentation liquid reached 40.14 g/L at 20%solid content,which was 80.8%of the theoretical yield.2.Extraction and structure analysis of hemicellulose in corn fiberHemicellulose in corn fiber is one of the main obstacles limiting enzymatic hydrolysis of corn fiber because of its high content,complex sugar compositions and structural properties.A clear understanding of the structural characteristics of hemicellulose in corn fiber can theoretically guide the screening of enzymes required for degradation and optimization of highefficient degrading enzyme systems.Firstly,an improved extraction method was established for obtaining the hemicellulose samples with intact structure and high purity to meet the need for hemicellulose structure research,which was composed of enzymatic de-starching.PAA(peroxyacetic acid)de-lignification.DMSO(dimethyl sulfoxide)extraction and ethanol precipitation.Using this method,hemicelluloses were successfully extracted from corn fiber,a starch-rich cereal seed coat-like biomass.The molecular structure characteristics of hemicelluloses in corn fiber were determined by monosaccharide compositions analysis,molecular weights and its distribution determination,functional group analysis,methylation analysis,and NMR(nuclear magnetic resonance)spectroscopy,and molecular structures models of hemicellulose in corn fiber were constructed.It was shown that the hemicellulose in corn fiber consisted of glucuronoarabinoxylan(about 80%)and xyloglucan(about 20%).Among them,xylose(about 46%)and arabinose(about 32%)were the main sugar components of glucuronoarabinoxylan.More than half of the xylose units of the glucuronoarabinoxylan backbone was replaced by various monomeric or oligomeric side chains at O-2 and/or O-3,including arabinose,xylose,galactose,mannose,glucuronic acid,etc.The terminal substituents of these side chains were mainly arabinose residues,xylose residues,and galactose residues.Compared to the structure of glucuronoarabinoxylan reported in the literature,glucuronoarabinoxylan in corn fiber hemicellulose had up to 16 types of side chains and a higher proportion of oligomeric side chains,thus a more complex enzymatic system was required for achieving its degradation.The main chain of xyloglucan was a β-1,4-linked glucan,and about 75%of glucose residues at O-6 position were substituted by α-D-xylose,but it was not further replaced by other sugar residues.The small amount of xyloglucan cross-linked with glucuronoarabinoxylan,resulted in more difficult to degrade corn fiber with enzymatic hydrolysis.Based on the structural characteristics of hemicellulose in corn fiber,the enzymes that may be required to hydrolyze hemicellulose in corn fiber were predicted.The effects of the enzymes on enzymatic hydrolysis of corn fiber were researched by adding the enzymes to three different sources cellulase systems.On the other hand,by comparing the degradation effects of different sources cellulase systems,the P.oxalicum MCAX cellulase system was finally identified as the basic enzyme system and its dosage was 5 mg protein/g DM.Further,optimization of the basic enzyme system was carried out.3.Structural analysis of hemicellulose in solid residue from enzymatic hydrolysis of corn fiber with Penicillium oxalicum MCAX cellulaseIt was found that,after enzymatic hydrolysis of corn fiber with the P.oxalicum MCAX cellulase system,there were still more hemicelluloses in the solid residue.The hemicelluloses that was resistant to enzymatic degradation need to be further degraded to increase sugar yield in enzymatic hydrolysis of corn stover.In order to further optimize the degrading enzyme system to degrade the hemicelluloses,the hemicellulose fractions with intact structure and high purity were extracted according to above improved extraction method,and its structure characteristics were investigated.It was showed that,similar to the sugar compositions and structure of hemicellulose in corn fiber,the hemicelluloses in the residue were also composed of glucuronoarabinoxylan and xyloglucan.However,the