| Currently, the global energy-sources shortage and environmental pollution are worsening and one effective solulution of these problems is depended on the exlporation and utilization of renewable resource. Cellulose is the most abundant renewable resources on this planet. Solely efficient cellulose-degrading enzyme is cellulase. But cellulases currently used are defective in some characters, such as low activity and poor temperature adaptability, and etc. To screen microorganisms with high activity of cellulose-degrading, chatacterize the cellulase and clone and express cellulose gene are still important to study.Inner Mongolia, located in the temperate regions, the perennial average annual temperature is low and low-temperatre season is long. Therefore, based on regional characteristics, this study isolated cellulose-degarading microbes from soil of different regions of Inner Mongolia under low temperatures and room temperature conditions, performed cellulase feature analysis and gene cloning and expression. This work may provide the basis for resolving the relationship between the structure and function of cellulase and supply cellulose-degarading strins for wasted material reclamation. The main results and conclusions of this study were as followings:1)From the samples collected from different locations of rotten woods and underneth soil of Alxa, Daxinganling and Chifeng decadent leaves and soil, we obtained a total of 246 cellulose degrading isolats, including bacterial strains of 100 at 28℃,82 ones at 10℃ temperature and fungi 64 at 28℃. Bacterial strains, isolated at 28℃, belonged to five phylums, Actinobacleria (34%), Proteobacteria (α-Proleobacteria 9%; β-Proteobacteria 7%; γ-Proteohacteria 25%), Firmicutes (19%), Bacteroidetes (5%), Acidobacteria (1%), Bacterial strains, isolated at 10℃, belonged to four phylums, Actinobacleria (21%), Proteohacteria (α-Proteobacteria 11%;β-Proleobacteria 7%; γ-Proteohacteria 40%), Firmicutes (5%), Bacteroidetes (16%), Among 64 isolated cellulose degrading fungus, there are 54 belong to the Ascomycota (84%) of the groups:other strains belonged to Basidiomycota (8%). Glomeromycota (6%), Chytridiomycota (2%).2)3.5-dinitrosalicylic acid method was applied to analyze the the activity of endoglucanase (carboxymethyl cellulase, CMCase).Among all of isolates, fungus strain the CMCase activity of DAZJ13 was highest. Growth temperature rang of was 25℃-35℃, and in which it could produce CMCase. Cultured for four days at 30 ℃, the highest activity was observed; pH effects on the activity showed that the fastest rate of CMC utilization was at pH 6; when yeast extract, beef extract, and ammonium sulfate were used as a nitrogen source, strain DAZJ13 cltured at different pH and incubation time, the highest activity occued in the presence of yeast extract. So optimum fermentation conditions of strain DAZJ13:pH value of 6,30℃ for 4 days, yeast extract as nitrogen source.3)The similarity of 16S rRNA gene sequence of strain DW12 determined by Blast-n program showed a higest value of 94% to that of type species, Sphingobacterium spiritivorvm JCM 1277T. Biolog Microbial Identification System of the identification results demonstrated the closest relative to DW12 was Sphingobacterium thalpophilum, but the SIM value is only 0.582. These results suggested that DW12 belonged to Sphingomonasand was a potential novel species.The statistics of DW12 genome sequencing:total reads number 6154720 x 2, read length of 101 bp, total bases 1243253440 bp, library with an average insert length of 300 bp, the average coverage of 233.7 x; G+C content of 41.02% of the total bases; scaffolds number 73, number of large scaffords(> 1000 bp) has 42; Scaffold N50 is 421003 bp; Scaffold N90 is 95730 bp; contigs number 101, number of large contigs(> 1000 bp) has 46; Contig N50 is 421003 bp; Contig N90 is 93754 bp. DW12 genome annotation showed that there are 101 Contigs,4429 coding sequences,54 RNA. Note showed interest in this experiment cellulose degradation genes have six.Strain DW12 gene cloning expression:E3565 gene heterologous expression in Eschrichia Coli BL21, the expression of the protein product of the predicted molecular size matches 30kD. And to determine the optimum conditions for inducing IPTG concentration of 1.0mmol/L, about 16℃ culture, a lot of cellulase gene expression after induction 5h; recombinase for cellulosic substrate highest activity is microcrystalline cellulose, the temperature stability of the purified protein showed good stability at below 50℃, the optimum temperature is 40℃. more stable pH8.0-10.6. the optimum pH of 9. |
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