Nam-Soo Han

Solid-phase refolding of cyclodextrin glycosyltransferase adsorbed on cation-exchange resin.

Expression with a fusion partner is now a popular scheme to produce a protein of interest because it provides a generic tool for expression and purification. In our previous study, a strong polycationic tail has been harnessed for an efficient purification scheme. Here, the same polycation tail attached to a protein of interest is shown to hold versatility for a solid‐phase refolding method that utilizes a charged adsorbent as a supporting material. Cyclodextrin glycosyltransferase (CGTase) fused with 10 lysine residues at the C‐terminus (CGTK10ase) retains the ability to bind to a cation exchanger even in a urea‐denatured state. When the denatured and adsorbed CGTK10ase is induced to refold, the bound CGTK10ase aggregates little even at a g/L range. The renatured CGTK10ase can also be simply recovered from the solid support by adding high concentration of NaCl. The CGTK10ase refolded on a solid support retains specific enzyme activity virtually identical to that of the native CGTK10ase. Several factors that are important in improving the refolding efficiency are explored. Experimental results indicate that nonspecific electrostatic interactions between the charge of the ion exchanger and the local charge of CGTase other than the polycationic tag should be reduced to obtain higher refolding yield. The solid‐phase refolding method utilizing a strong polycationic tag resulted in a remarkable increase in the refolding performance. Taken together with the previous report in which a series of polycations were explored for efficient purification, expression of a target protein fused with a strong polycation provides a straightforward protein preparation scheme.

Characterization of polycationic amino acids fusion systems for ion-exchange purification of cyclodextrin glycosyltransferase from recombinant Escherichia coli.

Fusion proteins with charged polycationic amino acid tails were constructed for the purpose of simple ion‐exchange purification with high purity. A number of positively charged lysine and arginine tails were fused to the C‐terminus of cyclodextrin glycosyltransferase (CGTase) derived from Bacillus macerans and expressed in Escherichia coli. The ionic binding forces provided by the tails allowed the selective recovery of CGTase from recombinant E. coli cell extracts, while CGTase by itself could not bind to the cation exchanger at neutral pH. The type of amino acids used and the length of the tail directly affected the purification factors. Most intracellular proteins of E. coli adsorbed on the cation exchanger could be removed by washing with 400 mM NaCl solution at pH 7.4, suggesting that a fusion partner suitable for purification purpose should be provided with high binding strength and the maintenance of adsorption by washing with NaCl solution. Among the fusion CGTases constructed, the CGTK10ase containing 10 lysine residues provided sufficiently high binding strength to allow purification to its homogeneity through simple ion‐exchange chromatography.

Optimization of electrotransformation conditions for Leuconostoc mesenteroides subsp. mesenteroides ATCC8293

Aims:  To establish an efficient genetic transformation protocol for Leuconostoc species, methods for competent‐cell preparation and electroporation conditions were optimized.

Leuconostoc citreum HJ‐P4 (KACC 91035) regulates immunoglobulin E in an ovalbumin‐induced allergy model and induces interleukin‐12 through nuclear factor‐kappa B and p38/c‐Jun N‐terminal kinases signaling in macrophages

Leuconostoc citreum (L. citreum) HJ‐P4 (KACC 91035) is one of the major predominant species in kimchi fermentation in Korea. The purpose of the present study was to test the immunomodulatory capacity of L. citreum to modulate the IgE‐mediated allergic response and to examine the involvement of NF‐κB and MAPK in IL‐12 production in macrophages. Balb/c mice were sensitized with OVA/alum and oral administration of L. citreum to the mice began before or after the OVA sensitization. Protein and mRNA expression of Th1 cytokines in splenocytes by L. citreum in vitro was measured. The role of NF‐κB and MAPK such as p38, ERK1/2 and JNK in L. citreum‐induced IL‐12 was investigated in peritoneal macrophages and RAW264.7 cell lines. L. citreum inhibited the serum levels of total IgE, IgG1 and IgG2a altogether and increased OVA‐specific IFN‐γ production in splenocytes from pre‐ and post‐sensitized animals. However, the downregulation of IL‐4 and IL‐5 production was observed only in the pre‐sensitization group. The ability of L. citreum to stimulate IFN‐γ was dependent on its induction of IL‐12. NF‐κB, p38 and JNK were mainly involved in L. citreum‐induced IL‐12 production. In conclusion, the current study demonstrated that L. citreum is able to regulate serum IgE generation at the induction and effector phases of allergic response through overall control over antibody production and that its involvement of IL‐12 production was mediated through NF‐κB and p38/JNK. Taken together, the use of L. citreum can be useful in preventing the development and progression of IgE production.

