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.
Liu
Je-Ruei
JR
Department of Biotechnology, National Formosa University, Yunlin, Taiwan, ROC.
Yu
Bi
B
Lin
Shiou-Hua
SH
Cheng
Kuo-Joan
KJ
Chen
Yo-Chia
YC
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D004271:DNA, Fungal / Q000235:genetics; D043364:Endo-1,4-beta Xylanases / Q000235:genetics* / Q000378:metabolism; D005658:Fungi / Q000201:enzymology; D007778:Lactobacillus / Q000235:genetics* / Q000502:physiology; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D012417:Rumen / Q000382:microbiology*
D004271:DNA, Fungal; D043364:Endo-1,4-beta Xylanases