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The metagenome-derived enzyme RhaB opens a new subclass of bacterial B type α-l-rhamnosidases

Paper ID Volume ID Publish Year Pages File Format Full-Text
22996 43409 2014 8 PDF Available
Title
The metagenome-derived enzyme RhaB opens a new subclass of bacterial B type α-l-rhamnosidases
Abstract

•Novel bacterial B type α-l-rhamnosidase from glycoside hydrolase family GH78.•Enzyme comprising α-l-rhamnosidase domain and GDSL-like lipase domain.•Multidomain hydrolytic enzyme from intestinal microbiome.

A combined sequence- and function-based analysis of a metagenomic library DNA derived from elephant feces led to the identification of a novel bacterial α-l-rhamnosidase belonging to glycoside hydrolase family 78 (GH78). The gene was designated rhaB (4095 bp) and encoded for a putative protein of 1364 amino acids. The C-terminal part of the enzyme revealed an amino acid (AA) sequence identity of 58% to a predicted bacterial α-l-rhamnosidase from Bacteroides nordii. Interestingly, the N-terminal region of the deduced enzyme RhaB contained a GDSL-like lipase motif and an acetyl-xylan esterase (DAP2) motif. While heterologous expression of the complete rhaB failed, subcloning of the gene identified the most active open reading frame (ORF) to be of 3081 bp, which we designated rhaB1. The enzyme RhaB1 was overexpressed in Escherichia coli BL21 (DE3) and was purified to an amount of 75 mg/L of culture medium. In accordance to the intestinal origin, RhaB1 showed a preference for mesophilic conditions with an optimum activity at a temperature TOpt of 40 °C and a pHOpt of 6.5, respectively. The recombinant protein had a Km value of 0.79 mM and a specific activity vmax of 18.4 U for pNP-α-l-rhamnose, a calculated Km of 6.36 mM and vmax of 2.9 × 10−3 U for naringin, and a Km of 6.75 mM and specific activity vmax of 8.63 × 10−2 U for rutin, respectively. Phylogenetic analysis and amino acid domain architecture comparison revealed that RhaB1 belongs to a new subclass of bacterial B type α-l-rhamnosidases of GH 78. To our knowledge RhaB1 is the first biochemically-characterized enzyme of this subclass.

Keywords
α-l-rhamnosidase; Glycoside hydrolase family 78; Metagenome analysis; Intestinal bacteria; Microbiome
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The metagenome-derived enzyme RhaB opens a new subclass of bacterial B type α-l-rhamnosidases
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Publisher
Database: Elsevier - ScienceDirect
Journal: Journal of Biotechnology - Volume 191, 10 December 2014, Pages 38–45
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
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Full-text PDF Download
Online Support
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