Diversity Analysis Of Cellulase Genes In Changbai Mountain Forest Soil And Expression Of The Genes Cloned Directly From The Soil Metagenomic DNA

Looking for the clean and renewable alternative energy is an important research and development direction in the 21st century. Biodegradation of cellulose is an ideal means to produce clean renewable biofuels. Screening for new cellulases with excellent properties has important ecological significance and application value. Natural environment, especially soil environment, as the most abundant microbial resource, could be one of the important screening sources for cellulase genes. Well-preserved Changbai mountain nature reserve is an ideal soil sample collection zone. Using homologous amplification and TAIL-PCR, we studied the diversity of Changbai mountain forest soil cellulase genes, screening new cellulase genes successfully, and completed the prokaryotic expression, purification and characterization of the recombinases. This study provided the experimental data about the research between cellulase structure and function, and provided necessary theoretical basis to help people to better use and modification about cellulases. The results of this research are as follows:1. The Changbai mountain forest soil GH7_cbhI and GH48 family cellulase gene libraries were constructed, and the genetic diversity of two libraries were studied.Using two pairs of degenerate primers, the Changbai mountain forest soil cellulase GH7_cbhI and GH48 family gene libraries were constructed respectively, and analyzed the diversity and phylogenetic of gene families through lots of sequencing. The results shown that the genetic diversity of GH7_cbhI family is higher than GH48 family, the sequence similarity was between 99-38%,95% gene sequences which with the highest genetic similarity were from culture-independent fungi of Ascomycetes and Basidiomycete. GH48 family gene library sequences shown "unipolarization", that is more than 80% sequences are very conservative, which with the highest sequence similarity with enduglucanase sequence of Herpetosiphon aurantiacus. Compared with the reported gene libraries, the genetic diversity of the Changbai mountain soil gene library are similar to corn straw library, and different with marine sediment library respectively. The great genetic diversity proved that Changbai mountain forest soil is a potential repository of cellulase gene.2. Obtained multiple GH48 and GH5 family cellulase gene sequences by using the methods of homologous amplification and TAIL-PCR, and the sequences structure were analyzed.Two GH48 endoglucanase gene sequences, six GH5 endoglucanase gene sequences were cloned. Using the methods of homologous amplification and TAIL-PCR, the GH48 cellulase gene cel48_hm01 was cloned from the natural soil environment for the first time. The sequence had the highest similarity （62%） with endoglucanase sequence from Herpetosiphon aurantiacus [WP₀12187855.1]. cel48_hm01 sequence included the binding domain CBM2 and the catalytic domain of GH48. Then the gene cel48_hmO2 was cloned, which included single catalytic domain of GH48.Using the method of homologous primers amplification from soil samples genomic DNA directly, six GH5 endoglucanases gene sequences were amplified: egl01, egl02, egl03, egl04, egl05, egl06. egl01 sequence was shorter 17 amino acid than egl02 on N-terminal sequence only, but these two sequences were all contains N-terminal catalytic domain GH5 and C-terminal binding domain CBM3. These two sequences all had highest similarity with enduglucanase sequence from Bacillus licheniformis,99% and 99% respectively. egl03, egl04, egl05 and egl06 were all different between egl02 in some amino acid sites, while all had 99% similarity with Bacillus licheniformis cellulase sequence. These genes, could be ideal natural mutants provided for research about the relationship of GH5 cellulase protein structure and function.3. The GH48 family cellulase genes cloned from the soil environmental samples directly were expression and purification for the first time, enzymology properties of recombinases were detected and homologous models were builted.The GH48 family cellulase genes cloned from the soil environmental samples directly were prokaryotic expression and purification for the first time. Characterization of recombinases proved the acidophily and great thermal stability of recombinant Ce148_hm01 and Ce148_hmO2. The optimal pH and reaction temperature of recombinases were 6.0,50℃. In pH 8.0-10.0 range and dealed 3 h under 70℃, recombinases can kept more than 50% activity. A certain concentration of metal ions Na+, K+, Ca2+, Fe3+ can significantly promoted the activity, which means recombinases also have great substrate specificity. Homology modeling results shown that recombinases Ce148 hm01 and Ce148 hm02 had a model of Clostridium cellulolyticum enduglucanase CelF （PDB:lfbw）, the protein advanced structural homology were above 58%. Recombinases Cel48_hm01 and Cel48_hm02 all that the recombinases may also adopted the same catalytic mechanism with GH48 cellulase from Fusobacterium sp.. The results above provided experimental data to study the relationship of GH48 family protein structure and function.4. The GH5 family cellulase EgI01 was prokaryotic expressed and charactered, some properties of structure-domain mutants of EgI01 were detected, and activity of site-directed mutants of EgI01 were studied.Characterization shown that the highest activity of EgI01 was at the condition of pH 5.0,50℃. Under the condition of pH 3.0-9.0,4-50℃, the activity was stable. In the presence of high concentrations of metal ions, organic reagents, surfactants and salt ions, EgI01 activity was still very stable.3 M KC1 and 2 M NaCl increased the activity to 135% and 135% respectively. In addition, under the condition of 1-4 M KCl/NaCl for 6 d, the activity remained over 70%. EgI01 could hydrolyzed carboxymethyl cellulose, beechwood xylan, corn stover, laminarin, filter paper and crystalline cellulose, hinted that it had a broad substrate specificity.Based on the sequence analysis of EgI01, a short peptide of C-terminal-NGKLIWGTEPK, may be associated with the acidophily of EgI01. After deleting the short peptide, the acidophily of mutant did not change significantly, which shown that this short peptide was not the directly structure foundation of EgI01. After deleting the binding domain CBM, the substrate specificity of mutants changed dramatically, which lost the hydrolysis ability of insoluble substrates. The result proved the integrity of the structure of CBM is important to the activity of EgI01.The natural mutants of EgI01 were expressed and charactered, the result shown that the properties did not change significantly, which also means these mutant sites are not the key sites of GH5 family cellulase. BLASTed with available cellulases of GH5 family, five key amino acid sites in the groove structure may be related to the substrate combination of cellulase. The researches about thses site-directed mutants shown that these amino acid sites do play an important role in cellulase activity, which the change of a single site can cause significant reduction of activity or even loss. These results provided the information for the research of modification and action mechanism of GH5 family cellulase.