| Xylooligosaccharide(XOS)is a kind of low molecular polymer with a variety of biological activities,among which xylobiose and xylotriose are widely used in food,health care and medical fields due to their unique prebiotic,antioxidant and other activities.In the current XOS production process,enzymatic degradation has been extensively applied due to its efficiency,environmental friendliness,and economic advantages.As a glycosylase capable of specifically and efficiently degrading xylan,xylanases play a crucial role in the enzymatic preparation of XOS.To obtain more efficient xylanases,current research on enzymatic XOS production mainly focuses on discovering new xylanases,with little attention paid to the impact of molecular improvement of xylanases on product composition.This study cloned and expressed two thermostable and high-specific-activity xylanases from the 10 th family(GH10)from microbial resources,rationally designed modifications by analyzing the regional flexibility,energy optimization,and substrate-binding site optimization of xylanases,and investigated the impact of key amino acid sites on xylanase degradation product composition through molecular improvement.This aims to obtain xylanases with high production of xylobiose and xylotriose,providing technical support for the development and utilization of feed enzymes.The main research results are as follows:(1)Based on flexible region modification,a xylanase mutant S21 Y / N318 W was screened to catalyze the degradation of beech xylan to produce xylobiose and xylotriose.The GH10 high-temperature xylanase xylanase Hwxyl10 A was excavated and expressed from the fungu Hortaea werneckii,and the proportion of xylobiose and xylotriose in the product composition of the catalytic degradation of beech xylan was up to 30%.To further improve the enzyme performance,rational design was employed to enhance the rigidity of two key flexible regions S21 and N318,yielding the advantageous mutant S21Y/N318 W.Enzymatic properties and sugar composition of the mutant were measured,revealing that the total proportion of X2 and X3 in the degradation products of beechwood xylan by S21Y/N318 W reached 75.2%,a 45% increase compared to the wild-type.Additionally,the specific activity of S21Y/N318 W is 1901 U/mg,which was higher than the wild-type(p<0.05).Furthermore,the half-life(102 min)of S21Y/N318 W at 75 °C was 16 times higher than that of the wild-type,indicating better thermostability(p<0.05).The mutant S21Y/N318 W has reduced the flexibility of the key loop region of catalytic activity,enhanced its structural stability,and changed the product specificity.Therefore,the proportion of xylobiose and xylotriose in the products of catalytic degradation of beech xylan has been greatly increased.(2)Based on the optimization of substrate binding sites,a xylanase mutant S265 A was screened to catalyze the degradation of beech xylan to produce xylobiose and xylotriose.The GH10 high-temperature xylanase xylanase Ccxyl10 B was excavated and expressed from the fungu Cladophialophora carrionii.Ccxyl10 B degraded beechwood xylan with X2 and X3 accounting for 47% of the product composition and specific activity of 2145 U/mg.The rational design was used to identify key substrate-binding amino acid residues in the Ccxyl10 B catalytic channel,yielding the advantageous mutant S265 A.Enzymatic properties and sugar composition of the mutant were measured,revealing that the total proportion of X2 and X3 in the degradation products of beechwood xylan by S265 A reached 65%,a 20%increase compared to the wild-type.Furthermore,almost no X4 or larger oligo xylose was present in the product.Meanwhile,the specific activity(3277 U/mg)of S265 A increased by53%.Molecular dynamics simulation analysis shows that the central group of the catalytic channel of mutant S265 A has an enhanced affinity with the high degree of polymerization xylooligosaccharide substrate in the degradation product of beech wood xylan,promoting the degradation of the high degree of polymerization components into the low degree of polymerization components such as xylobiose and xylotriose.This is manifested by a decrease in the hydrolysis components of xylooligosaccharides,which facilitates product separation.(3)Based on the overall energy optimization,a xylanase mutant V126 F with a high yield of xylobiose and xylotriose was obtained.Based on the homologous modeling structure of Ccxyl10 B,the key amino acid sites affecting its overall energy were subjected to virtual saturation mutation,and the dominant mutant V126 F was screened.Molecular dynamics simulation results showed that the root means square deviation(RMSD)of V126 F was lower than that of the wild type,the overall energy of the enzyme decreased,the structural stability was stronger,and the residue changes in V216 F made the product release faster.HPLC analysis of the products showed that the total proportion of X2 and X3 in the products of beech xylan degradation by V126 F reached 74%,an increase of 27% compared to the wild type.The enzymatic properties and sugar composition of the mutant were measured,showing that compared with the wild type,the half-life(t1/2)of V126 F was 27 minutes,3.3 times that of the wild type(8 minutes).When using beech xylan as the substrate,the specific activity(2359 U/mg)of the mutant V126 F increased by 25%compared to the wild type.Molecular dynamics simulation analysis showed that the overall root mean square deviation of the mutant V126 F was lower than that of the wild type,the overall energy of the enzyme decreased,the steric hindrance of the catalytic active channel decreased,the structural stability was strengthened,and the change of its residues made the product release faster,so its catalytic degradation of beech xylan to produce xylobiose and xylotriose was more efficient.(4)The process of seawater pretreatment biomass combined with xylanase mutant and cellulase catalytic degradation of the mulberry branch to produce xylooligosaccharides was constructed.Mulberry branches were pretreated by seawater immersion,and the catalytic degradation of mulberry branches was carried out by synergistic treatment of xylanase and cellulase.The degree of synergy(DS)and reducing sugar production were used as indicators to analyze the actual performance of xylanase in the catalytic degradation of mulberry branches.The results showed that the combination of V126F+cellulase reached the highest sugar production of 18.89 μmol/m L in the shortest time(8 h),and the synergy degree reached its highest(DS 2.99)at 2 h,which was 59% higher than that of the wild type and cellulase.In the product composition,the proportion of X2 and X3 in the degradation products of mulberry branches by the mutant V126 F was the highest,reaching 71.5%.In the existing reports on the degradation of mulberry branches to produce xylooligosaccharides,the highest proportion of xylobiose and xylotriose was achieved.Compared with the production of xylooligosaccharides,the treatment method of mulberry branches in this paper is simpler,cheaper,and more efficient.Therefore,the mutant V126 F and cellulase synergistically catalyze the hydrolysis of mulberry branches to produce specific xylooligosaccharides,which helps to improve the production efficiency of functional xylooligosaccharides and improve the utilization rate and added value of mulberry resources.In summary,this thesis molecularly modified two thermostable xylanases,Hwxyl10 A,and Ccxyl10 B,from fungi and successfully constructed three advantageous mutants,S21Y/N318 W,V126F,and S265 A.Through basic enzymatic properties and mulberry branch hydrolysis analysis,the GH10 family xylanase V126 F,which is promising for degrading mulberry branches to produce specific xylooligosaccharides,was selected,providing a reference for exploring the improvement of xylanase sugar production characteristics and the diversified utilization of mulberry branch resources. 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