Earticle

Human breast milk contains full of bioactive components to support the healthy growth and development of newborn babies in their early stage of life. These bioactive components are consisted of essential microbes, human milk oligosaccharides (HMOs), immunoglobulins, lactoferrin, cytokines, and fatty acids. These factors harmonize with gut bacteria to establish the intestinal microbiome of babies. Human milk oligosaccharides (HMOs) are the third most abundant solid components after lactose and lipids. The structural nature of HMOs are complex mixtures of unconjugated glycans with backbone of lactose linked with fucose, sialic acids and/or N-acetyl glucosamine. HMOs are known to have over 200 derivatives resulting from the combination of sugar moiety and linkages. There have been intensive studies on the utilization of HMOs by gut microbiota. Bifidobacterium species are actively involved in the digestion of major HMOs compounds. B. infantis, B. longum, B. breve, and B. bifidum are well known Bifidobacterium species equipped with the enzymes required for the digestion of complex glycans of mother's milk. There is a strong evidence that the establishment of a healthy microbiome in neonates are very crucial for the immune development and prevention of pathogen infections. HMOs are specific and selective prebiotics for Bifidobacterium and play major roles for the growth of B. infantis in the newborn babies' intestine. Potentials of HMOs as antivirals are being studied in several research groups. It is considered that HMOs may mimic structures of viral receptors and block adherence to target cells thus preventing infection. Some of recent review on the glycan binding studies for several viruses will be introduced briefly. Advanced Protein Technologies Corporation (APTech) is working on the development of series of major HMOs. APTech is operating HMO manufacturing plant in Hwasung city for the production of 2’-fucosyllactose (2’-FL). The company got FSSC22000 certificate in October, 2020. APTech is expecting the approval of 2’-FL as food ingredient in Korea and US FDA GRAS notification early next year. EU novel food application is also underway and will be finalized within 2021. References 1. Wicinski M., Sawicka E., Gebalski J., Kubiak K., and Malinowski B., Human Milk Oligosaccharides: Health Benefits, Potential Applications in Infant Formulas, and Pharmacology (2020), Nutrients 12(1):266 2. Sakanaka M., Gotoh A., Yoshida K., Odamaki T., Koguchi H., Xiao J., Kitaoka M. and Katayama T., Varied Pathways of Infant Gut-Associated Bifidobactirium to Assimilate Human Milk Oligosaccharides: Prevalence of the Gene Set and Its Correlation with Bifidobacteria-Rich Microbiota Formation (2020), Nutrients 12 (1):71 3. Morozov V., Hansman G., Hanisch F., Schroten H. and Kunz C., Human milk oligosaccharides as promising antivirals (2018), Mol Nutr Food Res 62(6): 1700679

차세대 올리고당으로 일컬어지는 모유 올리고당 (Human Milk Oligosaccharides)에 관하여 국내 외에서 진행되고 있는 개발 및 연구 현황을 파악하고, HMO가 적용된 제품군을 확인하였으며, 향 후 연구개발 방향을 예측하여 보았음. 모유는 아기에게 필요한 영양성분을 제공하는 급원으로, 모 유 중 탄수화물은 100 mL당 약 7.1 g으로 고형분 기준으로 함량이 가장 높음. 탄수화물의 대부 분을 차지하고 있는 lactose는 HMO 구성 기본 뼈대로 glucose, galactose, fucose 등이 이에 다 양한 형태로 결합하여 약 200여종의 HMO를 이룸. 여러 종의 HMO 중 가장 많은 함량을 차지하 는 것은 2'-fucosyllactose (2‘-FL)로 현재 글로벌 기업에서 미생물 발효 공법을 통하여 대량생 산되어 주로 영유아 조제분유 제품에 적용되고 있음. 국내외에서 생산되고 있는 galactosyllactose (GL)도 조제분유 제품에 적용되고 있는 추세임. 자사에서도 충남대와 협업하여 한국인 모유 내 3'-GL, 4'-GL, 6'-GL 함량을 분석하고, 모유의 GL 프로파일과 일치하는 자재를 분석 및 선정하 여 제품 설계의 근거로 활용함. HMO 섭취에 따른 기능성은 본 소재를 함유한 조제분유 수유 시 장내 미생물 균총이 모유수유군과 유사한 결과가 나타난 영유아 대상 임상연구로 검증된 바 있으 나, 향후 전연령층을 대상으로 하는 여러 제품에 HMO가 적용된다면 관련 전임상연구 및 임상연 구가 활발해질 것으로 예상됨.

