skip to main content
DOE PAGES title logo U.S. Department of Energy
Office of Scientific and Technical Information

Title: Accurate Quantification of Laminarin in Marine Organic Matter with Enzymes from Marine Microbes

Abstract

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 withmore » 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.« less

Authors:
; ; ; ;
Publication Date:
Research Org.:
Massachusetts Inst. of Technology (MIT), Cambridge, MA (United States)
Sponsoring Org.:
USDOE Office of Science (SC)
OSTI Identifier:
1536860
Grant/Contract Number:  
SC0008743
Resource Type:
Accepted Manuscript
Journal Name:
Applied and Environmental Microbiology
Additional Journal Information:
Journal Volume: 83; Journal Issue: 9; Journal ID: ISSN 0099-2240
Publisher:
American Society for Microbiology
Country of Publication:
United States
Language:
English
Subject:
Biotechnology & Applied Microbiology; Microbiology

Citation Formats

Becker, Stefan, Scheffel, André, Polz, Martin F., Hehemann, Jan-Hendrik, and Vieille, Claire. Accurate Quantification of Laminarin in Marine Organic Matter with Enzymes from Marine Microbes. United States: N. p., 2017. Web. doi:10.1128/aem.03389-16.
Becker, Stefan, Scheffel, André, Polz, Martin F., Hehemann, Jan-Hendrik, & Vieille, Claire. Accurate Quantification of Laminarin in Marine Organic Matter with Enzymes from Marine Microbes. United States. doi:10.1128/aem.03389-16.
Becker, Stefan, Scheffel, André, Polz, Martin F., Hehemann, Jan-Hendrik, and Vieille, Claire. Fri . "Accurate Quantification of Laminarin in Marine Organic Matter with Enzymes from Marine Microbes". United States. doi:10.1128/aem.03389-16. https://www.osti.gov/servlets/purl/1536860.
@article{osti_1536860,
title = {Accurate Quantification of Laminarin in Marine Organic Matter with Enzymes from Marine Microbes},
author = {Becker, Stefan and Scheffel, André and Polz, Martin F. and Hehemann, Jan-Hendrik and Vieille, Claire},
abstractNote = {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.},
doi = {10.1128/aem.03389-16},
journal = {Applied and Environmental Microbiology},
number = 9,
volume = 83,
place = {United States},
year = {2017},
month = {2}
}

Journal Article:
Free Publicly Available Full Text
Publisher's Version of Record

Save / Share:

Works referenced in this record:

The Biochemistry and Structural Biology of Plant Cell Wall Deconstruction
journal, April 2010


Enzymatic assembly of DNA molecules up to several hundred kilobases
journal, April 2009

  • Gibson, Daniel G.; Young, Lei; Chuang, Ray-Yuan
  • Nature Methods, Vol. 6, Issue 5, p. 343-345
  • DOI: 10.1038/nmeth.1318

Protein production by auto-induction in high-density shaking cultures
journal, May 2005