Characterisation of a phylogenetically distinct PL25 family ulvan lyase from a seaweed biomass enriched metagenome

Abstract

Ulvan is a polysaccharide most abundant in green macroalgae biomass. Investigation of ulvan confirmed the potential of the polysaccharide for food, pharmaceutical and chemistry applications, emphasising the beneficial properties of ulvan oligosaccharides. Efficient production of oligosaccharides requires action of ulvan lyases capable of ensuring polysaccharide enzymatic depolymerisation. The armoury of available ulvan lyases was expanded by characterisation of SH2L_Ulv3 ulvan lyase, which was found to be phylogenetically distinct from previously characterised lyases attributed to PL25 family. A gene encoding a novel ulvan lyase was identified among sequences from a seaweed biomass metagenome enriched in an intertidal coastal hot spring. Identified ulvan lyase was most similar to a hypothetical protein from a Bacteroidales bacterium. Recombinant SH2L_Ulv3 was heterologously (over)produced in Escherichia coli at a high yield, remaining soluble in the expression host as well as after affinity purification. Ulvan lyase active as a 48.6 kDa monomer with evaluated activity optimum pH 7.5 and 200 mm NaCl at 25 °C demonstrated broad substrate specificity. SH2L_Ulv3 degraded ulvan from blade-thallus as well as tubular-thallus morphology algae species, efficiently producing three different DP4 and DP2 unsaturated oligosaccharides. The kinetic parameters of SH2L_Ulv3 were K_M 3.63 ± 0.12 mg·mL^-1, V_max 1.78 ± 0.04 μmol·min^-1·mL^-1 and k_cat 1.46 ± 0.04 s^-1. Magnesium ion stimulated SH2L_Ulv3 activity. The characterised enzyme was not thermostable, displaying T_m 42 °C. The computationally modelled structure of SH2L_Ulv3 revealed structural organisation and active site architecture as well as ligand substrate binding and zinc ion coordinating residues typical for PL25 lyases; however, with a larger central active site cleft facilitating ulvan polysaccharide degradation.

Keywords: PL25 family; enriched metagenome; ulvan enzymatic depolymerisation; ulvan lyase; ulvan oligosaccharides.