Polysaccharide quantification using microbial enzyme cocktails

Abstract

AbstractPolysaccharide quantification is crucial for studying ecological and nutritional processes in microbes, plants, and animals, as well as their role in carbon cycling and sequestration in aquatic environments. Traditional methods hydrolyze polysaccharides into monomers for quantification but are limited to chemically hydrolysable polysaccharides or require characterized recombinant enzymes. Here we use undefined mixtures of enzymes that are secreted by marine microbes or seawater communities to hydrolyze polysaccharides, focusing on colloidal chitin and laminarin as model marine polysaccharides. Colloidal chitin was digested using an enzyme cocktail from a chitin-degradingPsychromonas sp. bacterial isolate. By quantifying mono- and oligomers with a 3,5-dinitrosalicylic acid reducing sugar assay and/or liquid chromatography-mass spectrometry, chitin could be quantified down to 62 mg/L and 15 mg/L, respectively, enabling us to monitor microbial chitin degradation. Laminarin was digested using enzymes from an undefined marine microbial community with a lower detection limit of 30 mg/L using a reducing sugar assay. Our findings demonstrate that individual microbes or undefined communities can be readily harnessed as enzyme sources, offering a flexible method for polysaccharide quantification that bypasses limitations in chemical hydrolysis and availability of characterized recombinant enzymes.