Fiber Heap Capsule Bacteria So9733-1 Xylanase Gene Cloning, Characterization And Heterologous Expression Analysis

Out of 11 cultivable myxobacterial genera, Sorangium is the only one that is able to degrade cellulosic materials. Till now, Studies on Sorangium have mainly been carried out on its intriguing, novel and diverse bioactive metabolites and the genes for synthesis of these metabolites. However, to our knowledge no studies on the cellulolytic activity, the unique and basic property of Sorangium among myxobacteria have been reported yet.Our previous studies show that sorangium cellulosum can be cultivated on simple inorganic salt medium with sterilized filter paper (CNST) as the only carbon sources and can finally degrade the filter paper significantly. The results from enzymatic activity assay studies indicate that the cellulolytic activities are linked to cellular surfaces and organized into a large complex, which contains at least cellulase and xylanase activities. And maybe just for this reason, the enzyme activity of the separated and purified complex components is usually too low to do further biochemical research. In this study the enzyme component was studied using molecular biological methods of gene cloning , gene characterization and gene expression etc. The work has established a primary foundation for further research on the cellulolytic system of Sorangium cellulosum .After enzymatic analysis of the sorangium cellulosum isolated from soil samples by our lab, we find that cellulolytic activity is the common property among them and the cellulolytic ability is different among different strains.Based on the upper results and the previous studies, we selected Sorangium cellulosum 9733-1 (So9733-1) as a model strain and selected the molecular biological study of xylanase as the start of our subsequent work for studying cellulose-degrading systems of sorangium. After Polymerase Chain Reaction (PCR) using ten different primer pairs, we found that a 422bp gene fragment encoding xylanase could be cloned successfully from So733-1 genomeDNA by PCR method with primer pair GHlO-f/GHlO-r. Sequence analysis with BLAST tools showed that the gene fragment cloned belongs to Glycoside Hydrolase family F/10. and xylanase fragment of this kind with different length and amino acid composition was also cloned from other sorangium cellulosum using the same method. The universality and diversity of these xylanase gene fragemts are consistent with the upper results of cellulolytic activity. Based on the sequences of F/10 xylanase genes in GenBank, neighbor-joining method was adopted to examine the phylogenetic relationships among other bacteria and fungi. The similar trees showed that the cloned xylanase fragment locates in the separate branche and should be a novel F/10 xylanase.After fully digestion of So9733-1 genomic DNA with Sal I ,Using the 422bp fragment as a probe and with the methods of southern blotting> DNA library' construction and dot blotting, we finally succeed to get two complete xylanase genes named xynA and xynB. Their open reading frame were 1302bp> 1197bp encoding 433 ^ 398 amino acids respectively. In comparison with GenBank data, the similarities of amino sequence were 60% and 70% respectively.Based on the multiple cloning sites(MCS) of expression vector, two expression primers, upstream primer containing a Ndel restriction site and downstream primer including a Xliol restriction site, were designed corresponding to open reading frame of the cloned xynA and xynB genes.The amplified PCR fragments were double digested with restriction endonucleases, and target fragments were recovered. Then they were inserted into pET-22b(+) vector at Ndel and Xhol restriction sites, respectively. The recombinant expression plasmids were transformed into E.coli BL21(DE3). Two positive recombinants BL-pET-xynA and BL-pET-xynB were both expressed as inclusion bodies after induced by IPTG . After refolded by dialysis, part of the purified inclusion bodies became soluble with enzyme activity of xylanase.But the enzyme activity of BL-pET-xynA was too low to do further enzymatic study. Some properties of xylanase expressed from recombinant BL-pET-xynB hadbeen studied.Km is 1.2mg/ml when the substrate is xylan. Optimum temperature of the xylanase is 45 °C , and it almost retaines none of the original activity at temperature higher than 55XU for 30min, not heat-resistant. Optimum pH is 6.5, and the xylanase showes to have a good activity over a broad pH range from 4.0 to 8.0. The effects of various metal ion on the xylanase activity are different, the xylanase activity is inhibited by 5mmol/l Zn2+% Cu2+ and improved by lmmol/1 Na+ ^ K+. The result from TLC indicates that the xylanase can hydrolyze xylans into short chain xylo-oligosaccharides (mainly xylobiose-, xylotriose and xylotetraose ) and it should belong to endo-xylanase.