Saccharification protocol for small-scale lignocellulosic biomass samples to test processing of cellulose into glucose

Second generation biofuels are derived from inedible lignocellulosic biomass of food and non-food crops. Lignocellulosic biomass is mainly composed of cell walls that contain a large proportion of cellulosic and hemicellulosic polysaccharides. An interesting route to generate biofuels and bio-based materials is via enzymatic hydrolysis of cell wall polysaccharides into fermentable sugars, a process called saccharification. The released sugars can then be fermented to fuels, e.g. by use of yeast. To test the saccharification efficiency of lignocellulosic biomass on a lab-scale, a manual saccharification protocol was established that uses only small amounts of biomass and a low concentration of enzyme. This protocol can be used for different plant species like Arabidopsis thaliana, tobacco, maize and poplar. The low enzyme concentrations make it possible to detect subtle improvements in saccharification yield and to analyze the speed of hydrolysis. Although a specific acid and alkali pretreatment were included, the saccharification step can be preceded by any other pretreatment. Because no advanced equipment is necessary, this protocol can be carried out in many laboratories to analyze saccharification yield. The protocol was initially described in Van Acker et al. (2013)

Human gut Bacteroidetes can utilize yeast mannan through a selfish mechanism

Yeasts, which have been a component of the human diet for at least 7,000 years, possess an elaborate cell wall α-mannan. The influence of yeast mannan on the ecology of the human microbiota is unknown. Here we show that yeast α-mannan is a viable food source for the Gram-negative bacterium Bacteroides thetaiotaomicron, a dominant member of the microbiota. Detailed biochemical analysis and targeted gene disruption studies support a model whereby limited cleavage of α-mannan on the surface generates large oligosaccharides that are subsequently depolymerized to mannose by the action of periplasmic enzymes. Co-culturing studies showed that metabolism of yeast mannan by B. thetaiotaomicron presents a ‘selfish’ model for the catabolism of this difficult to breakdown polysaccharide. Genomic comparison with B. thetaiotaomicron in conjunction with cell culture studies show that a cohort of highly successful members of the microbiota has evolved to consume sterically-restricted yeast glycans, an adaptation that may reflect the incorporation of eukaryotic microorganisms into the human diet

Synthesis and evaluation of 2-deoxy-2-amino-β-cellobiosides as cellulase inhibitors

The cellulase mixture of Hypocrea jecorina (formerly Trichoderma reesei) contains a variety of exo- and endoglucanases that belong to different structural families. As such, these enzymes form an interesting model system to study the enzyme-ligand interactions in glycoside hydrolases. The nucleophilic carboxylate of retaining -glycosidases is believed to form a hydrogen bond with the 2-hydroxyl group of their substrate. Consequently, replacing this hydroxyl group with an amino group should result in a stronger electrostatic interaction and thus an increased affinity for the ligand. In this study, several modified cellobiosides were synthesized and evaluated as cellulase inhibitors. The introduction of an amino group was found to have an unpredictable effect on the inhibitory power of the ligands. However, the enzymes display a very high affinity for the corresponding 2-azido compounds, precursors in the synthetic route. The new ligand m-iodobenzyl 2-deoxy-2-azido--cellobioside even is the strongest inhibitor of cellobiohydrolase I known to date (KI= 1 M)

An elaboration on the syn-anti proton donor concept of glycoside hydrolases: electrostatic stabilisation of the transition state as a general strategy

AbstractAn in silico survey of all known 3D-structures of glycoside hydrolases that contain a ligand in the −1 subsite is presented. A recurrent crucial positioning of active site residues indicates a common general strategy for electrostatic stabilisation directed to the carbohydrate’s ring-oxygen at the transition state. This is substantially different depending on whether the enzyme’s proton donor is syn or anti positioned versus the substrate. A comprehensive list of enzymes belonging to 42 different families is given and selected examples are described. An implication for an early evolution scenario of glycoside hydrolases is discussed

Synthesis of isofagomine and a new C-6 pyrrolidine azasugar with potential biological activity

An efficient asymmetric synthesis of isofagomine, based on a precursor containing three differentiated hydroxyl functions, is described. The side product in the key alkylation step is converted into (2S,3R,4R)-2,4-bis(hydroxymethyl)-3-hydroxypyrrolidine, a new C-6 pyrrolidine azasugar, which inhibits alpha-glucosidase from yeast

Airtight storage of moist wheat grain improves bioethanol yields

Abstract Background Drying is currently the most frequently used conservation method for cereal grain, which in temperate climates consumes a major part of process energy. Airtight storage of moist feed grain using the biocontrol yeast Pichia anomala as biopreservation agent can substantially reduce the process energy for grain storage. In this study we tested the potential of moist stored grain for bioethanol production. Results The ethanol yield from moist wheat was enhanced by 14% compared with the control obtained from traditionally (dry) stored grain. This enhancement was observed independently of whether or not P. anomala was added to the storage system, indicating that P. anomala does not impair ethanol fermentation. Starch and sugar analyses showed that during pre-treatment the starch of moist grain was better degraded by amylase treatment than that of the dry grain. Additional pre-treatment with cellulose and hemicellulose-degrading enzymes did not further increase the total ethanol yield. Sugar analysis after this pre-treatment showed an increased release of sugars not fermentable by Saccharomyces cerevisiae. Conclusion The ethanol yield from wheat grain is increased by airtight storage of moist grain, which in addition can save substantial amounts of energy used for drying the grain. This provides a new opportunity to increase the sustainability of bioethanol production.</p

