Saeyoung Lee

An expansin-like protein from Hahella chejuensis binds cellulose and enhances cellulase activity

Molecular function of the expansin superfamily has been highlighted for cellulosic biomass conversion. In this report, we identified a new bacterial expansin subfamily by analysis of related bacterial sequences and biochemically examined a member of this new subfamily from Hahella chejuensis (HcEXLX2). Among the various complex polysaccharides tested, HcEXLX2 bound most efficiently to cellulose. The relative binding constant (Kr) against Avicel was 2.1 L g−1 at pH 6.0 and 4°C. HcEXLX2 enhanced the activity of cellulase, producing about 4.6 times more hydrolysis product after a 36 h reaction relative to when only cellulase was used. The extension strength test on filter paper indicated that HcEXLX2 has a texture loosening effect on filter paper, which was 53% of that observed for 8 M urea treatment. These activities, compared with a cellulose binding domain from Clostridium thermocellum, implied that the synergistic effect of HcEXLX2 comes from not only binding to cellulose but also disrupting the hydrogen bonds in cellulose. Based on these results, we suggest that the new bacterial expansin subfamily functions by binding to cell wall polysaccharides and increasing the accessibility of cell wall degrading enzymes.

The complete enzymatic saccharification of agarose and its application to simultaneous saccharification and fermentation of agarose for ethanol production.

A sugar platform equipped with acetic acid, multiple agarases and neoagarobiose hydrolase (NABH) converted recalcitrant agar polysaccharide into monosugars, which was evaluated by simultaneous saccharification and fermentation (SSF). The sugar platform was divided into chemical liquefaction and enzymatic saccharification. The chemical liquefaction was carried out in mild conditions (using a dilute acetic acid at 80°C for 1-6h) to avoid the production of fermentation inhibitors and hence the highest degree of liquefaction of 95.6% (w/w) was obtained. We mimicked the natural agarolytic pathway using three microbial agarases (Aga16B, Aga50D and DagA) and NABH, and the enzyme system converted 79.1% of agarose to monosugars. The chemical liquefaction and SSF of 30 g/l agarose resulted in 4.4 g/l ethanol concentration and 49.3% of the theoretical ethanol yield to d-galactose. This is the first report on the complete enzymatic conversion of agarose into its monosugars and the SSF of agarose into ethanol.

Crystal structure of a key enzyme in the agarolytic pathway,α-neoagarobiose hydrolase from Saccharophagus degradans 2–40

In agarolytic microorganisms, α-neoagarobiose hydrolase (NABH) is an essential enzyme to metabolize agar because it converts α-neoagarobiose (O-3,6-anhydro-alpha-l-galactopyranosyl-(1,3)-d-galactose) into fermentable monosaccharides (d-galactose and 3,6-anhydro-l-galactose) in the agarolytic pathway. NABH can be divided into two biological classes by its cellular location. Here, we describe a structure and function of cytosolic NABH from Saccharophagus degradans 2-40 in a native protein and d-galactose complex determined at 2.0 and 1.55 Å, respectively. The overall fold is organized in an N-terminal helical extension and a C-terminal five-bladed β-propeller catalytic domain. The structure of the enzyme-ligand (d-galactose) complex predicts a +1 subsite in the substrate binding pocket. The structural features may provide insights for the evolution and classification of NABH in agarolytic pathways.

The novel catabolic pathway of 3,6‐anhydro‐L‐galactose, the main component of red macroalgae, in a marine bacterium

The catabolic fate of the major monomeric sugar of red macroalgae, 3,6-anhydro-L-galactose (AHG), is completely unknown in any organisms. AHG is not catabolized by ordinary fermentative microorganisms, and it hampers the utilization of red macroalgae as renewable biomass for biofuel and chemical production. In this study, metabolite and transcriptomic analyses of Vibrio sp., a marine bacterium capable of catabolizing AHG as a sole carbon source, revealed two key metabolic intermediates of AHG, 3,6-anhydrogalactonate (AHGA) and 2-keto-3-deoxy-galactonate; the corresponding genes were verified in vitro enzymatic reactions using their recombinant proteins. Oxidation by an NADP(+) -dependent AHG dehydrogenase and isomerization by an AHGA cycloisomerase are the two key AHG metabolic processes. This newly discovered metabolic route was verified in vivo by demonstrating the growth of Escherichia coli harbouring the genes of these two enzymes on AHG as a sole carbon source. Also, the introduction of only these two enzymes into an ethanologenic E. coli strain increased the ethanol production in E. coli by fermenting both AHG and galactose in an agarose hydrolysate. These findings provide not only insights for the evolutionary adaptation of a central metabolic pathway to utilize uncommon substrates in microbes, but also a metabolic design principle for bioconversion of red macroalgal biomass into biofuels or industrial chemicals.