As an alternative to biosugar production from biomass resources, extraction of cellulose fibers is getting highlighted. Understanding of the surface morphologies and properties of nanocellulose could possibly provide valuable information for enhancing applicability of nanocellulose as a potent food- or bio- material. At first part of this talk, the surface morphologies, heterogeneity, and accessibilities changes in different types of nanocellulose such as cellulose microfibrils and cellulose nanocrystals from lignocelluosic biomass during manufacturing process were compared. At second part of this talk, after obtaining nanocellulose from Gelidium amansii, one of the representative marine biomass, the feasibility of using the nanocellulose as a anti-inflammatory material was investigated.

Arabinans are unique hemicellulosic homo-polymers in nature which consists of α -(1,5)-linked main backbone and α-(1,2)- and/or α-(1,3)-linked branches of L-arabinoses. Based on the genomes of Bacillus species, the gene clusters encoding various arabinan-degradaing enzymes such as exo-acting α-L-arabino- furanosidases (AFs) and endo-α-(1,5)-L-arabinanases (ANs). Their functional expression and characterization revealed that these hydrolases possess distinct hydrolytic mode of actions and work synergistically with each other. According to the comparative genomics study, the probable roles of each hydrolase for the efficient arabinan-degradation and utilization in Bacillus species as the model bacterial systems have been proposed in this study.

In this study, a novel sugar fatty acid ester (SFAE), maltoheptaose-palmitate ester (G7-PA), was synthesized using commercially available 3 lipases, free or immobilized form, as catalysts. The central composite design of RSM based on three-factor (DMSO concentration, the molar ratio of substrates, and enzyme amount) at five-level was used to optimize the G7-PA synthesis. Among these lipases, the immobilized CALB with the greatest esterification/hydrolysis activity ratio was the best enzyme for the G7-PA synthesis. The optimal conditions for achieving 92.75% conversion yield was determined as 10% DMSO, 0.2 of G7/PA molar ratio, 1g of the immobilized CALB per 0.128 g of PA at 60°C for 24 h. G7-based sugar esters using capric (C10:0), lauric (C12:0), myristic (C14:0) acid as substrates were also synthesized at the same optimal condition. Among the G7-FAEs, G7-PA exhibited improved properties as a biosurfactant in O/W emulsion compared with a commercial sucrose-PA (S-PA). However, emulsions prepared with the other G7-FAEs were unstable at pH 7. At the low surfactant concentration (0.1%), G7-PA emulsion exhibited a droplet distribution similar to 0.2% surfactant condition, while S-PA emulsion was quickly destabilized. Intriguingly, the G7-PA showed better emulsifying properties than the S-PA at the acidic condition (pH 3). Flocculation and phase separation observed at the S-PA emulsion, but that of G7-PA was stable for 7-day. In thermostability tests, G7-PA and S-PA both were stable at the pasteurization and the boiling treatment. Conclusively, the novel bio-surfactant G7-PA could expand the application fields of sugar esters based on its superior properties.

