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

Paper ID Volume ID Publish Year Pages File Format Full-Text
16998 42630 2014 5 PDF Available
Title
Characterization of two truncated forms of xylanase recombinantly expressed by Lactobacillus reuteri with an introduced rumen fungal xylanase gene
Abstract

•Two truncated xylanases with different lengths were expressed by Lactobacillus reuteri and confirmed.•The truncation of expressed product affects the hydrophobicity and secondary structure of recombinant xylanases.•The enzymes properties of recombinant xylanase were also changed due to the proteolysis of C-terminal residues by L. reuteri.

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.

Keywords
Gene modified lactobacilli; Heterogeneous expression; Lactobacillus reuteri; Xylanase
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Characterization of two truncated forms of xylanase recombinantly expressed by Lactobacillus reuteri with an introduced rumen fungal xylanase gene
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Publisher
Database: Elsevier - ScienceDirect
Journal: Enzyme and Microbial Technology - Volumes 64–65, October 2014, Pages 6–10
Authors
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Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering
Get Full-Text Now
Don't Miss Today's Special Offer
Price was $35.95
You save - $31
Price after discount Only $4.95
100% Money Back Guarantee
Full-text PDF Download
Online Support
Any Questions? feel free to contact us