Isothermal and short mashing versus traditional mashing

In 16 case studies the traditional infusion mashing method was compared with isothermal mashing at 60-65°C combined with short mashing at half of the normal mashing time. Also, the effect of mash pH adjustment (5.4-5.6) and application of exogenous enzymes (proteases, glucanases, amylases, and amyloglucosidases) was studied. The results indicated that isothermal short mashing is a potential economic mashing process. Adjustment of the mash pH had a positive effect on the mashing performance. The use of exogenous enzymes gave an increase in the amino acid concentration but this is not an essential advantage for the later fermentation process

Detection, separation and sequencing of oligosaccharides in beer

De volmondigheid van een bier wordt ondermeer bepaald door de oligosachariden van het restextact (1). Drie krachtige methoden werden gecombineerd om het complexe sacharideprofiel in bier weer te geven: HPAEC-PAD (High pH Anion Exchange Chromatography with Pulsed Amperometric detection), DSA-FACE (Fluorescent Assisted Carbohydrate Electrophoresis uitgevoerd op een capillaire array DNA Sequenator) en MALDI TOF MS (Matrix Assisted Laser Desorption/Ionisation with Time-of-Flight Mass Spectrometer). De sacharidesamenstelling van verschillende bieren werd met elkaar vergeleken en oligosachariden uit één type bier werden preparatief gescheiden aan de hand van Carbograph-SPE (Solid Phase Extraction) en gelfiltratie. De bekomen sachariden werden enzymatisch gesequeneerd. Met HPAEC-PAD worden sachariden gescheiden op basis van hun lading en structuur. De detectie gebeurt zonder voorafgaande derivatisering wat massaspectrometrische analyse van de sachariden toelaat na de scheiding. De detectiegrens voor malto-oligosachariden bedraagt 10 pmol en de resolutie voor de malto-oligosachariden in bier met DP<10 is zeer goed (Figuur 1, links). Carbohydraatelektroforese op DNA sequeneringsapparatuur laat toe om met zeer hoge gevoeligheid en resolutie glycaanmengsels te "fingerprinten". Deze analysetechniek werd oorspronkelijk geperfectioneerd voor complexe N-glycaanmengsels (2) en werd in dit werk toegepast op de oligosachariden in bier. De suikers worden vooraf gederivatiseerd met het fluorescent label APTS (9-aminopyrene-1,4,6-trisulfonaat). De detectiegrens voor de malto-oligosachariden bedraagt 10 fmol en de resolutie blijft bewaard met stijgende grootte van de sachariden (Figuur 2, rechts). Met MALDI-TOF MS wordt de massa van de oligosachariden bepaald zonder risico tot fragmentatie. De onderzochte bieren verschillen sterk in concentraties aan hogere malto-oligosachariden en isomalto-oligosachariden (Figuur 1). Sommige bieren bevatten veel α(1→4) gebonden en weinig α(1→6) vertakte sachariden, voor andere geldt het omgekeerde. De profielen vertonen niet echt een tendens per type bier. Deze verschillen kunnen hun weerslag hebben op de organoleptische eigenschappen van het bier en in het bijzonder op de volmondigheid. Uit één type bier dat veel α(1→6) vertakte sachariden bevat, werden de oligosachariden met een DP hoger dan 5 preparatief gescheiden op basis van hun moleculair gewicht. De oligosachariden werden geadsorbeerd op koolstof gepakt in een solid phase extraction kolom en geëlueerd met een stapgradiënt van water en n-butanol gesatureerd water (3). De oligosachariden elueerden in stijgende grootte en werden geïsoleerd in batch. De fractie geëlueerd met 1% butanol bestaat bijvoorbeeld uit vier isomalto-oligosachariden. MALDI-TOF MS analyse wijst op een mengsel van een hexa-, hepta-, octa- en nonasachariden. Deze vier oligosachariden elueren als enkelvoudige pieken voor de α(1→4) gebonden malto-oligosachariden (M6-M9) bij HPAEC-PAD analyse. DSA-FACE analyse toont echter dat iedere piek bestaat uit verschillende oligosachariden, waarschijnlijk positie-isomeren met dezelfde DP (Figuur 2). De oligosachariden werden verder gescheiden met Biogel P4 gelfiltratie chromatografie en de scheiding werd opnieuw gecontroleerd met HPAEC-PAD. De afzonderlijke oligosachariden werden enzymatisch gesequeneerd: specifieke enzymen werden gebruikt om de structuur van de oligosachariden op te helderen. De gevormde producten werden geanalyseerd met HPAEC-PAD en DSA-FACE. Zo werd een isomalto-oligosacharide met DP9 (drie pieken op DSA-FACE) na een pullulanase-behandeling enkel omgezet tot maltohexaose en maltotriose. Hieruit kan geconcludeerd worden dat dit DP9-suiker een α(1→4) maltohexaose eenheid is die α(1→6) gesubstitueerd is met een maltotriose eenheid op de O-6 positie van drie verschillende α(1→4) gelinkte glucose residu's. Referenties (1) Van Landschoot, A., Vanbeneden, N., Machtelinckx, M., Stals, I. & Claeyssens, M. (2005) Pecularities of seven refermented Belgian strong ales and their corresponding industrial yeast. Cerevisia, 30, 3, 181-188. (2) Laroy W., Contreras R. and Callewaert N. (2006) Glycome mapping on DNA sequencing equipment. Nature Protocols 397-405. (3) Redmond J.W. and Packer N.H. (1999) The use of solid-phase extraction with graphitised carbon for the fractionation and purification of sugars. Carbohydrate Research 319, 74-79