Owing to our recent understanding of the fundamental glycobiology describing crucial roles of glycan chains attached to gycoconjugates in many important physiological and pathogenic processes, such as cell surface recognition, signal transduction, tumor metastasis, etc., and significant advances in carbohydrate analysis, synthesis, and medical applications over the past decades, carbohydrate-based therapeutic drugs are suddenly on attracting menu in the biomedical field. Polysaccharides are widely distributed in nature and show diversity in their structures, physico-chemical properties, and various interesting bioactivities, offering a variety of potential applications in the fields of foods, cosmetics, drug-delivery, tissue engineering and regenerative medicine, vaccines, and pharmaceutical industries. For example, the capsular polysaccharide of Haemophilus influenzae type b (Hib), a major causative of bacterial meningitis, was successfully developed as a Hib vaccine. The anticoagulant heparin and anti-diabetic drug acarbose have long been the number-one-selling drugs in the world. Sulfated polysaccharides (SPSs) having sulfate esters on their sugar residues and their lower molecular weight oligosaccharide derivatives from marine seaweeds such as brown, red, and green algae, have been shown to possess a variety of biological and pharmacological activities, including anticancer, immunostimulatory, anti-inflammatory, anti-coagulant, antioxidant, antibacterial, and antiviral activities. It is now generally recognized that biological and pharmacological activities, including antiviral activity, of SPSs depend on a complex interplay of structural features including the degree of sulfation (charge density), distribution of sulfate groups on their sugar residues, molecular weight, monosaccharide composition, and stereochemistry. We have isolated and purified polysaccharides, a sulfated galactofucan (tentatively named MF) from the sporophill of Korean brown alga Undaria pinnatifida and a sulfated glucuronorhamnoxylan (named SPS-CF) from a green alga Capsosiphon fulvescens, and determined their chemical structures. We also prepared lower-sized polysaccharides (LMMGFs, 1 ~ 4 kDa) from the high-molecular weight MF (~ 2,000 kDa) by enzymatic digestion. This talk focuses on the antiviral and anticancer activities of these polysaccharides and discuss the effects of their molecular weight and degree of sulfation on these bioactivities. On the other hand, flavonoids and anthraquinones are the most abundant natural polyphenolic comounds in plants and have diverse health beneficial effects including anti-cancer, anti-inflammatory, antioxidant, anti-obesity, anti-diabetic, anti-osteoporotic, and immunostimulatory activities. Interestingly, these phenolic compounds observed in nature almost all exist in glycosylated forms in plants, with sugar groups such as glucose, galactose, rhamnose, arabinose, xylose, and rutinose. Glycosylation of phenolic compounds has been reported to increase water-solubility and bioavailability, reduce toxicity, and sometimes give improved or novel pharmacological activities. We have evaluated and compared the bioactivities of various non-glycosylated (aglycone) and glycosylated phenolic compounds against several disease models in vitro and in vivo. This talk also discuss the beneficial effects of glycosylation of phenolic compounds especially on their immunostimulating and anti-obesity activities.

Sleep is an evolutionarily conserved physiological process implicated in the consolidation of learning and memory (L/M). Here, we hypothesize that sleep deprivation (SD)-induced cognitive deficits in zebrafish and mouse are mediated through reduction in O-GlcNAcylation in the brain. At the molecular level, SD downregulated mRNA of key enzymes of HBP metabolism and O-GlcNAc transferase (OGT) along with increased O-GlcNAcase (OGA) expression. Fear conditioning (FC) induced an increase in the expression or activities of proteins associated with long term memory (LTM), and at the same time, OGT protein and O-GlcNAcylation were significantly increased in the normalbut not in the SD-group of zebrafish. Suppression of HBP by the GFAT inhibitor, diazo-oxo-norleucine (DON), impaired L/M function and FC-mediated activation of PKA/CREB signaling. Conversely, enhancement of HBP by glucosamine significantly restored cognitive deficits and increased PKA/CREB signaling in the SD group. To our knowledge, the current study has provided valuable insights into the molecular and biochemical changes associated with L/M at the whole-brain level using the zebrafish and mouse system for the first time. Our findings highlight the role of the HBP during the L/M process and provide potential therapeutic targets for cognitive defects.

