A novel ulvan lyase family with broad-spectrum activity from the ulvan utilisation loci of Formosa agariphila KMM 3901

Venkat Rao Konasani,Chunsheng Jin,Eva Albers,Niclas G Karlsson

doi:10.1038/s41598-018-32922-0

Venkat Rao Konasani, Chunsheng Jin + Show 2 more

Open Access

https://doi.org/10.1038/s41598-018-32922-0

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Abstract

Ulvan, which is one of the major structural polysaccharides of the cell walls of green macroalgae, is degraded by ulvan lyases via the β-elimination mechanism with the release of oligosaccharides that have unsaturated 4-deoxy-L-threo-hex-4-enopyranosiduronic acid (∆) at the non-reducing end. These ulvan lyases belong to the PL24 or PL25 or PL28 family in the CAZy database. In this study, we identify and biochemically characterise a periplasmic novel broad-spectrum ulvan lyase from Formosa agariphila KMM 3901. The lyase was overexpressed in Escherichia coli, and the purified recombinant enzyme depolymerised ulvan in an endolytic manner with a Km of 0.77 mg/ml, and displayed optimum activity at 40 °C and pH 8. This lyase also degraded heparan sulphate and chondroitin sulphate. Detailed analyses of the end-products of the enzymatic degradation of ulvan using 1H- and 13C-NMR and LC-MS revealed an unsaturated disaccharide (∆Rha3S) and a tetrasaccharide (∆Rha3S-Xyl-Rha) as the principal end-products. In contrast to the previously described ulvan lyases, this novel lyase is mostly composed of α-helices that form an (α/α)6 incomplete toroid domain and displays a remarkably broad-spectrum activity. This novel lyase is the first member of a new family of ulvan lyases.

Highlights

PSI-BLAST revealed the presence of many homologs of Cdf79930 in the two major phyla found in the human gut microbiota, i.e., Bacteroides and Firmicutes (Fig. S1). This is the first report on the presence of homologs of ulvan lyase in the human gut microbiota, as the PSI-BLAST search for homologs of the previously identified ulvan lyases did not reveal any homologs in the human gut microbiota
In Bacteroides, interestingly, the homolog of Cdf79930 is located in the polysaccharide utilisation loci (PUL) for heparin, whereas in firmicutes it lies in proximity to the genes that encode chondroitin catabolizing enzymes
Among the 41 proteins encoded by this PUL, except the extracellular ulvan lyase, the majority of the proteins were predicted to be localised to the outer membrane or periplasm

Summary

Introduction

The Cdf79930 protein does not share homology with the previously characterised ulvan lyases; instead, forms an evolutionarily distinct new family (Fig. 2A). In the size exclusion chromatography, ulvan lyase activity was eluted as a single peak, and the eluted protein was homogeneous, as observed using denaturing polyacrylamide gel electrophoresis (Fig. 3A). 0.77 mg ml−1 540 μg of glucose ml/min/mg protein 40 °C 8.0 No effect incubation with a two-fold concentration of enzyme, the ulvan was completely depolymerised to small oligosaccharides (Fig. S2).

Results

Conclusion