Identification and characterization of a novel fumarase gene by metagenome expression cloning from marine microorganisms

Abstract

Background: Fumarase catalyzes the reversible hydration of fumarate to L-malate and is a key enzyme in the tricarboxylic acid (TCA) cycle and in amino acid metabolism. Fumarase is also used for the industrial production of L-malate from the substrate fumarate. Thermostable and high-activity fumarases from organisms that inhabit extreme environments may have great potential in industry, biotechnology, and basic research. The marine environment is highly complex and considered one of the main reservoirs of microbial diversity on the planet. However, most of the microorganisms are inaccessible in nature and are not easily cultivated in the laboratory. Metagenomic approaches provide a powerful tool to isolate and identify enzymes with novel biocatalytic activities for various biotechnological applications.

Results: A plasmid metagenomic library was constructed from uncultivated marine microorganisms within marine water samples. Through sequence-based screening of the DNA library, a gene encoding a novel fumarase (named FumF) was isolated. Amino acid sequence analysis revealed that the FumF protein shared the greatest homology with Class II fumarate hydratases from Bacteroides sp. 2_1_33B and Parabacteroides distasonis ATCC 8503 (26% identical and 43% similar). The putative fumarase gene was subcloned into pETBlue-2 vector and expressed in E. coli BL21(DE3)pLysS. The recombinant protein was purified to homogeneity. Functional characterization by high performance liquid chromatography confirmed that the recombinant FumF protein catalyzed the hydration of fumarate to form L-malate. The maximum activity for FumF protein occurred at pH 8.5 and 55°C in 5 mM Mg(2+). The enzyme showed higher affinity and catalytic efficiency under optimal reaction conditions: K(m) = 0.48 mM, V(max) = 827 μM/min/mg, and k(cat)/K(m) = 1900 mM/s.

Conclusions: We isolated a novel fumarase gene, fumF, from a sequence-based screen of a plasmid metagenomic library from uncultivated marine microorganisms. The properties of FumF protein may be ideal for the industrial production of L-malate under higher temperature conditions. The identification of FumF underscores the potential of marine metagenome screening for novel biomolecules.

Figure 1

Sequence alignment of FumF protein with other fumarases. Fumarases are identified by their GenBank accession numbers. Sequence similarity searches were performed with the BLAST 2.0 program. Amino acid sequence alignment of the target putative protein with homologous proteins was performed with the Align × program, a component of the Vector NTI suite (Informax, North Bethesda, MD, USA), using the blosum62mt2 scoring matrix.

Figure 2

Phylogenetic relationship of FumF protein with other fumarases. Sequence alignment was performed using ClustalW version 1.81, and the phylogenetic tree was constructed by the neighbor-joining method using MEGA version 4.0 [30]. Boot-strapping values were used to estimate the reliability of phylogenetic reconstructions (1,000 replicates). The numbers associated with the branches refer to bootstrap values (confidence limits) representing the substitution frequencies per amino acid residue. Fumarases are identified by their GenBank accession numbers.

Figure 3

SDS-PAGE of recombinant FumF protein. Proteins were separated by 12% (w/v) SDS-PAGE and then stained with Coomassie brilliant blue G-250. Lane 1, molecular weight standards; Lane 2, total protein of E. coli BL21(DE3)pLysS harboring empty pETBlue-2 (control); Lane 3, total protein of E. coli BL21(DE3)pLysS harboring the recombinant fumF in pETBlue-2 without induction by IPTG; Lane 4, total protein of E. coli BL21(DE3)pLysS harboring the recombinant fumF in pETBlue-2 induced by addition of 0.5 mM IPTG; Lane 5, sample purified by the Ni-NTA column method. The recombinant FumF protein is indicated by the black arrow.

Figure 4

Effects of pH on the enzymatic activity of recombinant FumF protein. The buffers used were 50 mM citric acid/100 mM Na₂HPO₄ buffer (black regular triangle) (pH 3.0-8.0) and 100 mM glycine/NaOH buffer (black square) (pH 8.0-9.5). Relative activities represent enzyme activities at each pH divided by maximal activity.

Figure 5

Effects of temperature on the enzymatic activity of recombinant FumF protein. Relative activities are the raw enzyme activities at each temperature divided by the maximal activity.

Figure 6

The thermostability of recombinant FumF protein. The enzyme was pre-incubated at temperature ranging from 15 to 70°C at optimum pH. Subsequently, the residual activity was determined with fumarate as the substrate at 55°C in 100 mM glycine/NaOH buffer (pH 8.5).