Since conventional secretory pathway from ER to Golgi is involved in many essential functions of a cell, it is an important to understand how transport vesicles are regulated. The pathway is initiated by ER’s making transport vesicles at ER exit sites (ERES) with specific coat protein complexes such as COPII vesicles. Our previous study showed that O-GlcNAcylation (O-GlcNAc) on Sec31A, a main component for COPII vesicle, accelerates the anterograde transport of vesicles in ER-Golgi networks. In the present study, we focused on the effect of Ab, a crucial factor in the pathogenesis of Alzheimer’s disease (AD), on the formation of COPII vesicles at ERES. Ab-induced disrupted intracellular calcium levels affected the formation of COPII vesicles at ERES through O-GlcNAcylation of Sec31A in neuronal cells. In addition, Ab caused Golgi fragmentation which was rescued by up-regulation of O-GlcNAcylation using Thiamet G, an OGA inhibitor. Overall, Ab impaired ERES formation through altered Sec31A O-GlcNAcylation triggered by disruption of intracellular calcium homeostasis, suggesting that protection of ERES or Sec31 O-GlcNAcylation may be a promising novel avenue for the development of AD therapeutics.

Intracellular transport is fundamental for the proper functions of the cell, as proteins and membranes must be localized properly to perform their function. The Golgi complex has a central role in secretory pathway. Following synthesis in the endoplasmic reticulum (ER), cargo molecules are transported to the Golgi, so that they can be converted to their proper glycosylation form and transported to their final destinations. From the structural perspective, the Golgi is a unique organelle consisting of a series of membrane stacks, as known as cisternae. As such, a longstanding challenge has been to understand how transport of cargoes through the Golgi is accomplished. Cisternal maturation has been thought to explain anterograde transport at the Golgi, but more recently, transport through tubules that connect the stacks has also been suggested. Here, we find that Rho GTPase, Cdc42 increases the anterograde transport by promoting the formation of the Golgi tubular structure. However, retrograde transport at the Golgi using COPI vesicle is regulated not only by Cdc42 but also by key lipids with different geometries. We further found that the polarized transport at the Golgi contributes to tumor invasiveness by promoting the formation of invadopodia. These findings advance the understanding of how cargo transported through the Golgi is accomplished, and also helps to explain how secretory pathway promotes tumor progression.

Lysosomal storage diseases (LSDs), a group of inherent diseases, are caused by hydrolase deficiency, which results in the accumulation of undigested metabolites in the lysosome. For the treatment of LSDs, enzyme replacement therapies with recombinant enzymes have been used successfully. Most of the therapeutic enzymes, except for the ones for Gaucher disease, require mannose-6-phosphate (M6P) glycans that are recognized by mannose-6-phosphate receptors on the plasma membrane for cellular uptake and targeting to lysosomes. Therefore, M-6-P content is a key factor for efficient MPR binding and lysosomal delivery, which is essential for clearance of the accumulated molecules. Several glyco-engineering strategies for increasing M6P glycan content have been designed to produce “Biobetter” enzymes for LSDs [1]. We constructed the glyco-engineered yeast harboring high content of mannosylphosphorylated glycans, which can be converted M6P glycans by uncapping of the outer mannose residue. The short glycopeptides containing M6P glycans (M6PgPs) were prepared from cell wall mannoproteins of the glyco-engineered yeast by using hot-citrate buffer extraction and pronase digestion. The purified M6PgPs were successfully conjugated to a therapeutic enzyme, recombinant acid a-glucosidase (rGAA) used for treatment of Pompe disease, by two-step reactions using two hetero-bifunctional corsslinkers. The M6PgP-conjugated rGAA had 18-fold higher content of M6P glycan than rGAA and displayed greatly increased its uptake by Pompe disease patient fibroblasts [2]. For the first time, a strategy for in vitro mannosyl-phosphorylation of high-mannose type N-glycans has been established employing a recombinant Mnn14 protein (rM14) derived from Saccharomyces cerevisiae. In vitro reaction using rM14 mannosyl-phosphorylated the glycans on rhGAA, which can be converted to have high M6P glycans contents. It will promise the generation of “Biobetter” therapeutic enzymes with improved lysosomal targeting capability.

