Cloning And Heterologous Expression Of ?-Mannanase Genes From Aspergillus Niger NG1306

Dendrobium polysaccharides are not easily digested and absorbed by the intestinal tract due to their large molecular weight,high polarity and poor oral absorption.?-Mannanase hydrolyzes them into mannan oligosaccharides,which can significantly improve their bioavailability.The Aspergillus niger NG1306 isolated in the previous work of the thesis was used as the starting strain.A single univariate method was used to explore the optimal conditions for solid-state fermentation to produce?-mannase,and the TLC method was used to analyze the product types of polysaccharides hydrolyzed by the crude enzyme solution.Dendrobium polysaccharide was used as the sole carbon source to induce enzyme production by Aspergillus niger.The differentially expressed genes were analyzed by transcriptome sequencing technology,and the screened?-mannanase genes were cloned and heterologously expressed,and the enzymatic properties of the recombinase were studied.The results showed that the most suitable fermentation medium was perlite:dendrobium residue=1:1(m/m),2.0%dendrobium polysaccharide,2.0%(NH₄)₂SO₄,and initial moisture content of 75%.Under these conditions,the protein concentration of the crude enzyme solution obtained by fermentation was 2.5 mg/m L,and the enzyme activity was 150.75 U/m L.TLC analysis showed that after reaction at 50°C for 2 h,dendrobium polysaccharides were not completely hydrolyzed,and the products were mainly oligosaccharides such as mannobiose,trisaccharides,tetrasaccharides,and hexasaccharides;when the reaction was 6 h,dendrobium polysaccharides were completely hydrolyzed to produce a large amount of mannobiose,a small amount of monosaccharides and other oligosaccharidesBased on transcriptome analysis,six mannanase genes ABL-00818,ABL-03475,ABL-03917,ABL-06152,ABL-09166 and ABL-10129 were successfully cloned from Aspergillus niger,among which ABL-03475,ABL-03917 and ABL-09166 were successfully expressed in prokaryotic hosts,but only ABL-03475 was active.The analysis of enzymatic properties shows that the optimum temperature of ABL-03475 is35°C,the optimum p H is 8.0,and the highest enzyme activity is 68 U/m L.TLC analysis shows that ABL-03475 can hydrolyze dendrobium polysaccharides into mannhexaose,tetrasaccharide,trisaccharide,disaccharide and a small amount of monosaccharide..In this study,by optimizing the medium conditions of solid-state fermentation,the optimal conditions for the production of?-mannanase by solid-state fermentation of Aspergillus niger were obtained.Based on transcriptome analysis,we successfully cloned the?-mannanase gene from Aspergillus niger,explored its enzymatic properties through heterologous expression,and analyzed the main types of hydrolysates by TLC,which will lay the foundation for the efficient preparation of dendrobium oligosaccharides in subsequent experiments.