Expression of Bacillus macerans cyclodextrin glucanotransferase gene in Saccharomyces cerevisiae

Cyclodextrin glucanotransferase (CGTase) gene of Bacillus macerans was subcloned down-stream of yeast ADH1 promoter and expressed in Saccharomyces cerevisiae. Most of the CGTase expressed was in the extracellular medium with a maximum activity of about 0.28 unit ml−1 after 48 h cultivation. The recombinant CGTase was secreted as an N-linked-glycosylated form and predominantly produced α-cyclodextrin from starch.

Comparison of Chemical and Microbiological Characteristics of Commercial Kimchi Products in Korea and Japan

Jinmi Company LimitedAbstract The goal of this study was to investigate the chemical and microbiological characteristics of kimchi productsdistributed in Japan (5 brands, J-products) and Korea (2 brands, K-products). When their average analyses were compared,J-products showed higher values in pH, total sugar and acetic acid contents, while K-products showed higher values innumber of lactic acid bacteria, sugar alcohol and lactic acid contents including textural hardness or chewiness. In addition,the analysis showed great variation in composition levels regarding pH, total sugar and acetic acid contents of J-products,and this fact revealed that different manufacturing processes are being attempted in Japan. Interestingly, some J-productshad high concentrations of acetic acid with little mannitol, as this result implies that some manufacturers in Japan producekimchi by adding acetic acid or lactic acid to control the rate of lactic acid fermentation. The result of this study elucidatesthe Japanese consumer’s taste preference as well as the manufacturing processes in Japanese companies.Keywords: kimchi, chemical characteristic, microbiological characteristic, Japan, Korea

Production of natural antimicrobial compound d-phenyllactic acid using Leuconostoc mesenteroides ATCC 8293 whole cells involving highly active d-lactate dehydrogenase

Phenyllactic acid (PLA) is an antimicrobial compound naturally synthesized in various fermented foods and its D‐form of PLA is known to be more active than the L‐isomer. In this study, Leuconostoc mesenteroides ATCC 8293 cells, elaborating d‐lactate dehydrogenase (d‐ldh) were used to produce d‐PLA from phenylpyruvic acid (PPA). When cultured in the presence of PPA (≤50 mmol l−1), growing cells produced a maximum yield of 35 mmol l−1 of d‐PLA, and the yields were between 75·2 and 83·3%. Higher conversion yields were obtained at pH 6·0–7·0 when growing cells were used, while the optimum pH range was broader for resting cells. The time required for the complete conversion of PPA into PLA could be shortened to 3 h using resting cells. d‐ldh, an enzyme encoded by the LEUM_1756 gene of Leuc. mesenteroides ATCC 8293, was found to be responsible for the conversion of PPA into PLA. The Km and kcat values of the enzyme for PPA were found to be 15·4 mmol l−1 and 5645 s−1, respectively. The conditions required for the efficient production of d‐PLA were optimized for both growing and resting cells of Leuc. mesenteroides, with special emphasis on achieving high stereoselectivity and conversion yield.

Construction of a dextran-free Leuconostoc citreum mutant by targeted disruption of the dextransucrase gene.

Leuconostoc citreum is an important lactic acid bacterium in fermented foods, but dextran production often causes undesired ropiness. To prevent this side effect, a dextran‐free mutant needs to be created.

The anti-aging properties of a human placental hydrolysate combined with dieckol isolated from Ecklonia cava

BackgroundsIn the present study, we aimed to examine the anti-aging properties of human placental hydrolysate (HPE) and dieckol (DE) from Ecklonia cava against free radical scavenging, muscle hypertrophy-related follistatin mRNA expression, amelioration of cognition-related genes and proteins, inhibition of collagenase-regulating genes, and elastinase activity.MethodsThe anti-aging effects were examined in human fibroblast (CCD986sk), mouse myoblast (C2C12), and neuroblastoma (N2a) cell models, by employing various assays such as 2,2-diphenyl-1-picrylhydrazyl hydrate (DPPH) scavenging, hydroxyl radical-mediated oxidation, quantitative real-time polymerase chain reaction, enzyme activity, and immunocytochemistry observation.ResultsOur results show that HPE combined with DE (HPE:DE) strongly scavenged DPPH radicals and protected proteins against degradation by hydroxyl radical attack. HPE:DE effectively inhibited matrix metalloproteinase-1 expression, protein kinase C alpha expression, and elastinase activity. Furthermore, HPE:DE improved the expression of cognition-related genes (choline acetyltransferase and vesicular acetylcholine transporter). These events may proactively contribute to retard the aging processes and the abrupt physiological changes probably induced by mitochondrial dysfunction with aging.ConclusionsBased on these findings, we conclude that the combined treatment of HPE:DE may be useful for anti-aging therapy in which the accumulation of oxidative damage is the main driving force.