Sun Bok Lee

Aptamers as functional nucleic acids:In vitro selection and biotechnological applications

Aptamers are functional nucleic acids that can specially bind to proteins, peptides, amino acids, nucleotides, drugs, vitamins and other organic and inorganic compounds. The aptamers are identified from random DNA or RNA libraries by a SELEX (systematic evolution of ligands by exponential amplification) process. As aptamers have the advantage, and potential ability to be released from the limitations of antibodies, they are attractive to a wide range of therapeutic and diagnostic applications. Aptamers, with a high-affinity and specificity, could fulfil molecular the recognition needs of various fields in biotechnology. In this work, we reviewed some aptamer selection techniques, properties, medical applications of their molecules and their biotechnological applications, such as ELONA (enzyme linked oligonucleotide assay), flow cytometry, biosensors, electrophoresis, chromatography and microarrays.

Soluble expression of archaeal proteins in Escherichia coli by using fusion-partners.

Expression of archaeal proteins in soluble form is of importance because archaeal proteins are usually produced as insoluble inclusion bodies in Escherichia coli. In this study, we investigated the use of soluble fusion tags to enhance the solubility of two archaeal proteins, d-gluconate dehydratase (GNAD) and 2-keto-3-deoxy-D-gluconate kinase (KDGK), key enzymes in the glycolytic pathway of the thermoacidophilic archaeon Sulfolobus solfataricus. These two proteins were produced as inclusion bodies in E. coli when polyhistidine was used as a fusion tag. To reduce inclusion body formation in E. coli, GNAD and KDGK were fused with three partners, thioredoxin (Trx), glutathione-S-transferase (GST), and N-utilization substance A (NusA). With the use of fusion-partners, the solubility of the archaeal proteins was remarkably enhanced, and the soluble fraction of the recombinant proteins was increased in this order: Trx>GST>NusA. Furthermore, In the case of recombinant KDGKs, the enzyme activity of the Trx-fused proteins was 200-fold higher than that of the polyhistidine-fusion protein. The strategy presented in this work may contribute to the production of other valuable proteins from hyperthermophilic archaea in E. coli.

Computer‐Aided Control of Water Activity for Lipase‐Catalyzed Esterification in Solvent‐Free Systems

A computer system for on‐line monitoring and control of the water activity (aw) in solvent‐free media has been developed. The performance of this system was investigated by carrying out the lipase‐catalyzed esterification of n‐capric acid with n‐decyl alcohol. A humidity sensor measured the relative humidity in the reactor headspace, which was then transmitted electrically to a digital computer that was used as a feedback controller. The water activity control was achieved by sparging either humidified air or dried air through the reaction medium at a flow rate determined by the digital feedback controller. The use of humid air and dry air for aw control made it possible to induce a larger aw gradient and thereby higher water transfer rate. As a result, the water activity quickly reached the desired aw values. We tested whether water activity in the reaction medium can be monitored by measuring relative humidity in the headspace. When the water activity in the liquid phase was determined from measurements of water content in the medium and compared to that measured directly with the humidity sensor, the aw in the reaction medium did not differ significantly from that in the headspace. This indicates that there is a near‐equilibrium between the liquid medium and the exit air stream. Water activity was also successfully maintained close to the set point despite the massive production of water during the esterification process. Thus, the control system developed in this study is particularly useful for systems where large amounts of water are produced and where conventional methods make it difficult to control water activity as a result of a low water transfer rate. The effects that computer control of the water activity had on the reaction rate and yield were also examined. The reaction yield was significantly improved with water activity control. The conversions obtained at 28 h without and those with water activity control were 70% and 96%, respectively. In addition, from the fact that the final yields increased with decreasing aw, computer‐aided water activity control was performed with a set‐point change. By controlling aw at 0.55 during initial reaction phase, followed by a step change of aw from 0.55 to 0 after 11 h of reaction, it was possible to enhance the final conversion to 100%.

Purification and characterization of glycerate kinase from the thermoacidophilic archaeonThermoplasma acidophilum: An enzyme belonging to the second glycerate kinase family

Thermoplasma acidophilum is a thermoacidophilic archaeon that grows optimally at 59°C and pH 2. Along with another thermoacidophilic archaeon,Sulfolobus solfataricus, it is known to metabolize glucose by the non-phosphorylated Entner-Doudoroff (nED) pathway. In the course of these studies, the specific activities of glyceraldehyde dehydrogenase and glycerate kinase, two enzymes that are involved in the downstream part of the nED pathway, were found to be much higher inT. acidophilum than inS. solfataricus. To characterize glycerate kinase, the enzyme was purified to homogeneity fromT. acidophilum cell extracts. TheN-terminal sequence of the purified enzyme was in exact agreement with that of Ta0453m in the genome database, with the removal of the initiator methionine. Furthermore, the enzyme was a monomer with a molecular weight of 49 kDa and followed Michaelis-Menten kinetics withKm values of 0.56 and 0.32 mM forDL-glycerate and ATP, respectively. The enzyme also exhibited excellent thermal stability at 70°C. Of the seven sugars and four phosphate donors tested, onlyDL-glycerate and ATP were utilized by glycerate kinase as substrates. In addition, a coupled enzyme assay indicated that 2-phosphoglycerate was produced as a product. When divalent metal ions, such as Mn2+, Co2+, Ni2+, Zn2+, Ca2+, and Sr2+, were substituted for Mg2+, the enzyme activities were less than 10% of that obtained in the presence of Mg2+. The amino acid sequence ofT. acidophilum glycerate kinase showed no similarity withE. coli glycerate kinases, which belong to the first glycerate kinase family. This is the first report on the biochemical characterization of an enzyme which belongs to a member of the second glycerate kinase family.

