Younho Song

Efficient approach for bioethanol production from red seaweed Gelidium amansii

Gelidium amansii (GA), a red seaweed species, is a popular source of food and chemicals due to its high galactose and glucose content. In this study, we investigated the potential of bioethanol production from autoclave-treated GA (ATGA). The proposed method involved autoclaving GA for 60min for hydrolysis to glucose. Separate hydrolysis and fermentation processing (SHF) achieved a maximum ethanol concentration of 3.33mg/mL, with a conversion yield of 74.7% after 6h (2% substrate loading, w/v). In contrast, simultaneous saccharification and fermentation (SSF) produced an ethanol concentration of 3.78mg/mL, with an ethanol conversion yield of 84.9% after 12h. We also recorded an ethanol concentration of 25.7mg/mL from SSF processing of 15% (w/v) dry matter from ATGA after 24h. These results indicate that autoclaving can improve the glucose and ethanol conversion yield of GA, and that SSF is superior to SHF for ethanol production.

Enhanced lignocellulosic biomass hydrolysis by oxidative lytic polysaccharide monooxygenases (LPMOs) GH61 from Gloeophyllum trabeum

Lignocellulose is a renewable resource that is extremely abundant, and the complete enzymatic hydrolysis of lignocellulose requires a cocktail containing a variety of enzyme groups that act synergistically. The hydrolysis efficiency can be improved by introducing glycoside hydrolase 61 (GH61), a new enzyme that belongs to the auxiliary activity family 9 (AA9). GH61was isolated from Gloeophyllum trabeum and cleaves the glycosidic bonds on the cellulose surface via oxidation of various carbons. In this study, we investigated the properties of GH61. GtGH61 alone did not exhibit any notable activity, but the synergistic activity of GtGH61 with xylanase (GtXyl10G) or cellulase (GtCel5B) showed efficient bioconversion rates of 56 and 174% in pretreated kenaf (Hibiscus cannabinus L.) and oak (Quercus spp.), respectively. Furthermore, the GtGH61 activity was strongly accelerated in the presence of cobalt Co(2+). Enzyme cocktails (GtXyl10G, GtCel5B, and GtGH61) increased the amount of sugar released by 7 and 6% for pretreated oak and kenaf, respectively, and the addition of Co(2+) stimulated bioconversion by 12 and 11% in pretreated oak and kenaf, respectively.

High Cadmium-Binding Ability of a Novel Colocasia esculenta Metallothionein Increases Cadmium Tolerance in Escherichia coli and Tobacco

Experimental evidence in vivo as to the functional roles and binding properties to cadmium (Cd) of type-2 plants metallothionein (MT) has been limited thus far. We investigated the biological role of metallothionein from Colocasia esculenta (CeMT2b) in Escherichia coli and tobacco, and developed a new model for the relationship between Cd tolerance and Cd-binding ability. Heterologous expression of CeMT2b in Escherichia coli greatly enhanced Cd tolerance and accumulated Cd content as compared to control cells. The molecular weight of CeMT2b increased with Cd, and CeMT2b bound up to 5.96±1 molar ratio (Cd/protein). Under Cd stress, transgenic tobacco plants displayed much better seedling growth and high Cd accumulation than the wild type. The presence of an extra CXC motif in CeMT2b contributed to the enhanced Cd-tolerance. The present study provides the first insight into the ability of type-2 plant MT to bind physiological Cd.

Heterologous expression of endo-1,4-β-xylanase A from Schizophyllum commune in Pichia pastoris and functional characterization of the recombinant enzyme.

Endo-1,4-β-xylanase A (XynA) from Schizophyllum commune was cloned into pPCZαA and expressed in Pichia pastoris GS115. The open reading frame of the xynA gene is composed of 684 bp, encoding 278 amino acids with a molecular weight of 26 kDa. Based on sequence similarity, XynA belongs to the CAZy glycoside hydrolase family 11. The optimal activity of XynA was at pH 5 and 50 °C on beechwood xylan. Under these conditions, the K(m), V(max) and specific activity of XynA were 5768 units mg(-1), 4 mg ml(-1) and 9000 μmol min(-1)mg(-1), respectively. XynA activity was enhanced in the presence of cations, such as K(+), Na(+), Li(2+), Cd(2+), and Co(2+). However, in the presence of EDTA, Hg(2+) and Fe(3+), xylanase activity was significantly inhibited. This enzyme effectively degraded approximately 45% of unsubstituted xylans in the cell wall from poplar stems. The high level of XynA activity might increase the yield of enzyme hydrolysis from biomass. Thus, XynA could be used as a major component of a lignocellulosic degrading enzyme cocktail.

Cellulosic bioethanol production from Jerusalem artichoke (Helianthus tuberosus L.) using hydrogen peroxide-acetic acid (HPAC) pretreatment.

Jerusalem artichoke (JA) is recognized as a suitable candidate biomass crop for bioethanol production because it has a rapid growth rate and high biomass productivity. In this study, hydrogen peroxide-acetic acid (HPAC) pretreatment was used to enhance the enzymatic hydrolysis and to effectively remove the lignin of JA. With optimized enzyme doses, synergy was observed from the combination of three different enzymes (RUT-C30, pectinase, and xylanase) which provided a conversion rate was approximately 30% higher than the rate with from treatment with RUT-C30 alone. Fermentation of the JA hydrolyzates by Saccharomyces cerevisiae produced a fermentation yield of approximately 84%. Therefore, Jerusalem artichoke has potential as a bioenergy crop for bioethanol production.

