Screening Of Cellulase-producing Bacteria From Mangrove Habitat And The Recombinental Expression Of Functional Genes

Cellulases have an important role in biological degradation of plant cell wall polysaccharides.These plant cell wall polysaccharides are comprised of cellulose(35-50%),hemicellulose(20-35%)and lignin(5-30%),hemicellulose(20-35%)and lignin(5-30%).Cellulose is the major carbohydrate synthesized by plants.Therefore,the degradation of cellulosic is an important part of the carbon cycle within the biosphere.In this process,cellulase plays a very important role.Biotechnologists are interested in using cellulolytic enzymes to degrade and treat cellulose in industrial production to obtain the required carbohydrate.However,there are many problems in industrial cellulase,such as low enzyme activity and high cost.Therefore,it is of great practical significance to utilize and excavate microorganisms that can degrade cellulose efficiently.Mangrove forests are regularly soaked in seawater and empty in space due to their special geographical location.With the accumulation of a large number of plant residues and fallen leaves,it continuously provides nutrients such as cellulose for microorganisms.Therefore,obtaining Cellulase Producing Microorganisms from mangrove habitat has a broad research prospect.In this paper,two strains of Bacillus sp.were obtained by screening cellulose degrading bacteria,and the recombinant expression of endoglucanase and its enzymatic propertie were studied.With the help of whole genome sequencing,the important functional proteins and the gene resources of Bacillus sp.Cellulolyticus were excavated.The results are as follows:1.Screening,identification and characterization of Bacillus sp.producing enzymeIn this study,two Bacillus sp.strains of cellulose degradation were successfully screened from the soil samples in Mangrove Natural Reserve,Zhangzhou,which were ms-2 and ms-22,respectively.The isolates were identified as Bacillus subtilis ms-2 and Bacillus xiamenensis ms-22 according to its morphological,physiological and biochemical characteristics and the 16S rDNA sequencing result.The highest enzyme activity of Bacillus subtilis ms-2 was 0.6557 U/mL in LB medium for 24 hours.The optimum reaction temperature and pH of cellulase produced by ms-2 were 65? and 6.0,respectively.The enzyme has a wide range of pH tolerance and is suitable for reaction in neutral and slightly acidic conditions.The hydrolysis circle of Bacillus xiamenensis ms-22 was found in the primary screening plate for cellulase production with CMC-Na as the sole carbon source.However,when it contains other carbon sources or CMC-Na as the only carbon source liquid medium,the bacteria often do not grow or self dissolve earlier.The specific mechanism should be further studied.2.Expression of endoglucanase gene,purification and characterization of recombinant enzymesThe primers were designed according to the published cellulase gene sequence of Bacillus sp.on NCBI GenBank.Endoglucanase gene was successfully amplified from genomic DNA of Bacillus subtilis ms-2 and Bacillus xiamenensis ms-22,respectively.By analysis,the length of ms-2 gene fragment is 1542 bp,encoding 508 amino acids,N-terminal has signal peptide,and the expression product Celms2 has GH5/CBM3 structure and the size is about 56.5 kDa.The optimum reaction temperature and pH of Celms2 were 60? and 5.0,respectively,and the specific activity of cellulase was 20184.66 U/g.The enzyme has good heat-resistant property and poor tolerance to strong acid,so it is suitable to react in neutral and slightly acidic conditions,The length of ms-22 is 2398 bp,encoding 616 amino acids and no signal peptide.The expression product Celms22 has GH9/CBM3 structure and the size is about 69.6 kDa.The optimum reaction temperature and pH of Celms22 were 70? and 6.0,respectively,and the specific activity of cellulase was 7892,38 U/g.It has a good thermophilic activity and a wide temperature tolerance range.The enzyme has a poor tolerance to strong acid and is suitable for reaction under neutral and slightly acidic conditions.3.Whole genome sequencing of target strainsBacillus subtilis ms-2 and Bacillus xiamenensis ms-22 were used as the starting strains to complete genome sequencing.The results showed that the chromosome of ms-2 was circular,the size was 4 126 713 bp,the content of G+C was 43.79%,and there were 4103 predicted genes.It contains 458 carbohydrate active enzymes.After preliminary analysis,the chromosome of ms-22 was circular,the size was 3 662 658 bp,the content of G+C was 41.51%,and there were 3490 predicted genes.It contains 428 carbohydrate active enzymes.It was found that both strains had a highly efficient carbohydrate metabolism pathway,and the annotation results showed that there were a large number of glycoside hydrolase genes.Combined with the results of KEGG annotation,the related metabolic pathway map was drawn,and many enzymes(especially carbohydrate substances)related to carbohydrate metabolism were shown.4.Comparative genomic analysis of Bacillus sp.The phylogenetic relationship of ms-2 and ms-22 was confirmed by comparative genomic analysis of 6 Bacillus sp.,which indicated that ms-2 belonged to Bacillus subtilis and ms-22 belonged to Bacillus xiamenensis.The results showed that there were some homologies among the same species of Bacillus sp.,and the strains also had their own specificity.There are some enzyme genes related to lignocellulose degradation in this strain.It mainly includes the genes involved in the decomposition of cellulose and hemicellulose,and there are relatively few genes related to lignin degradation.Based on the isolation and identification of Bacillus subtilis ms-2 and Bacillus xiamenensis ms-22 from the soil samples in Mangrove Natural Reserve,Zhangzhou.The characteristics of cellulase produced by wild-type strains and endoglucanase expressed by engineering strains were analyzed.At the same time,the whole genome of ms-2 and ms-22 were sequenced and analyzed,and the genes related to carbohydrate active enzyme were deeply mined.To a certain extent,it promotes our efficient utilization and control of Bacillus sp..In addition,it provides us with convenience to understand the biological mechanism of Bacillus sp.strains.