Characterization of two truncated forms of xylanase recombinantly expressed by Lactobacillus reuteri with an introduced rumen fungal xylanase gene.

The xylanase R8 gene (xynR8) from uncultured rumen fungi was cloned and successfully expressed in Lactobacillus reuteri. A xylanase activity of 132.1 U/mL was found in the broth of L. reuteri R8, the transformant containing pNZ3004 vector with xynR8 gene insertion. Two distinct forms of recombinant xylanase with different hydrophobicities and molecular weights were found in the broth after purification. According to the results of Western blotting, only the T7-tag, fused in the N-terminus of XynR8, could be bound to the expressed proteins, which indicated that the C-terminus of XynR8 had been truncated. These results, combined with tryptic digestion and mass spectrometry analyses, allow us to attribute the two xylanase forms to an optional cleavage of C-terminal sequences, and XynR8A, a 13 amino acid residues truncated form, and XynR8B, a 22 amino acid residues truncated form, were the main products in the extracellular fraction of L. reuteri R8. The specific activities of XynR8A and R8B were 1028 and 395 U/mg protein. Both forms of recombinant xylanase displayed a typical endoxylanase activity when they were reacted with xylan, but XynR8A demonstrated a better specific activity, catalytic efficiency and thermostability than XynR8B according to the results of enzyme characterization. These changes in enzyme properties were highly possibly caused by the present of the ß-sheet in the C-terminal undeleted fragment of XynR8A. This study demonstrates that modified forms with different enzyme properties could be produced when a gene was recombinantly expressed by a L. reuteri transformant.

Direct cloning of a xylanase gene from the mixed genomic DNA of rumen fungi and its expression in intestinal Lactobacillus reuteri.

A relatively newly defined xylanase gene, xynR8, was obtained directly from a mixed DNA sample prepared from unpurified rumen fungal cultures by PCR amplification. The DNA sequence of xynR8 revealed that the gene was 884 bp in size and encoded amino acid sequences with a molecular weight of 27.9 kDa. XynR8 belonged to glycosyl hydrolase family 11, and the catalytic site residues were also found in its amino acid sequence. The main hydrolysis products of XynR8 were xylobiose, xylotriose and xylotetrose, which indicated that it belonged to the endoxylanases. The xynR8 gene was constructed so as to express and secrete under the control of the Lactococcus lactis lac A promoter and its secretion signal, and was transformed into L. reuteri Pg4, a strain isolated from the gastrointestinal tract of broiler chickens. The L. reuteri transformants harboring xynR8 not only acquired the capacity to break down xylan, but also maintained their high adhesion efficiency to mucin and mucus and their resistance to bile salts and acid.