title: The metagenome-derived enzyme RhaB opens a new subclass of bacterial B type α-L-rhamnosidases.
abstract: 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 (4095bp) 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 3081bp, which we designated rhaB1. The enzyme RhaB1 was overexpressed in Escherichia coli BL21 (DE3) and was purified to an amount of 75mg/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 0.79mM and a specific activity vmax of 18.4U for pNP-α-l-rhamnose, a calculated Km of 6.36mM and vmax of 2.9×10(-3)U for naringin, and a Km of 6.75mM 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.
authors.affiliation: Abteilung für Mikrobiologie und Biotechnologie, Biozentrum Klein Flottbek, Universität Hamburg, Ohnhorststraße 18, D-22609 Hamburg, Germany.
authors.forename: U
authors.initials: U
authors.lastname: Rabausch
authors.affiliation: Abteilung für Mikrobiologie und Biotechnologie, Biozentrum Klein Flottbek, Universität Hamburg, Ohnhorststraße 18, D-22609 Hamburg, Germany.
authors.forename: N
authors.initials: N
authors.lastname: Ilmberger
authors.affiliation: Abteilung für Mikrobiologie und Biotechnologie, Biozentrum Klein Flottbek, Universität Hamburg, Ohnhorststraße 18, D-22609 Hamburg, Germany. Electronic address: wolfgang.streit@uni-hamburg.de.
authors.forename: W R
authors.initials: WR
authors.lastname: Streit
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D003001:Cloning, Molecular; D004606:Elephants / Q000382:microbiology; D005243:Feces / Q000382:microbiology; D006026:Glycoside Hydrolases / Q000737:chemistry* / Q000145:classification / Q000235:genetics* / Q000302:isolation & purification; D054892:Metagenome; D010802:Phylogeny; D017422:Sequence Analysis, DNA; D013329:Structure-Activity Relationship
chemical_list: D006026:Glycoside Hydrolases; C022525:alpha-L-rhamnosidase
keywords: Glycoside hydrolase family 78; Intestinal bacteria; Metagenome analysis; Microbiome; α-l-rhamnosidase