title: Accurate Quantification of Laminarin in Marine Organic Matter with Enzymes from Marine Microbes.
abstract: Marine algae produce a variety of glycans, which fulfill diverse biological functions and fuel the carbon and energy demands of heterotrophic microbes. A common approach to analysis of marine organic matter uses acid to hydrolyze the glycans into measurable monosaccharides. The monosaccharides may be derived from different glycans that are built with the same monosaccharides, however, and this approach does not distinguish between glycans in natural samples. Here we use enzymes to digest selectively and thereby quantify laminarin in particulate organic matter. Environmental metaproteome data revealed carbohydrate-active enzymes from marine flavobacteria as tools for selective hydrolysis of the algal β-glucan laminarin. The enzymes digested laminarin into glucose and oligosaccharides, which we measured with standard methods to establish the amounts of laminarin in the samples. We cloned, expressed, purified, and characterized three new glycoside hydrolases (GHs) ofFormosabacteria: two are endo-β-1,3-glucanases, of the GH16 and GH17 families, and the other is a GH30 exo-β-1,6-glucanase.Formosasp. nov strain Hel1_33_131 GH30 (FbGH30) removed the β-1,6-glucose side chains, andFormosa agariphilaGH17A (FaGH17A) and FaGH16A hydrolyzed the β-1,3-glucose backbone of laminarin. Specificity profiling with a library of glucan oligosaccharides and polysaccharides revealed that FaGH17A and FbGH30 were highly specific enzymes, while FaGH16A also hydrolyzed mixed-linked glucans with β-1,4-glucose. Therefore, we chose the more specific FaGH17A and FbGH30 to quantify laminarin in two cultured diatoms, namely,Thalassiosira weissflogiiandThalassiosira pseudonana, and in seawater samples from the North Sea and the Arctic Ocean. Combined, these results demonstrate the potential of enzymes for faster, stereospecific, and sequence-specific analysis of select glycans in marine organic matter.IMPORTANCEMarine algae synthesize substantial amounts of the glucose polymer laminarin for energy and carbon storage. Its concentrations, rates of production by autotrophic organisms, and rates of digestion by heterotrophic organisms remain unknown. Here we present a method based on enzymes that hydrolyze laminarin and enable its quantification even in crude substrate mixtures, without purification. Compared to the commonly used acid hydrolysis, the enzymatic method presented here is faster and stereospecific and selectively cleaves laminarin in mixtures of glycans, releasing only glucose and oligosaccharides, which can be easily quantified with reducing sugar assays.
authors.affiliation: Center for Marine Environmental Sciences, University of Bremen, Bremen, Germany.|Max Planck Institute for Marine Microbiology, Bremen, Germany.
authors.forename: Stefan
authors.initials: S
authors.lastname: Becker
authors.affiliation: Max Planck Institute of Molecular Plant Physiology, Potsdam-Golm, Germany.
authors.forename: André
authors.initials: A
authors.lastname: Scheffel
authors.affiliation: Department of Civil and Environmental Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA.
authors.forename: Martin F
authors.initials: MF
authors.lastname: Polz
authors.affiliation: Center for Marine Environmental Sciences, University of Bremen, Bremen, Germany jhhehemann@marum.de.|Max Planck Institute for Marine Microbiology, Bremen, Germany.
authors.forename: Jan-Hendrik
authors.initials: JH
authors.lastname: Hehemann
mesh_terms: D059001:Aquatic Organisms / Q000201:enzymology* / Q000235:genetics; D002623:Chemistry Techniques, Analytical; D003001:Cloning, Molecular; D017377:Diatoms / Q000737:chemistry*; D041965:Flavobacteriaceae / Q000201:enzymology* / Q000235:genetics; D015870:Gene Expression; D005936:Glucans / Q000032:analysis*; D006026:Glycoside Hydrolases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D009659:North Sea; D052638:Particulate Matter / Q000737:chemistry; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012623:Seawater / Q000737:chemistry; D013379:Substrate Specificity
chemical_list: D005936:Glucans; D052638:Particulate Matter; D011994:Recombinant Proteins; C008247:laminaran; D006026:Glycoside Hydrolases
keywords: algal bloom; carbon cycle; diatoms; glycobiology; glycoside hydrolase; laminarin; laminarinase; marine microbes; organic matter; β-glucan