Title: Understanding the structure and composition of recalcitrant oligosaccharides in hydrolysate using high-throughput biotin-based glycome profiling and mass spectrometry

Abstract

Novel Immunological and Mass Spectrometry Methods for Comprehensive Analysis of Recalcitrant Oligosaccharides in AFEX Pretreated Corn Stover. Lignocellulosic biomass is a sustainable alternative to fossil fuel and is extensively used for developing bio-based technologies to produce products such as food, feed, fuel, and chemicals. The key to these technologies is to develop cost competitive processes to convert complex carbohydrates present in plant cell wall to simple sugars such as glucose, xylose, and arabinose. Since lignocellulosic biomass is highly recalcitrant, it must undergo a combination of thermochemical treatment such as Ammonia Fiber Expansion (AFEX), dilute acid (DA), Ionic Liquid (IL) and biological treatment such as enzyme hydrolysis and microbial fermentation to produce desired products. However, when using commercial fungal enzymes during hydrolysis, only 75–85% of the soluble sugars generated are monomeric sugars, while the remaining 15–25% are soluble recalcitrant oligosaccharides that cannot be easily utilized by microorganisms. Previously, we successfully separated and purified the soluble recalcitrant oligosaccharides using a combination of charcoal and celite-based separation followed by size exclusion chromatography and studies their inhibitory properties on enzymes. We discovered that the oligosaccharides with higher degree of polymerization (DP) containing methylated uronic acid substitutions were more recalcitrant towards commercial enzyme mixtures thanmore » lower DP and neutral oligosaccharides. Here, we report the use of several complementary techniques that include glycome profiling using plant biomass glycan specific monoclonal antibodies (mAbs) to characterize sugar linkages in plant cell walls and enzymatic hydrolysate, matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) using structurally-informative diagnostic peaks offered by negative ion post-secondary decay spectra, gas chromatography followed by mass spectrometry (GC–MS) to characterize oligosaccharide sugar linkages with and without derivatization. Since oligosaccharides (DP 4–20) are small, it is challenging to mobilize these molecules for mAbs binding and characterization. To overcome this problem, we have applied a new biotin-coupling based oligosaccharide immobilization method that successfully tagged most of the low DP soluble oligosaccharides on to a micro-plate surface followed by specific linkage analysis using mAbs in a high-throughput system. This new approach will help develop more advanced versions of future high throughput glycome profiling methods that can be used to separate and characterize oligosaccharides present in biomarkers for diagnostic applications.« less

Authors:

Xue, Saisi ^[1]; Pattathil, Sivakumar ^[2]; da Costa Sousa, Leonardo ^[1]; Ubanwa, Bryan ^[3]; Dale, Bruce ^[1]; Jones, A. Daniel ^[1]; Balan, Venkatesh ^[3]

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+ Show Author Affiliations

1. Michigan State University, East Lansing, MI (United States)
2. Mascoma LLC, Lebanon, NH (United States); University of Georgia, Athens, GA (United States)
3. University of Houston, Sugarland, TX (United States)

Publication Date:

Tue Feb 15 00:00:00 EST 2022

Research Org.:

Univ. of Wisconsin, Madison, WI (United States); University of Houston, Sugarland, TX (United States)

Sponsoring Org.:

USDOE Office of Science (SC), Biological and Environmental Research (BER); University of Houston; State of Texas; US Department of Agriculture (USDA)

OSTI Identifier:

1904710

Grant/Contract Number:  

FC02-07ER64494; AC05-00OR22725

Resource Type:

Accepted Manuscript

Journal Name:

Scientific Reports

Additional Journal Information:

Journal Volume: 12; Journal Issue: 1; Journal ID: ISSN 2045-2322

Publisher:

Nature Publishing Group

Country of Publication:

United States

Language:

English

Subject:

59 BASIC BIOLOGICAL SCIENCES; biological techniques; biomarkers; biotechnology; energy science and technology; engineering; environmental sciences; molecular biology; plant sciences