percentage of mono-substituted xylose residues(1,3,4-Xylp)in backbone xylose units of the glucuronoarabinoxylan of hemicellulose in the residue increased significantly while the percentage of un-substituted xylose residues(1,4-Xylp)decreased significantly,indicating that the un-substituted xylose residues in glucuronoarabinoxylan were easily broken during enzymatic hydrolysis,while the side chain structures attached to the remaining mono-substituted xylose residues and double-substituted xylose residues(1,2,3,4-Xylp)were resistant to enzymatic hydrolysis.For xyloglucan,the degree of substitution at the O-6 position of the backbone glucose residues of the xyloglucan was significantly reduced after enzymatic hydrolysis with the P.oxalicum MCAX cellulase system.By comparing the changes in the structure of hemicellulose before and after enzymatic hydrolysis and combining the types and ratios of terminal substituents in the hemicellulose from residue obtained from enzymatic hydrolysis,five hemicellulases that could enhance enzymatic performance of P.oxalicum MCAX cellulase system were predicted,namely α-Larabinofuranosidases(GH43 and GH51),α-D-galactosidase(GH27),β-D-galactosidase(GH35),and α-D-xylosidase(GH31).4.Construction and evaluation of an high-efficiency enzyme system for enzymatic hydrolysis of corn fiber based on Penicillium oxalicum cellulase systemThe five hemicellulases predicted above were heterologously expressed in Pichiapastoris GS115 and Trichoderma reesei QMP.respectively,and their pure enzyme fractions were respectively obtained by fermentation and purification.The enzymatic properties of the five recombinant hemicellulases were studied.including the optimum temperature,optimum pH,stability of temperature,and stability of pH.The five hemicellulases were added individually to the P.oxalicum MCAX cellulase system to hydrolyze corn fiber.and found that,compared to the single P.oxalicum MCAX cellulase system,the addition of α-L-arabinofuranosidase rPoAbf51A,α-D-galactosidase rPoGal27A,and α-D-xylosidase rAnXyl3lA promoted the enzymatic hydrolysis of corn fiber,in especial,the rAnXyl31A showed the most obvious promotion effect.but the addition of rPoAbf43C and rAnGal35A had little impact on enzymatic hydrolysis of corn fiber.It was also found that the rPoAbf51A,the rPoGal27A,and the rAnXyl31A had synergistic effects for enzymatic hydrolyze of corn fiber.The optimal amounts of the three hemicellulases were optimized by RSM.which was 1.98 mg/g DM for rPoAbf51 A.1.99 mg/g DM for rAnXyl31A.and 1.54 mg/g DM for rPoGal27A,respectively.Using the optimized cellulase system containing the three hemicellulases,the yield of total sugars reached to 486.75 mg/g corn fiber at 72 h of enzymatic hydrolysis,and the yields of different monosaccharides were 81.82%for glucose,36.02%for xylose,34.88%for galactose,54.89%for arabinose,and 52.02%for mannose,respectively.These exceed the highest levels of sugar yields reported in the literature so far for unpretreated corn fiber hydrolyzed using cellulase alone.Compared to the P.oxalicum MCAX cellulase system,the optimized enzyme system resulted in the yield increase of 19.18%for total sugars,8.83%for glucose,27.12%for xylose,39.85%for galactose,15.68%for arabinose,and 198.07%for mannose,respectively.The total sugar yield of 661.58 mg/g corn fiber was achieved after 72 h of enzymatic hydrolysis with the optimized cellulase system when using pretreated corn fiber by dilute acid under optimum pretreatment conditions,and the monosaccharide yields were 93.76%for glucose,65.43%for xylose,63.10%for galactose,78.94%for arabinose,and 68.97%for mannose,respectively.Compared to the P.oxalicum MCAX enzyme system(the yield of total sugars was 579.11 mg/g corn fiber and the yield of xylose,galactose,arabinose,and mannose was 51.08%,52.60%,62.60%,and 59.45%,respectively),the optimized cellulase enzyme system could effectively increase the yield of total sugars,especially the yields of xylose,galactose,arabinose,and mannose after enzymatic hydrolysis of corn fiber.