Molecular Cloning and Biochemical Characterisation of a Novel Acidic Laminarinase Derived from Jermuk Hot Spring Metagenome

Abstract

Laminarinase, an enzyme with a specific affinity for laminarin--a complex polysaccharide found in the cell walls of brown algae and select marine organisms--was investigated in this study. We cloned and characterised a gene encoding a putative glycoside hydrolase family 16 (GH16) laminarinase from the Jermuk hot spring metagenome by heterologous expression in Escherichia coli. The resulting product, named Jermuk-LamM, represents a novel endo-1,3-{beta}-D-glucanase (EC 3.2.1.39) with only 48.1 % amino acid sequence similarity to previously characterised GH16 family members catalogued in the NCBI database. To date, this stands as the sole described endo-1,3-{beta}-D-glucanase within the Marinimicrobia phylum. Jermuk-LamM, identified as an acidic laminarinase, exhibits robust enzymatic activity at pH 5.0 and a temperature of 55 {degrees}C, maintaining its function for a duration of at least 7 hours. Notably, this enzyme effectively catalyses the hydrolysis of both soluble and insoluble (1,3)-{beta}-D-glucans, as well as (1,3;1,4)-{beta}-D-glucans, displaying a pronounced preference for laminarin. The specificity of Jermuk-LamM lies in its cleavage of 1,3-{beta}-D-glucosidic linkages, yielding monosaccharides, disaccharides, and oligosaccharides. These breakdown products hold the potential for conversion into energy carriers, including alcohols, methane, and hydrogen. The enzymes exceptional specific activities, coupled with its resistance to various additives, render Jermuk-LamM a promising candidate for various industrial applications, encompassing the realms of biofuel and pharmaceutical production.