Direct cloning of genes encoding novel xylanases from the human gut.
The aim of this study was to identify a novel 1,4-beta-xylanase gene from the mixed genome DNA of human fecal bacteria without bacterial cultivation. Total DNA was isolated from a population of bacteria extracted from fecal microbiota. Using PCR, the gene fragments encoding 5 different family 10 xylanases (xyn10A, xyn10B, xyn10C, xyn10D, and xyn10E) were found. Amino acid sequences deduced from these genes were highly homologous with those of xylanases from anaerobic intestinal bacteria such as Bacteroides spp. and Prevotella spp. Self-organizing map (SOM) analysis revealed that xynA10 was classified into Bacteroidetes. To confirm that one of these genes encodes an active enzyme, a full-length xyn10A gene was obtained using nested primers specific to the internal fragments and random primers. The xyn10A gene encoding the xylanase Xyn10A consists of 1146 bp and encodes a protein of 382 amino acids and a molecular weight of 43,552. Xyn10A was a single module novel xylanase. Xyn10A was purified from a recombinant Escherichia coli strain and characterized. This enzyme was optimally active at 40 degrees C and stable up to 50 degrees C at pH 6.5 and over the pH range 4.0-11.0 at 25 degrees C. In addition, 2 ORFs (ORF1 and ORF2) were identified upstream of xyn10A. These results suggested that many unidentified xylanolytic bacteria exist in the human gut and may contribute to the breakdown of xylan which contains dietary fiber.
Hayashi
Hidenori
H
Microbe Division/Japan Collection of Microorganisms, RIKEN BioResource Center, Wako, Saitama. hayashi@jcm.riken.jp
Abe
Takashi
T
Sakamoto
Mitsuo
M
Ohara
Hiroki
H
Ikemura
Toshimichi
T
Sakka
Kazuo
K
Benno
Yoshimi
Y
D000465:Algorithms; D000595:Amino Acid Sequence; D001421:Bacteria, Anaerobic / Q000201:enzymology* / Q000235:genetics; D003001:Cloning, Molecular; D019295:Computational Biology / Q000379:methods; D004269:DNA, Bacterial / Q000032:analysis / Q000302:isolation & purification*; D004064:Digestive System / Q000382:microbiology*; D043364:Endo-1,4-beta Xylanases / Q000737:chemistry / Q000235:genetics*; D005243:Feces / Q000382:microbiology; D006801:Humans; D008969:Molecular Sequence Data; D016571:Neural Networks, Computer; D010802:Phylogeny; D016133:Polymerase Chain Reaction; D017422:Sequence Analysis, DNA
D004269:DNA, Bacterial; D043364:Endo-1,4-beta Xylanases