title: Characterization of two truncated forms of xylanase recombinantly expressed by Lactobacillus reuteri with an introduced rumen fungal xylanase gene.
abstract: 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 β-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.
authors.affiliation: Department of Biological Science & Technology, National Pingtung University of Science and Technology, Pingtung 912, Taiwan, ROC.
authors.forename: Hsueh-Ling
authors.initials: HL
authors.lastname: Cheng
authors.affiliation: Department of Nutrition and Health Science, Fooyin University, Taliao Hsiang, Kaohsiung 831, Taiwan, ROC.
authors.forename: Chun-Yi
authors.initials: CY
authors.lastname: Hu
authors.affiliation: Department of Biological Science & Technology, National Pingtung University of Science and Technology, Pingtung 912, Taiwan, ROC.
authors.forename: Shiou-Hua
authors.initials: SH
authors.lastname: Lin
authors.affiliation: Department of Biological Science & Technology, National Pingtung University of Science and Technology, Pingtung 912, Taiwan, ROC.
authors.forename: Jing-Ya
authors.initials: JY
authors.lastname: Wang
authors.affiliation: Institute of Biotechnology and Department of Animal Science, National Taiwan University, Taipei 106, Taiwan, ROC.
authors.forename: Je-Ruei
authors.initials: JR
authors.lastname: Liu
authors.affiliation: Department of Biological Science & Technology, National Pingtung University of Science and Technology, Pingtung 912, Taiwan, ROC. Electronic address: ox@mail.npust.edu.tw.
authors.forename: Yo-Chia
authors.initials: YC
authors.lastname: Chen
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D004795:Enzyme Stability; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D005800:Genes, Fungal; D006868:Hydrolysis; D057927:Hydrophobic and Hydrophilic Interactions; D007700:Kinetics; D052200:Limosilactobacillus reuteri / Q000201:enzymology* / Q000235:genetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017433:Protein Structure, Secondary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012417:Rumen / Q000382:microbiology*; D014995:Xylosidases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D005656:Fungal Proteins; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D014995:Xylosidases
keywords: Gene modified lactobacilli; Heterogeneous expression; Lactobacillus reuteri; Xylanase