Cloning And Expression Of Xylanase Gene Of Uncultured Microorganism From Hu Sheep

Metagenome has been a most interesting area since most microorganisms are unculturable.A Fosmid metagenomic library of uncultured microorganism from Hu sheep rumen was constructed,which contained 12,000 clones and the capacity of inserted DNA was 3.6×10~8 bp.Two clones expressing endo-1,4-beta-xylanase activity were isolated and one clone designated UMXYL2 was characterized by subcloning and sequencing.Three new putative endo-1,4-beta-xylanase gene were cloned which were named as UMXYL2-1-1,UMXYL2-1-2 and UMXYL2-6-1 respectively,for further study of the degradation mechanism of xylanase and to build a xylanase library for different sources of xylan.The UMXYL2-1-1 gene was comprised of 1,086 bp with a G + C content of 53.78%and a melting temperature at 86.49℃.It encodes a protein of 361 amino acids with a molecular mass of 38,974.2 daltons, and its pI is 8.56.The UMXYL2-1-2 gene was comprised of 849 bp with a G + C content of 52.41%and a melting temperature at 85.80℃.It encodes a protein of 282 amino acids with a molecular mass of 31,039.2 daltons,and its pI is 8.52.The UMXYL2-6-1 gene was comprised of 1,278 bp with a G + C content of 50.23%and a melting temperature at 85.11℃.It encodes a protein of 425 amino acids with a molecular mass of 45,777.8 daltons,and its pI is 8.72.Deduced amino acid sequence analysis of three genes indicated that all of them were from glycosyl hydrolases family 11.The amino acid sequence have significant similarly with xylanase from Fibrobacter succinogenes(accession no.AAA21848.1),with identies of 67%,45% and 62%respectively.They shared great similarity in the middle region of the sequence,and was identified as the activity center.Since gene UMXYL2-1-1 and UMXYL2-1-2 were from the same subclone UMXYL2-1 and they were only 205bp away,they were properly existed as the functional gene cluster.Phylogenetic analysis shows that UMXYL2-1-1,UMXYL2-1-2 and UMXYL2-6-1 might come from Fibrobacter succinogenes,or a uncultured species of Fibrobacter.DNA sequences containing the DNA region encoding the catalytic domain of UMXYL2-1-1 and UMXYL2-1-2 were amplified by PCR, cloned into expression vector pET 28a(+) respectively,and successfully expressed in E.coli BL21.The conditions for UMXYL2-1-2 expression in BL21 and its enzyme properties have been investigated.The result shows its optimal temperature for induction was 25℃.The optimal pH was 4.5, and the optimal temperature was 40℃.The enzyme activity at pH 4.5 and 40℃was 24.63 u/mL.