Identification and characterization ofThermoplasma acidophilum 2-keto-3-deoxy-D-gluconate kinase: A new class of sugar kinases

The thermoacidophilic archaeonThermoplasma acidophilum has long been known to utilized-glucosevia the non-phosphorylated Entner-Doudoroff (nED) pathway. We now report the identification of a gene encoding 2-keto-3-deoxy-d-gluconate (KDG) kinase. The discovery of this gene implies the presence of a glycolysis pathway, other than the nED pathway. It was found that Ta0122 in theT. acidophilum genome corresponded to KDG kinase. This enzyme shares no similarity with known KDG kinases, and belongs to a novel class of sugar kinases. Of the five sugars tested only KDG was utilized as a substrate.

Metabolic pathway of 3,6-anhydro-L-galactose in agar-degrading microorganisms

Recently, agarose-containing macroalgae have gained attention as possible renewable sources for bioethanol-production because of their high polysaccharide content. Complete hydrolysis of agarose produces two monomers, D-galactose (D-Gal) and 3,6-anhydro-L-galactose (L-AnG). However, at present, bioethanol yield from agarophyte macroalgae is low due to the inability of bioethanolproducing microorganisms to convert non-fermentable sugars, such as L-AnG, to bioethanol. Therefore, to increase the bioethanol productivity of agarophytes, it is necessary to determine how agar-degrading microorganisms metabolize L-AnG, and accordingly, construct recombinant microorganisms that can utilize both D-Gal and L-AnG. Previously, we isolated a novel microorganism belonging to a new genus, Postechiella marina M091, which hydrolyzes and metabolizes agar as the carbon and energy source. Here, we report a comparative genomic analysis of P. marina M091, Pseudoalteromonas atlantica T6c, and Streptomyces coelicolor A3(2), of the classes Flavobacteria, Gammaproteobacteria, and Actinobacteria, respectively. In this bioinformatic analysis of these agarolytic bacteria, we found candidate common genes that were believed to be involved in L-AnG metabolism. We then experimentally confirmed the enzymatic function of each gene product in the L-AnG cluster. The formation of two key intermediates, 2-keto-3-deoxy-L-galactonate and 2-keto-3-deoxy-D-gluconate, was also verified using enzymes that utilize these molecules as substrates. Combining bioinformatic analysis and experimental data, we showed that L-AnG is metabolized to pyruvate and D-glyceraldehyde-3-phosphate via six enzymecatalyzed reactions in the following reaction sequence: 3,6-anhydro-L-galactose → 3,6-anhydro-L-galactonate → 2-keto-3-deoxy-L-galactonate → 2,5-diketo-3-deoxy-L-galactonate → 2-keto-3-deoxy-D-gluconate → 2-keto-3-deoxy-6-phospho-D-gluconate → pyruvate + D-glyceraldehyde-3- phosphate. To our knowledge, this is the first report on the metabolic pathway of L-AnG degradation.

In Situ Monitoring of Cell Concentration in a Photobioreactor Using Image Analysis: Comparison of Uniform Light Distribution Model and Artificial Neural Networks

Light intensity is a very important factor that determines the growth of photosynthetic cells. In this study, the light distribution in a photobioreactor was analyzed by processing the images captured with a digital camera. The contour images obtained by filtering the original images clearly showed the effects of the cell concentration and external light intensity on the light distribution. Image‐processing techniques were then applied to predict the cell density in the photobioreactor. To correlate the cell concentration with the light intensity in the photobioreactor, the captured images were processed using two different approaches. The first method involved the use of an average gray value after deriving a simplified model equation that could be related to the cell density. The second method involved the use of local points instead of a representative value. In this case, an artificial neural network model was adopted to infer the cell density from the information of the local points. By using these two methods, it was possible to relate the image data to the cell concentration. Finally, we compared these two methods with regard to their accuracy, easiness, and effectiveness.