Genome Sequence of Vibrio sp. Strain EJY3, an Agarolytic Marine Bacterium Metabolizing 3,6-Anhydro-l-Galactose as a Sole Carbon Source

The metabolic fate of 3,6-anhydro-L-galactose (L-AHG) is unknown in the global marine carbon cycle. Vibrio sp. strain EJY3 is an agarolytic marine bacterium that can utilize L-AHG as a sole carbon source. To elucidate the metabolic pathways of L-AHG, we have sequenced the complete genome of Vibrio sp. strain EJY3.

Crystallization and preliminary X-ray analysis of neoagarobiose hydrolase from Saccharophagus degradans 2-40

Many agarolytic bacteria degrade agar polysaccharide into the disaccharide unit neoagarobiose [O-3,6-anhydro-alpha-L-galactopyranosyl-(1-->3)-D-galactose] using various beta-agarases. Neoagarobiose hydrolase is an enzyme that acts on the alpha-1,3 linkage in neoagarobiose to yield D-galactose and 3,6-anhydro-L-galactose. This activity is essential in both the metabolism of agar by agarolytic bacteria and the production of fermentable sugars from agar biomass for bioenergy production. Neoagarobiose hydrolase from the marine bacterium Saccharophagus degradans 2-40 was overexpressed in Escherichia coli and crystallized in the monoclinic space group C2, with unit-cell parameters a = 129.83, b = 76.81, c = 90.11 A, beta = 101.86 degrees . The crystals diffracted to 1.98 A resolution and possibly contains two molecules in the asymmetric unit.

Model-Based Complete Enzymatic Production of 3,6-Anhydro-l-galactose from Red Algal Biomass

3,6-Anhydro-l-galactose (l-AHG) is a bioactive constituent of agar polysaccharides. To be used as a cosmetic or pharmaceutical ingredient, l-AHG is more favorably prepared by enzymatic saccharification of agar using a combination of agarolytic enzymes. Determining the optimum enzyme combination from the natural repertoire is a bottleneck for designing an efficient enzymatic-hydrolysis process. We consider all theoretical enzymatic-saccharification routes in the natural agarolytic pathway of a marine bacterium, Saccharophagus degradans 2-40. Among these routes, three representative routes were determined by removing redundant enzymatic reactions. We simulated each l-AHG production route with simple kinetic models and validated the reaction feasibility with an experimental procedure. The optimal enzyme mixture (with 67.3% maximum saccharification yield) was composed of endotype β-agarase, exotype β-agarase, agarooligosaccharolytic β-galactosidase, and α-neoagarobiose hydrolase. This approach will reduce the time and effort needed for developing a coherent enzymatic process to produce l-AHG on a mass scale.

3,6-Anhydro-l-galactonate cycloisomerase from Vibrio sp. strain EJY3: Crystallization and X-ray crystallographic analysis

3,6-Anhydro-L-galactonate cycloisomerase (ACI), which is found in the marine bacterium Vibrio sp. strain EJY3, converts 3,6-anhydro-L-galactonate into 2-keto-3-deoxygalactonate. ACI is a key enzyme in the metabolic pathway of 3,6-anhydro-L-galactose (AHG). Study of AHG metabolism is important for the efficient fermentation of agar and biofuel production, because AHG is a sugar that is non-fermentable by commercial microorganisms. The aci gene from Vibrio sp. strain EJY3 was cloned, and the recombinant protein was overexpressed and crystallized in order to determine the structure and understand the function of the protein. The crystals diffracted to 2.2 Å resolution and belonged to space group P41212 or P43212, with unit-cell parameters a = b = 87.9, c = 143.5 Å. The Matthews coefficient was 2.3 Å3 Da-1, with a solvent content of 47%.