Study On Heterologous Expression Of GH10 Family Thermophilic Xylanase Genes And Molecular Improvement Of Catalytic Efficiency At Animal Heat

As a kind of safe and efficient feed additive enzyme,xylanase can efficiently degrade xylan in plant feed.At present,studies on xylanase mainly focus on the catalytic efficiency and thermal stability of high-temperature xylanase,with little attention paid to the enzyme activity of high-temperature xylanase at animal heat(about 40?).The low activity of the enzyme seriously limits its application in feed industry.The purpose of this thesis is to obtain xylanase with high catalytic activity at animal heat and high temperature resistance,which provides guarantee for the large-scale industrial production and application of feed enzymes.Target xylanase was obtained with the method of exploring xylanase genes by using excellent properties(XYL10C?(?)N)from microbial resources and improving the catalytic activity of xylanase at animal heat by rational design.The main research results are as follows:(1)The GH10 family xylanase genes Hwxyl10a and Ccxyl10a were cloned from the fungi Hortaea werneckii and Cladophialophora carrionii,respectively,which expressed in yeast.With beech xylan as the substrate,the optimum temperature of Hw XYL10A and Cc XYL10A were 75°C and 80°C,respectively.The optimum p H of Hw XYL10A and Cc XYL10A were 4.0 and 4.5,respectively.Hw XYL10A and Cc XYL10A have good thermal stability,with T₅₀ values of 76°C and 86°C,respectively.The half-life(t_1/2)of Hw XYL10A at 80°C was 11 min,and that of Cc XYL10A at 90°C was 12 min.Compared with the template XYL10C?(?)N(the template T₅₀ value was 83°C),Cc XYL10A increased by 3°C,which was more stable than the template.Hw XYL10A decreased by 7°C,which was lower than the thermal stability of the template.Beech xylan used as substrate,the specific activities of Hw XYL10A,Cc XYL10A and XYL10C?(?)N at 40°C were 1056,1575 and 620 U/mg,respectively.The specific activity of Cc XYL10A was 2.5 times higher than that of XYL10C?(?)N.In conclusion,Cc XYL10A is not only an active xylanase with high specific activity at high temperature,but also 1.5 times higher than XYL10C?(?)N at 40?,which is expected to be used as a feed additive.(2)Through site-directed mutagenesis and combined mutation screening,a mutant xylanase M1 with high specific activity at high temperature and increased enzyme catalytic efficiency at 40?was obtained.Using the GH10 xylanase XYL10C?(?)N with a crystal structure report as the starting material,two mutants M1(MF53/54DS),M2(Q59Y)and a combined mutant M1/M2(MF53/54DS+Q59Y)were obtained through protein structure and sequence analysis and site-directed mutagenesis.At 40?,the catalytic efficiency of M1 was1.5 times higher than that of wild enzymes respectively(1300 vs.530 m L/s/mg),the specific activity was 2.1 times higher than the wild enzyme respectively(1900 vs.620 U/mg).In terms of enzyme thermal stability,the half-lives(t_1/2)of mutants M1 at 90?were 15 min,which were 1.5 times higher than the wild enzyme(6 min).The thermal stability of the enzyme was enhanced while the catalytic efficiency was improved at 40?and high temperature.In summary,the thermal stability of mutant M1 was increased,and at the same time the enzyme activity at 90?and 40?wass improved.Therefore,M1 is expected to become a more widely used xylanase with high specific activity at high temperature.(3)Take the lead in simulating the hydrolysis of xylanase with high specific activity at high temperature under animal heat.The xylanase M1,which had the highest enzyme activity at the animal heat,was hydrolyzed with cellulase and washed to the neutral dried mulberry bark.The experimental group added different enzymes,including xylanase and cellulase(cooperative treatment group),separate xylanase,cellulase alone,and control group.The amount of reducing sugar produced and the degree of synergy taken as indicators,the practical application performance of mutant M1 in hydrolyzing natural lignocellulose were analyzed.The results showed the mutant M1 and cellulase co-treatment group had the highest sugar production at 16 h,which was at 20?mol/m L,the highest synergy rate can reach 1.35,and the sugar production and synergy rate are both doubled compared to XYL10C?(?)N.In addition,when beech xylan was used as substrate,the hydrolysis ability of M1 to produce xylobiose was 3 times that of template.In conclusion,M1 is not only an active xylanase with high specific activity at high temperature,but also has high specific activity at 40?.At the same time,it has a good degradation effect on natural lignocellulose,and is a good material for feed additives.In summary,on the basis of mastering the structural information,biological information of the template XYL10C?(?)N in detail was analyzed.On the one hand,Hw XYL10A and Cc XYL10A from the fungal source were discovered,and on the other hand,three kinds of mutants(M1,M2 and M1/M2)were obtained by rational designs.After analyzing the enzymatic properties and mulberry bark hydrolysis of the obtained xylanase,the GH10 family xylanase M1,which is expected to be used in feed processing,was screened to explore the catalytic efficiency and structure of high-temperature xylanase under the conditions of animal body temperature.The correlation provides a good reference,and at the same time provides a new material for the research of high specific activity thermostable xylanases.