Isolation, Characterization And Related Gene Cloning Of Xylan-degrading Bacterias From Adult Sow Hindgut

Most xylanase products in feed show lower stablity in animal gastrointestine. It is very important that to exploit a new type of xylanase with high stablity in animal gastrointestine for improving feed enzyme application effects. In this thesis, isolatation xylan-degrading bacterias with excellent xylanase characteristies and related gene cloning were carried out to develop a new kind of xylanase for feed industry.Isolatation and identification of xylan-degrading bacterias. Two xylanase-secreting strains named ZF and MZ were isolated from the large intestine of high roughage-resistance Inner Mongolia black adult sows by clear zone methods using xylan as sole carbon source. ZF and MZ were Cellulomonas flavigena and Paenibacillus sp. respectively which are facultative anaerobe strains, identificated by morphology observation and 16S rDNA sequence analysis.The growth and enzyme production curve were researched and the NDF and ADF degradation of forages (alfalfa, hay and corn silage) by ZF and MZ and rumen liquid were also compared. Results show that, the maximum growth and xylanae activity time of ZF is 48h and 60h, and 24h and 60h for MZ, respectively. ZF and MZ showed higher xylanase production and were increased by 51.3 and 16.8 times with three kinds of forages (alfalfa, hay and corn silage) mixture as sole carben sources than with xylan. Rumen fluid showed higher NDF and ADF degradation rate of three forages than ZF and MZ, however, ZF shows higher NDF degradation ability of hay than rumen fluid microorganisms. There is no significant differences between ZF and MZ in NDF and ADF degradation of three forages.The characteristies of crude xylanase secreted by ZF and MZ was also investigated. The optimum temperature and pH of xylanase produced by ZF and MZ were 55℃and 60℃and 7.0 and 6.0, repctively. Both xylanases were stable over a broad range of pH from pH 3.0 to 9.0 and resistant to proteases (pepsine and trypsine) in gastrointestine and 80% of the activity remained when treated at 40℃for 60 min. The xylanase activity from MZ was strongly inhibited by Fe²⁺,Mn²⁺ in buffer at pH 6.0 and incubation temperature 40℃and inhibited by Cu²⁺,Zn²⁺, but strongly enhaced by Ca²⁺,Mg²⁺ . The xylanase activity from ZF was not affected by all metal ions.Two pairs of degenerate primer were designed based on the family 10 glycosyl hydrolase (GH10) and the family 11 glycosyl hydrolase (GH10) conserved domains. Touch-down PCR and thermal asymmertric interlaced (TAIL)-PCR were used to amplify full-length xylanase gene. Partial xylanse gene 11ZF-2 (1931 bp) from Cellulomonas flavigena stain ZF was showed 84% identity with Cellulomonas flavigena DSM 20109 family 11 glycosyl hydrolase. Partial xylanse gene 10MZ-2 (1860bp) from Paenibacillus sp. stain MZ was showed 78% identity with Paenibacillus sp.oral 786 str.D14 xylanase A which belongs to family 10 glycosyl hydrolase.