Substrate Specificity of the Bacillus licheniformis Lyxose Isomerase YdaE and Its Application in In Vitro Catalysis for Bioproduction of Lyxose and Glucose by Two-Step Isomerization

ABSTRACT Enzymatic processes are useful for industrially important sugar production, and in vitro two-step isomerization has proven to be an efficient process in utilizing readily available sugar sources. A hypothetical uncharacterized protein encoded by ydaE of Bacillus licheniformis was found to have broad substrate specificities and has shown high catalytic efficiency on d-lyxose, suggesting that the enzyme is d-lyxose isomerase. Escherichia coli BL21 expressing the recombinant protein, of 19.5 kDa, showed higher activity at 40 to 45°C and pH 7.5 to 8.0 in the presence of 1.0 mM Mn2+. The apparent Km values for d-lyxose and d-mannose were 30.4 ± 0.7 mM and 26 ± 0.8 mM, respectively. The catalytic efficiency (k cat/Km ) for lyxose (3.2 ± 0.1 mM−1 s−1) was higher than that for d-mannose (1.6 mM−1 s−1). The purified protein was applied to the bioproduction of d-lyxose and d-glucose from d-xylose and d-mannose, respectively, along with the thermostable xylose isomerase of Thermus thermophilus HB08. From an initial concentration of 10 mM d-lyxose and d-mannose, 3.7 mM and 3.8 mM d-lyxose and d-glucose, respectively, were produced by two-step isomerization. This two-step isomerization is an easy method for in vitro catalysis and can be applied to industrial production.

Isolation and characterization of lignin from the oak wood bioethanol production residue for adhesives.

Lignin was isolated from the residue of bioethanol production with oak wood via alkaline and catalyzed organosolv treatments at ambient temperature to improve the purity of lignin for the materials application. The isolated lignins were analyzed for their chemical composition by nitrobenzene oxidation method and their functionality was characterized via wet chemistry method, element analysis, (1)H NMR, GPC and FTIR-ATR. The isolated lignin by acid catalyzed organosolv treatment (Acid-OSL) contained a higher lignin content, aromatic proton, phenolic hydroxyl group and a lower nitrogen content that is more reactive towards chemical modification. The lignin-based adhesives were prepared and the bond strength was measured to evaluate the enhanced reactivity of lignin by the isolation. Two steps of phenolation and methylolation were applied for the modification of the isolated lignins and their tensile strengths were evaluated for the use as an adhesive. The acid catalyzed organosolv lignin-based adhesives had comparable bond strength to phenol-formaldehyde adhesives. The analysis of lignin-based adhesives by FTIR-ATR and TGA showed structural similarity to phenol adhesive. The results demonstrate that the reactivity of lignin was enhanced by isolation from hardwood bioethanol production residues at ambient temperature and it could be used in a value-added application to produce lignin-based adhesives.

Improving lignocellulose degradation using xylanase–cellulase fusion protein with a glycine–serine linker

The fungal hydrolytic system efficiently degrades lignocellulosics efficiently. We previously characterized two hydrolytic enzymes from Gloeophyllum trabeum, namely, endoglucanase (Cel5B) and xylanase (Xyl10g). To enhance lignocellulosic degradation, we designed a fusion protein (Xyl10g GS Cel5B) using a glycine-serine (GS) linker and expressed it in Pichia pastoris GS115, which produced a hydrolytic fusion enzyme for the degradation of lignocellulosics. Purified Xyl10g GS Cel5B protein has a molecular weight of approximately 97 kDa and shows a lower specific activity than Xyl10g or Cel5B. However, Xyl10g GS Cel5B can degrade popping-pretreated rice straw, corn stover, kenaf, and oak more efficiently than the mixture of Xyl10g and Cel5B, by about 1.41-, 1.37-, 1.32-, and 1.40-fold, respectively. Our results suggest that Xyl10g GS Cel5B is an efficient hydrolytic enzyme and a suitable candidate for degrading lignocellulosics to produce fermentable sugar.

Strategy for dual production of bioethanol and d-psicose as value-added products from cruciferous vegetable residue

In this study, fermentable sugars and d-fructose were produced from cruciferous vegetable residue by enzymatic method without the use of either chemical or mechanical mechanisms. Production of d-psicose was effectively converted from hydrolyzed d-fructose in cabbage residue by d-psicose-3 epimerase; the presence of the borate increased the conversion rate by about two fold, and ethanol production yield was 85.7% of the theoretical yield. Both products, bioethanol and d-psicose, were successfully separated and purified by pervaporation and cation exchange chromatography, and their recovery yields were approximately 87% and 86.2%, respectively.

Simultaneous production of bioethanol and value-added d -psicose from Jerusalem artichoke ( Helianthus tuberosus L.) tubers

In this study, the production of bioethanol and value added d-psicose from Jerusalem artichoke (JA) was attempted by an enzymatic method. An enzyme mixture used for hydrolysis of 100mgmL-1 JA. The resulting concentrations of released d-fructose and d-glucose were measured at approximately 56mgmL-1 and 15mgmL-1, respectively. The d-psicose was epimerized from the JA hydrolyzate, and the conversion rate was calculated to be 32.1%. The residual fructose was further converted into ethanol at 18.0gL-1 and the yield was approximately 72%. Bioethanol and d-psicose were separated by pervaporation. This is the first study to report simultaneous d-psicose production and bioethanol fermentation from JA.