Damao Wang

High temperature and low acid pretreatment and agarase treatment of agarose for the production of sugar and ethanol from red seaweed biomass

To obtain fermentable sugar from agarose, pretreatment of agarose by using acetic acid was conducted for short durations (10-30 min) at low acid concentrations (1-5% (w/v)) and high temperatures (110-130 °C). On testing the pretreated agarose by using an endo-β-agarase I (DagA), an exo-β-agarase II (Aga50D), and neoagarobiose hydrolase (NABH), we observed that the addition of the endo-type agarase did not increase the sugar yield. Use of the crude enzyme of Vibrio sp. EJY3 in combination with Aga50D and NABH including acetic acid pretreatment resulted in a 1.3-fold increase in the final reducing sugar yield (62.8% of theoretical maximum based on galactose and 3,6-anhydrogalactose in the initial agarose), compared to those obtained using Aga50D and NABH only after acetic acid pretreatment. The simultaneous saccharification and fermentation of pretreated agarose yielded ethanol of 37.1% theoretical maximum yield from galactose contained in the pretreated agarose.

A Novel Glycoside Hydrolase Family 5 β-1,3-1,6-Endoglucanase from Saccharophagus degradans 2-40T and Its Transglycosylase Activity

ABSTRACT In this study, we characterized Gly5M, originating from a marine bacterium, as a novel β-1,3-1,6-endoglucanase in glycoside hydrolase family 5 (GH5) in the Carbohydrate-Active enZyme database. The gly5M gene encodes Gly5M, a newly characterized enzyme from GH5 subfamily 47 (GH5_47) in Saccharophagus degradans 2-40T. The gly5M gene was cloned and overexpressed in Escherichia coli. Through analysis of the enzymatic reaction products by thin-layer chromatography, high-performance liquid chromatography, and matrix-assisted laser desorption ionization–tandem time of flight mass spectrometry, Gly5M was identified as a novel β-1,3-endoglucanase (EC 3.2.1.39) and bacterial β-1,6-glucanase (EC 3.2.1.75) in GH5. The β-1,3-endoglucanase and β-1,6-endoglucanase activities were detected by using laminarin (a β-1,3-glucan with β-1,6-glycosidic linkages derived from brown macroalgae) and pustulan (a β-1,6-glucan derived from fungal cell walls) as the substrates, respectively. This enzyme also showed transglycosylase activity toward β-1,3-oligosaccharides when laminarioligosaccharides were used as the substrates. Since laminarin is the major form of glucan storage in brown macroalgae, Gly5M could be used to produce glucose and laminarioligosaccharides, using brown macroalgae, for industrial purposes. IMPORTANCE In this study, we have discovered a novel β-1,3-1,6-endoglucanase with a unique transglycosylase activity, namely, Gly5M, from a marine bacterium, Saccharophagus degradans 2-40T. Gly5M was identified as the newly found β-1,3-endoglucanase and bacterial β-1,6-glucanase in GH5. Gly5M is capable of cleaving glycosidic linkages of both β-1,3-glucans and β-1,6-glucans. Gly5M also possesses a transglycosylase activity toward β-1,3-oligosacchrides. Due to the broad specificity of Gly5M, this enzyme can be used to produce glucose or high-value β-1,3- and/or β-1,6-oligosaccharides.

Effective production of fermentable sugars from brown macroalgae biomass

Brown macroalgae are renewable and sustainable biomass resources for the production of biofuels and chemicals, owing to their high levels of carbohydrates and low levels of lignin. To increase the biological usage of brown macroalgae, it is necessary to depolymerize the polysaccharides that generate macroalgal monomeric sugars or sugar derivatives and to convert them into fermentable sugars for the production of biofuels and chemicals. In this review, we discuss the chemical and enzymatic saccharification of the major carbohydrates found in brown macroalgae and the use of the resulting constituents in the production of biofuels and chemicals, as well as high-value health-benefiting functional oligosaccharides and sugars. We also discuss recently reported experimental results, novel enzymes, and technological breakthroughs that are related to polysaccharide depolymerization, fermentable sugar production, and the biological conversion of non-favorable sugars for fermentation using industrial microorganisms. This review provides a comprehensive perspective of the efficient utilization of brown macroalgae as renewable resources for the production of biofuels and chemicals.

Preparation of 4-Deoxy-L-erythro-5-hexoseulose Uronic Acid (DEH) and Guluronic Acid Rich Alginate Using a Unique exo-Alginate Lyase from Thalassotalea crassostreae

Marine multicellular algae are considered promising crops for the production of sustainable biofuels and commodity chemicals. However, their commercial exploitation is currently limited by a lack of appropriate and efficient enzymes for converting alginate into metabolizable building blocks, such as 4-deoxy-L-erythro-5-hexoseulose uronic acid (DEH). Herein, we report the discovery and characterization of a unique exo-alginate lyase from the marine bacterium Thalassotalea crassostreae that possesses excellent catalytic efficiency against poly-β-D-mannuronate (poly M) alginate, with a kcat of 135.8 s-1, and a 5-fold lower kcat of 25 s-1 against poly-α-L-guluronate (poly G alginate). We propose that this preference for poly M is due to a structural feature of the proteins active site. The mode of action and specificity of this enzyme has made it possible to design an effective and environmentally friendly process for the production of DEH and low molecular weight guluronate-enriched alginate.

A colorimetric assay to rapidly determine the activities of lytic polysaccharide monooxygenases

BackgroundLytic polysaccharide monooxygenase (LPMOs) are enzymes that catalyze the breakdown of polysaccharides in biomass and have excellent potential for biorefinery applications. However, their activities are relatively low, and methods to measure these activities are costly, tedious or often reflect only an apparent activity to the polysaccharide substrates. Here, we describe a new method we have developed that is simple to use to determine the activities of type-1 (C1-oxidizing) LPMOs. The method is based on quantifying the ionic binding of cations to carboxyl groups formed by the action of type-1 LPMOs on polysaccharides. It allows comparisons to be made of activities under different conditions.ResultsBased on the colorimetric detection and quantification of the pyrocatechol violet (PV)-Ni2+ complex, we have developed an assay to reliably detect and quantify carboxylate moieties introduced by type-1 LPMOs. Conditions were optimized for determining the activities of specific LPMOs. Comparisons were made of the activities against cellulose and chitin of a novel AA10 LPMO and a recently reported family AA11 LPMO. Activities of both LPMOs were boosted by hydrogen peroxide in the 1st hour of the reaction, with a 16-fold increase for the family AA11 LPMO, and up to a 34-fold increase for the family AA10 LPMO.ConclusionsWe developed a versatile colorimetric cation-based assay to determine the activities of type-1 LPMOs. The assay is quick, low cost and could be adapted for use in industrial biorefineries.