Polysaccharide quantification using microbial enzyme cocktails.

Abstract

Polysaccharide quantification plays a vital role in understanding ecological and nutritional processes in microbes, plants, and animals. Traditional methods typically hydrolyze these large molecules into monomers using chemical methods, but such approaches do not work for all polysaccharides. Enzymatic degradation is a promising alternative but typically requires the use of characterized recombinant enzymes or characterized microbial isolates that secrete enzymes. In this study, we introduce a versatile method that employs undefined enzyme cocktails secreted by individual microbes or complex environmental microbial communities for the hydrolysis of polysaccharides. We focus on colloidal chitin and laminarin as representative polysaccharides of ecological relevance. Our results demonstrate that colloidal chitin can be effectively digested with an enzyme cocktail derived from a chitin-degrading Psychromonas sp. isolate. Utilizing a 3,5-dinitrosalicylic acid reducing sugar assay or liquid chromatography-mass spectrometry for monomer and oligomer detection, we successfully determined chitin concentrations as low as 62 and 15 mg/l, respectively. This allows for effective monitoring of microbial chitin degradation. To extend the applicability of our method, we also leveraged complex, undefined microbial communities as sources of enzyme cocktails capable of degrading laminarin. With this approach, we achieved a detection limit of 30 mg/l laminarin through the reducing sugar assay. Our findings highlight the potential of utilizing enzyme cocktails from both individual microbes and, notably, from undefined microbial communities for polysaccharide quantification. This advancement addresses limitations associated with traditional chemical hydrolysis methods.