Tae-Su Kim

Enzymatic hydrolysis of aspen biomass into fermentable sugars by using lignocellulases from Armillaria gemina

A white rot fungus, identified as Armillaria gemina SKU2114 on the basis of morphological and phylogenetic analyses, was found to secrete efficient lignocellulose-degrading enzymes. The strain showed maximum endoglucanase, cellobiohydrolase, and β-glucosidase activities of 146, 34, and 15 U/mL, respectively, and also secreted xylanase, laccase, mannanase, and lignin peroxidase with activities of 1270, 0.16, 57, and 0.31 U/mL, respectively, when grown with rice straw as a carbon source. Among various plant biomasses tested for saccharification, aspen biomass produced the maximum amount of reducing sugar. Response surface methodology was used to optimize the hydrolysis of aspen biomass to achieve the highest level of sugar production. A maximum saccharification yield of 62% (429 mg/g-substrate) was obtained using Populus tomentiglandulosa biomass after 48 h of hydrolysis. A. gemina was shown to be a good option for use in the production of reducing sugars from lignocellulosic biomass.

A highly efficient sorbitol dehydrogenase from Gluconobacter oxydans G624 and improvement of its stability through immobilization

A sorbitol dehydrogenase (GoSLDH) from Gluconobacter oxydans G624 (G. oxydans G624) was expressed in Escherichia coli BL21(DE3)-CodonPlus RIL. The complete 1455-bp codon-optimized gene was amplified, expressed, and thoroughly characterized for the first time. GoSLDH exhibited Km and kcat values of 38.9 mM and 3820 s−1 toward L-sorbitol, respectively. The enzyme exhibited high preference for NADP+ (vs. only 2.5% relative activity with NAD+). GoSLDH sequencing, structure analyses, and biochemical studies, suggested that it belongs to the NADP+-dependent polyol-specific long-chain sorbitol dehydrogenase family. GoSLDH is the first fully characterized SLDH to date, and it is distinguished from other L-sorbose-producing enzymes by its high activity and substrate specificity. Isothermal titration calorimetry showed that the protein binds more strongly to D-sorbitol than other L-sorbose-producing enzymes, and substrate docking analysis confirmed a higher turnover rate. The high oxidation potential of GoSLDH for D-sorbitol was confirmed by cyclovoltametric analysis. Further, stability of GoSLDH significantly improved (up to 13.6-fold) after cross-linking of immobilized enzyme on silica nanoparticles and retained 62.8% residual activity after 10 cycles of reuse. Therefore, immobilized GoSLDH may be useful for L-sorbose production from D-sorbitol.

pH-rate profiles of L-arabinitol 4-dehydrogenase from Hypocrea jecorina and its application in L-xylulose production.

l-Arabinitol 4-dehydrogenase (LAD) from Hypocrea jecorina (HjLAD) was cloned and overexpressed in Escherichia coli BL21 (DE3). The kinetics of l-arabinitol oxidation by NAD(+), catalyzed by HjLAD, was studied within the pH range of 7.0-9.5 at 25°C. The turnover number (kcat) and the catalytic efficiency (kcat/Km) were 4200min(-1) and 290mM(-1)min(-1), respectively. HjLAD showed the highest turnover number and catalytic efficiency among all previously characterized LADs. In further application of HjLAD, rare l-sugar l-xylulose was produced by the enzymatic oxidation of arabinitol to give a yield of approximately 86%.

Characterization of a Mannose-6-Phosphate Isomerase from Bacillus amyloliquefaciens and Its Application in Fructose-6-Phosphate Production

The BaM6PI gene encoding a mannose-6-phosphate isomerase (M6PI, EC 5.3.1.8) was cloned from Bacillus amyloliquefaciens DSM7 and overexpressed in Escherichia coli. The enzyme activity of BaM6PI was optimal at pH and temperature of 7.5 and 70°C, respectively, with a kcat/Km of 13,900 s-1 mM-1 for mannose-6-phosphate (M6P). The purified BaM6PI demonstrated the highest catalytic efficiency of all characterized M6PIs. Although M6PIs have been characterized from several other sources, BaM6PI is distinguished from other M6PIs by its wide pH range and high catalytic efficiency for M6P. The binding orientation of the substrate M6P in the active site of BaM6PI shed light on the molecular basis of its unusually high activity. BaM6PI showed 97% substrate conversion from M6P to fructose-6-phosphate demonstrating the potential for using BaM6PI in industrial applications.

Immunological activities of cationic methylan derivatives

Methylan polysaccharide was aminated to add dialkylaminoethyl and free amino groups at hydroxyl sites in the methylan backbone, and these derivatives were quaternized to produce pH-independent cationic polyelectrolytes. The immunological activities of the resulting methylan derivatives were investigated. Diethylaminoethyl (DEAE)-methylan derivatives inhibited the classical pathway of the complement system in a dose-dependent way. Quaternized DEAE-methylan exhibited the highest anticomplementary activities among the all derivatives. Anticomplementary activities increased significantly as the cationic charge of the methylan derivatives increased via aminoderivatization followed by quaternization, indicating that there is an electrostatic interaction between the methylan derivatives and the negatively charged functional residues on the cell.