Won Cheol Shin

Immunostimulating activity by polysaccharides isolated from fruiting body of Inonotus obliquus

In this study, we investigated the immunostimulating activity of polysaccharides isolated from fruiting body of Inonotus obliquus (PFIO). Additionally, the signaling pathway of PFIO-mediated macrophage activation was investigated in RAW264.7 macrophage cells. We found that PFIO was capable of promoting NO/ROS production, TNF-α secretion and phagocytic uptake in macrophages, as well as cell proliferation, comitogenic effect and IFN-γ/IL-4 secretion in mouse splenocytes. PFIO was able to induce the phosphorylation of three MAPKs as well as the nuclear translocation of NF-κB, resulting in activation of RAW264.7 macrophages. PFIO also induced the inhibition of TNF-α secretion by anti-TLR2 mAb, consequently, PFIO might be involved in TNF-α secretion via the TLR2 receptor. In addition, our results showed that oral administration of PFIO suppressed in vivo growth of melanoma tumor in tumorbearing mice. In conclusion, our experiments presented that PFIO effectively promotes macrophage activation through the MAPK and NF-κB signaling pathways, suggesting that PFIO may potentially regulate the immune response.

In Vitro Skin Permeation of Cubosomes Containing Water Soluble Extracts of Korean Barberry

The monoolein (MO) cubic phases containing water soluble extract (WSE) from Berberis koreana (Korean barberry) were prepared by hydrating the molten MO with aqueous solutions of WSE (0.5, 1.0, and 1.5%). The phase transition temperature of cubic phase containing WSE (∼70°C) was almost the same as that of WSE-free MO cubic phase that indicates that WSE was immobilized in the water channels of the cubic phase and did not affect its structure. The release of WSE from the cubic phase fits the first order process. The cubosomes were obtained by micronizing the cubic phase in a sonicator using Pluronic F127 as a dispersant. The cubosomes were stable in size at the ethanol concentration ≲16%. When compared with WSE solution in phosphate-buffered saline (10 mM, pH 7.4), in vitro skin permeation of WSE in the cubosomes was enhanced by about two times.

Inonotus obliquus protects against oxidative stress-induced apoptosis and premature senescence.

In this study, we investigated the cytoprotective effects of Inonotus obliquus against oxidative stress-induced apoptosis and premature senescence. Pretreatment with I. obliquus scavenged intracellular ROS and prevented lipid peroxidation in hydrogen peroxide-treated human fibroblasts. As a result, I. obliquus exerted protective effects against hydrogen peroxide-induced apoptosis and premature senescence in human fibroblasts. In addition, I. obliquus suppressed UV-induced morphologic skin changes, such as skin thickening and wrinkle formation, in hairless mice in vivo and increased collagen synthesis through inhibition of MMP-1 and MMP-9 activities in hydrogen peroxide-treated human fibroblasts. Taken together, these results demonstrate that I. obliquus can prevent the aging process by attenuating oxidative stress in a model of stress-induced premature senescence.

Poly(vinyl alcohol) hollow microcapsules prepared by emulsification, salting out, and photo cross-linking method

Coumarin residues were conjugated to poly(vinyl alcohol) (PVA) by reacting epoxypropoxy coumarin (EPC) with the polymer. According to the peak areas on the 1H NMR spectrum, EPC was calculated to be conjugated to every 283 repeating units (vinyl alcohols). A cyclic photo-dimerization and dedimerization of EPC of PVA-EPC conjugate were observed under a cyclic irradiation of 365 nm and 254 nm. The salting-out of the conjugate significantly took a place in the range of 0–2.0M NaCl, and the phenomenon was observed at a lower concentration than that of unmodified PVA was. Oil-in-water emulsion was prepared as a template for the preparation of hollow microcapsules using chloroform as an oil phase and PVA-EPC as an emulsifier. The emulsion was stable for 24 hr in terms of droplet size. The wall surrounding droplets was built-up by the salting-out of PVA-EPC, and it was cross-linked by the irradiation of 365 nm. After chloroform was evaporated and salt was removed by a dialysis, hollow microcapsules were successfully obtained.

