Genetic determination of xylanases in rumen bacterium ruminococcus flavefaciens
The xylanases and xylanase genes in the rumen cellulolytic bacterium Ruminococcus flavefaciens were studied in this thesis. Based on the experiments carried out in this thesis, the following conclusions can be drawn: At least seven multiple xylanases are revealed in R.flavefaciens strain 17 and strain 007, and nine in strain FD-1 by the method used here for activity detection in gels. A im54 kDa constitutive xylanase appears in both strains 17 and 007, but not in FD-1. Straw and xylan are the best substrates for production of most xylanases in R.flavefaciens. Sequencing one of the four xylanase genes isolated from R.flavefaciens 17, xynA, shows a 2862 bp open reading frame initiating from a TTG start codon which is preceded by a Gram-positive Shine-Dalgarno sequence (robosome binding site) of AAAGGAG. The enzyme encoded by xynA (XYLA) is predicted to have 954 amino acids including a probable signal sequence at the amino terminus. XYLA is novel in its structure having two dissimilar catalytic domains (domain A, 248 amino acids; domain C, 332 amino acids) linked by a highly repetitive region (domain B, 374 amino acids) extremely rich in asparagine and glutamine residues. The two catalytic domains can be active independently and act on xylan differently, with domain C producing smaller end products than domain A from oat-spelt xylan. Amino acid sequence comparisons with other enzymes show that domain A and domain C are related to two different families of xylanases, G and F, respectively. Therefore, this thesis provides the first evidence of a bifunctional hemicellulase comprising two different catalytic domains. Antibodies raised separately against domains A and C of XYLA recognize common enzyme bands in Ruminococcus, ranging in apparent molecular mass from 110 kDa to 200 kDa. This tends to confirm that XYLA is produced as a high molecular weight polypeptide in R.flavefaciens, as predicted from the sequence. R.flavefaciens has strong preference of codon usage for several amino acid residues, eg. glutamate (GAG); glutamine (CAG); and lysine (AAG).