| Plant-derived oligosaccharides have many biological activities,such as probiotics,anti-cancer,anti-inflammation and so on which has attracted wide attention.Compared with the synthesis of nucleotides and proteins,the synthesis of polysaccharides is particularly difficult.so the utilization of polysaccharides or oligosaccharides must depend on the natural plant cell wall polysaccharides.Straw and other agricultural processing industry by-products are often burned,resulting in huge environmental pollution.Using agricultural wastes as raw materials to produce high-value-added functional sugar can improve the comprehensive utilization rate of by-products of agricultural by-products processing industry,reduce the production of wastes and make the recovery of high-value-added functional sugar possible,which has great economic and ecological benefits.The green biological sugar industry not only improves the value of raw materials and reduces the cost of production,but also saves water,reduces secondary pollution,and ultimately improves the industrial efficiency.At the same time,due to the substrate specificity of glycoside hydrolase and the heterogeneity of xylan,it is necessary to compound degrading enzymes with different substrates to achieve the goal of "customizing" high value-added functional sugars.In this study,the dynamic changes of lignocellulose degrading enzymes under different temperatures and carbon sources were studied based on comparative secretome analysis.The composition and degradation specificity of A.niger An76 xylan degrading enzymes were comprehensively studied.At the same time,the xylan degrading enzymes library of A.niger An76 was constructed.Based on the characteristics of substituents of xylan,the degradation of lignocellulose degrading enzymes was established different types of xylan substrates and the minimum optimal combination of enzymes for specific oligosaccharides were obtained.The specific research progress is as follows:1.The lignocellulose degradation characteristics of 31 fungi were analyzed by comparative genomics.It was found that the degradation of cellulose by different cellulose-degrading fungi was related to their niche.Based on comparative genomics,the composition and quantity of lignocellulose degradation related genes in 3 1 different strains of filamentous fungi,plant pathogens,human pathogens and yeasts were analyzed.It was found that the family distribution and active species of lignocellulose degradation enzymes genes of fungi were closely related to the niche of microorganisms.Among them,Aspergillus niger has only the second largest number of carbohydrate degrading enzymes and complete hemicellulose degrading enzymes.Analysis shows that A.niger An76,the most widely used industrial Aspergillus niger,has 28 xylan degrading related enzymes.2.Based on functional histology,the effects of external environment on the components of the secretory enzyme system of A.niger An76 were studied.It was found that the secretory sequence of different extracellular enzymes of A.niger An76 was temperature dependent and followed the glucose effect.It was found that the secretion of A.niger An76 glycoside hydrolase was influenced by temperature and extracellular carbon source,which could induce different extracellular glycoside hydrolase patterns.A.niger An76 grew best on a solid plate at 35?,but at 30?,its extracellular oligosaccharide content and extracellular protein secretion were the largest.The results showed that culture temperature could affect the secretion sequence of extracellular hydrolases of different components.Especially when three endoxylanases were cultured at different temperatures,the secretion sequence and expression amount showed significant differences.The secretion of An_XynB is not affected by temperature in the range of 25-35?.The secretion of An_XynC lags behind at 2? at low temperature,and the secretion of An_XynA lags behind at 35? at high temperature.Both high and low temperatures affect the secretion of Egl.It was found that glycerol did not induce the secretion of glycoside hydrolase in single carbon source culture.In the presence of glucose,endoxylanase and endocellulase were secreted only when the extracellular glucose was depleted,indicating that when the concentration of extracellular glucose was very low or depleted,the inhibition effect of carbon source repression of glucose would be removed and the glycoside hydrolase water would be secreted.Dissolve extracellular polysaccharides that are difficult to use.3.The expression characteristics of A.niger An76 xylan-degrading enzymes were investigated from genomic and proteomic perspectives.It was found that the production of various components of xylanase system was induced differently.The bioinformatics prediction of A.niger An76 xylan degrading enzyme genes showed that the location of A.niger An76 xylan degrading enzyme gene was concentrated on chromosome.Several xylan-degrading enzymes that play important roles in A.niger An76 genome were preliminarily identified based on the proteomic analysis of nine carbon sources.It was found that the xylan-degrading enzymes in this strain mainly come from 9 GH families and 2 CE families.Most of them are induced secretory proteins,and there may be gene co-expression.It was found that the expression of xylan main chain degrading enzymes on xylan substrates was higher than that on other substrates,but the induction of xylan side chain degrading enzymes had a preference for carbon sources.4.Construction of xylan degrading enzyme library,characterization of enzyme components and elaboration of its degradation mechanismThe xylan-degrading enzymes from A.niger An76 were cloned and purified to determine their enzymatic properties.Compared with GH11 family,GH10 family xylanases(An XynC)have more open fissures in active sites,which can degrade more complex substituents and produce smaller fragments of substituted xylo-oligosaccharides.Among the three GH11 family xylanases,An_XynB has the highest enzyme activity,but their degradation patterns are different.An_XynA and An_XynB can degrade large fragments of oligosaccharides(above xylopentaose)while An_XynD can't.An_X1nB is similar to An_AxhA in structure.An_XlnB is a bifunctional enzyme of a-L-arabinofuranosidase/?-xylosidase.An_XlnD is a single activity of ?-xylosidase.When the substrate is short-chain xylo-oligosaccharide or substituted xylo-oligosaccharide,its degradation efficiency is higher than that of An X nB.?-L-arabinofuranosidase An AbfB and An AxhA both have the ability to hydrolyze the a-1,5 between arabinofuranosides,but the efficiency of hydrolyzing the a-1,5 is low.However,the preference for substrates is different.An AbfB can hydrolyze monosubstituted arabinose side chains linked by a-1,2 or a-1,3,and has high activity for short-chain substituted xylo-oligosaccharides and pNPAf.An_AxhA hydrolyzes an arabinose side chain on the disubstituted xylan,which can not bind oligosaccharides,and may also have a-xylosidase activity.5.Construction of the optimum enzyme combination for different structures of xylan substratesThe mechanism of synergistic effect was gradually revealed and the synergistic degree of each degrading enzyme combination was studied for different substrates and different purposes.The study of synergistic hydrolysis showed that the complementary hydrolysis modes could produce good synergistic effect.There was a good synergistic effect between a-L-arabinofuranosidase and ?-1,4-endoxylanase and?-xylosidase.Side chain degrading enzymes are the speed-limiting bottleneck in the degradation process.Adding side chain degrading enzymes first can promote the enzymatic hydrolysis.However,the degree of side chain substitution and substituents of xylan substrates vary greatly.Single side chain degrading enzymes often can not meet the requirements of side chain degrading.In this paper,detailed synergistic combinations and synergistic sequences of xylan substrates with different degrees of substitution are given. |
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