Characterization of the starch degradation activity from newly isolated Rhizopus oryzae WCS-1 and mixed cultures with Saccharomyces cerevisiae for efficient ethanol production from starch

A starch degrading fungi was isolated from nuruk, a Korean traditional starter culture made with wheat flour. This newly isolated fungi was identified as Rhizopus oryzae WCS-1 via internal transcribed spacer region sequencing. Enzymatic assays for starch degradation activities indicated an optimum temperature and pH of 55°C and pH 4.0, respectively, using partially purified enzymes from R. oryzae WCS-1. Mixed cultures with S. cerevisiae CEN-PK 2-1D, L2612, BY4742, and D452-2 were performed. S. cerevisiae CEN-PK 2-1D has a maltose utilization pathway and showed the highest ethanol production rate with low maltose concentrations. Addition of 5 mM NaCl to mixed cultures of R. oryzae WCS-1 and S. cerevisiae CEN-PK 2-1D resulted in 21 and 33% improvements in the starch consumption rate and the ethanol production rate, respectively, because Na+ stimulated the starch degradation activity of partially purified enzymes from R. oryzae WCS-1.

Sequence analysis of KmXYL1 genes and verification of thermotolerant enzymatic activities of xylose reductase from four Kluyveromyces marxianus strains

Kluyveromyces marxianus has the capability of producing xylitol from xylose because of the endogenous xylose reductase (KmXYL1) gene. In this study, we cloned KmXYL1 genes and compared amino acid sequences of xylose reductase (XR) from four K. marxianus strains (KCTC 7001, KCTC 7155, KCTC 17212, and KCTC 17555). Four K. marxianus strains showed high homologies (99%) of amino acid sequences with those from other reported K. marxianus strains and around 60% homologies with that from Scheffersomyces stipitis. For XR enzymatic activities, four K. marxianus strains exhibited thermostable XR activities up to 45°C and K. marxianus KCTC 7001 showed the highest XR activity. When reaction temperatures were increased from 30 to 45°C, NADH-dependent XR activity from K. marxianus KCTC 7001 was highly increased (46%). When xylitol fermentations were performed at 30 or 45°C, four K. marxianus strains showed very poor xylitol production capabilities regardless fermentation temperatures. Xylitol productions from four K. marxianus strains might be limited because of low xylose uptake rate or cell growth although they have high thermostable XR activities.

Xylitol Production by Kluyveromyces marxianus 36907-FMEL1 at High Temperature was Considerably Increased through the Optimization of Agitation Conditions

Xylitol is usually used as a sugar substitute in food products because of low calorie content and used as the starting material for the production of valuable chemicals [1−4]. Xylitol is generally produced by biological methods for high production yield and environmental safety [5]. The XYL1 gene, coding for xylose reductase (XR), from Scheffersomyces stipitis has been mainly cloned and overexpressed among several reports for biological productions of xylitol by using recombinant yeast [6, 7]. Recently Kluyveromyces marxianus, a thermotolerant yeast, was engineered for xylitol production because thermotolerant capability from K. marxianus is more favored for a simultaneous saccharification and fermentation (SSF) process [8]. SSF process could enhance xylitol productivity from cellulosic biomass in practical aspect. However, non-thermotolerant yeast were not suitable for SSF process at high temperature [9, 10]. Previously, we performed a directed evolution and random mutagenesis with thermotolerant yeast K. marxianus ATCC 36907 for isolating the mutant K. marxianus 36907-FMEL1 having two fold improved xylose reductase activity as compared to the parental strain [11]. K. marxianus 36907-FMEL1 produced xylitol efficiently at 30°C, however, xylitol production was very poor at 40°C by flask fermentation experiments. As shown in Table 1, glucose consumption rate and ethanol production rate were not significantly changed by K. marxianus 36907-FMEL1, when fermentation temperatures were increased from 30°C to 40°C with 80 g/l of glucose as a sole carbon source. However, xylose consumption rate and xylitol production rate were highly reduced from when fermentation temperatures were increased from 30°C to 40°C with 80 g/l of xylose as a sole carbon source. At 40°C, relative xylose consumption and xylitol production rates were decreased to 69% and 47%, respectively, as compared to those from 30°C. Therefore it was speculated that that K. marxianus 36907-FMEL1 might possess low ATP regeneration capability from Recently, we isolated the mutant Kluyveromyces marxianus 36907-FMEL1, which demonstrated improved xylose reductase activity as compared to the parental strain, K. marxianus ATCC 36907. Effects of agitation conditions on xylitol production were verified using a bioreactor system. Under an agitation speed of 400 rpm, K. marxianus 36907-FMEL1 exhibited the highest xylitol yield (0.57 g/g) and productivity (0.64 g·l·h) at 30°C. When the fermentation temperature was increased to 40°C, interestingly, xylitol yield and productivity were found to be increased to 21% (0.64 g/g) and 58% (0.90 g·l·h), respectively, under the optimized agitation conditions.