Isolation Of Cellulose-degrading Microorganisms And Cellulase Genes' Cloning And Expression Based On Metagenomic

Global energy shortages,irrational utilization of resources,and environmental pollution continue to threaten the survival and development of human beings.Finding and developing renewable resources is an effective way to solve these problems.Cellulose is the most abundant and widely distributed renewable resources on the earth.It's hydrolysis is the key to solving the current crisis.However,due to the generally low cellulase activity and poor temperature adaptability.These have always restricted the industrial production of cellulase.In Inner Mongolia aera,there is abundant cellulose resources,and the average annual temperature is low and the cold season is long.To the characteristics of the region,the study collected forest soil from Jiufengshan,Daxinganling,and Saihanwula,determined the CMCase activity in the method of DNS,discoved and expressed cellulase gene based metagenome sequencing,to provide bacteria and genetic materials for cellulose resources utilization.The main results and conclusions of this study are as follows:1.A total of 217 strains of cellulose-degrading bacteria were obtained: 92 bacteria and 42 fungal strains at room temperatureand 83 bacteria and 0 fungal strains at low temperature.Cellulose-hydrolyzing bacteria of room temperature belong to 4categories,namely Proteobacteria(46%),Actinobacteria(38%),Firmicutes(11%),Bacteroidetes(5%).Cellulose-hydrolyzing fungi of room temperature belong to three categories,namely Ascomycota(84%),Basidiomycota(12%)and Glomeromycota(4%).Cellulose-hydrolyzing bacteria of low-temperature were distributed in 4categories,including Actinobacteria(54%),Proteobacteria(23%),Firmicutes(9%)and Bacteroidetes(14%).2.The CMC activity of low-temperature cellulose-degrading bacteria was generally low,with a maximum of only 21.63 U/m L.The cellulolytic enzyme activity of fungi of room temperature was generally high,with a maximum 112.76 U/m L.The activity of room temperature bacteria was between them.3.The results of metagenomic sequencing of low temperature-enriched soil showed that the most dominant bacteria was belonging to the Actinobacteria,totaly215,696 genes related to cellulose degradation were obtained through annotatedcarbohydrate active enzymes,accounting for 7.11% of the total sequences.4.Cloning and expression of cellulase genes: Four cellulase gene sequences were successfully subcloned and expressed.Recombinase of 23-1,93-5,23-5 and 103-2had the highest activity for the cellulose substrate CMC,p NPX,microcrystalline cellulose and p NPX,respectively,and the optimum temperatures were 50 ?,40 ?,55 ? and 40 ?,respectively;The appropriate p H is 5.0,6.0,9.0 and 5.0.