Winogradskyella damuponensis sp. nov., isolated from seawater

A novel bacterium, designated F081-2(T), isolated from seawater from Damupo beach in Pohang, Korea, was investigated using a polyphasic taxonomic approach. Cells were yellow-pigmented, strictly aerobic, motile by gliding, Gram-negative-staining and rod-shaped. The temperature, pH and NaCl ranges for growth were 4-35 °C, pH 5.5-9.5 and 1.0-5.0 %, respectively. Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain F081-2(T) belonged to a distinct lineage in the genus Winogradskyella of the family Flavobacteriaceae, sharing 93.7-98.1 % similarity with recognized members of the genus. Low levels of DNA-DNA relatedness values were found between strain F081-2(T) and Winogradskyella eximia KCTC 12219(T) (61.1 %), Winogradskyella thalassocola KCTC 12221(T) (47.0 %), Winogradskyella echinorum KCTC 22026(T) (39.3 %), Winogradskyella rapida CCUG 56098(T) (34.3 %) and Winogradskyella arenosi JCM 17633(T) (33.4 %). The major cellular fatty acids were iso-C(15 : 0) (25.3 %), iso-C(15 : 1) G (14.6 %), iso-C(17 : 0) 3-OH (9.3 %), anteiso-C(15 : 0) (7.8 %) and iso-C(15 : 0) 3-OH (7.6 %). The polar lipid profile was composed of phosphatidylethanolamine, one unidentified aminolipid, one unidentified phospholipid, one unidentified aminophospholipid and six unidentified lipids. The major respiratory quinone was menaquinone-6 and the DNA G+C content of the strain was 32.3 mol%. On the basis of phenotypic, phylogenetic and genotypic data, strain F081-2(T) represents a novel species within the genus Winogradskyella, for which the name Winogradskyella damuponensis sp. nov. is proposed. The type strain is F081-2(T) (=KCTC 23552(T) = JCM 17633(T)).

Algoriphagus jejuensis sp. nov., isolated from seawater

A gram-negative, pink-pigmented, non-motile, strictly aerobic rod, designated CNU040(T), was isolated from seawater from the coast of Jeju Island in Korea. The temperature, pH and NaCl ranges for growth were 4-30 °C, pH 5.5-10.0 and 0-5.0 % NaCl. Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain CNU040(T) belonged to a distinct lineage in the genus Algoriphagus and exhibited high sequence similarity with Algoriphagus terrigena DS-44(T) (98.3 %) and Algoriphagus alkaliphilus AC-74(T) (96.6 %) and lower sequence similarity (<96.0 %) with all other members of the genus Algoriphagus. DNA-DNA relatedness between strain CNU040(T) and A. terrigena KCTC 12545(T) was 44.5 %. The DNA G+C content of the isolate was 48.5 mol% and the major respiratory quinone was menaquinone-7. The major cellular fatty acids were iso-C(15 : 0) (28.6 %) and summed feature 3 (consisting of iso-C(15 : 0) 2-OH and/or C(16 : 1)ω7c; 24.0 %). The polar lipids consisted of diphosphatidylglycerol, phosphatidylethanolamine, one unknown amino lipid, one unknown aminophospholipid and three unknown polar lipids. On the basis of phenotypic, phylogenetic and genotypic data, strain CNU040(T) represents a novel species within the genus Algoriphagus, for which the name Algoriphagus jejuensis sp. nov. is proposed. The type strain is CNU040(T) ( = KCTC 22647(T)  = JCM 16112(T)).

Postechiella marina gen. nov., sp. nov., isolated from seawater.

A Gram-staining-negative, strictly aerobic, non-motile, yellow-pigmented bacterium, designated strain M091(T), was isolated from seawater at Damupo beach in Pohang, Republic of Korea, and investigated using a polyphasic taxonomic approach. The novel strain grew optimally at 25 °C, pH 7.0-8.0, and in the presence of 3% (w/v) NaCl. In a phylogenetic analysis based on 16S rRNA gene sequences, strain M091(T) formed a lineage within the family Flavobacteriaceae that was distinct from the most closely related genera of Flaviramulus (95.1% sequence similarity), Algibacter (94.9-93.9%), Mariniflexile (94.8-94.2%), Winogradskyella (94.8-93.2%), Lacinutrix (94.7-93.8%) and Tamlana (94.7-92.9%). The polar lipid profile of the novel strain comprised phosphatidylethanolamine, two unidentified aminolipids, one unidentified phospholipid and seven unidentified lipids. The predominant cellular fatty acids were iso-C(15:0) (20.5%), iso-C(17:0) 3-OH (15.4%), iso-C(15:0) 3-OH (12.4%), C(15:0) (10.9%) and iso-C(15:1) G (9.9%). The genomic DNA G+C content of strain M091(T) was 34.4 mol% and the major respiratory quinone was MK-6. Based on phenotypic and genotypic data, strain M091(T) represents a new genus and novel species in the family Flavobacteriaceae, for which the name Postechiella marina gen. nov., sp. nov. is proposed. The type strain of the type species is M091(T) (=KCTC 23537(T)=JCM 17630(T)).