The Hippo pathway plays a crucial role in maintaining tissue homeostasis. Generally, activated Hippo pathway effectors, YAP/TAZ, induce the transcription of their negative regulators, NF2 and LATS2, and this negative feedback loop maintains homeostasis of the Hippo pathway. However, YAP and TAZ are consistently hyperactivated in various cancer cells, enhancing tumor growth. Our study found that LATS2, a direct-inhibiting kinase of YAP/TAZ and a core component of the negative feedback loop in the Hippo pathway, is modified with O-GlcNAc. LATS2 O-GlcNAcylation inhibited its activity by interrupting the interaction with the MOB1 adaptor protein, thereby activating YAP and TAZ to promote cell proliferation. Thus, our study suggests that LATS2 O-GlcNAcylation is deeply involved in Hippo pathway dysregulation in cancer cells.

On-chip glycan biosynthesis is an effective strategy for preparing useful complex glycan sources and for applying glycan-involved applications simultaneously. However, current methods have some limitations of analyzing the biosynthesized glycans and optimizing the enzymatic reactions. This could result in undefined glycan structures on a surface, which leads to unequable and unreliable results. In this work, a novel glycan chip was developed by introducing pH-responsive i-motif DNA linker to control the immobilization and isolation of glycans on chip surface in pH-dependent manner. On-chip enzymatic glycosylations were optimized for uniformly biosynthesizing cancer-associated Globo H hexasaccharide and its related complex glycans through stepwise quantitative analyses of isolated products from the surface. Successful interaction analyses of anti-Globo H antibody and MCF-7 breast cancer cells with on-chip biosynthesized Globo H-related glycans demonstrated the feasibility of the structure switchable DNA linker-based glycan chip platform for on-chip complex glycan biosynthesis and glycan-involved applications.

Plants and plant cells are evolving with improved safety and production as attractive options for biopharmaceutical production. A significant barrier to the development of biopharmaceuticals in plants, however, lies in the fact that plant-derived N-glycans contain plant-specific sugar residues such as β1,2-xylose and α1,3-fucose bound to the pentasaccharide core (Man3GlcNAc2) as well as β1,3-galactose and α1,4-fucose involved in the formation of Lewis a (Lea) epitope that may cause allergic reactions in humans. Additionally, sugar residues such as α1,6-fucose, β1,4-galactose, and α2,6-sialic acid, which are believed to play important roles in biopharmaceutical action, storage, distribution, and half-life, are missing from the naturally occurring N-glycans in plants. To use plant cells as a means of producing biopharmaceuticals, it is essential to produce plants which contain N-glycan compatible with biopharmaceuticals. However, the structure of N-glycans appears to be related to hormone signalling and how the structure of N-glycans altered during glycoengineering influences plant production is still uncertain. Here, we suggest a strategy for producing customized N-glycans in plants and the related technological barriers.

Post-translational modifications, including O-GlcNAcylation, play fundamental roles in modulating cellular events, including transcription, signal transduction, and immune signaling. Several molecular targets of O-GlcNAcylation associated with pathogen-induced innate immune responses have been identified; however, the direct regulatory mechanisms linking O-GlcNAcylation with antiviral RIG-I-like receptor signaling are not fully understood. In this study, we found that cellular levels of O-GlcNAcylation decline in response to infection with Sendai virus. We identified a heavily O-GlcNAcylated serine-rich region between amino acids 249–257 of the mitochondrial antiviral signaling protein (MAVS); modification at this site disrupts MAVS aggregation and prevents MAVS-mediated activation and signaling. O-GlcNAcylation of the serine-rich region of MAVS also suppresses its interaction with TRAF3; this prevents IRF3 activation and production of interferon-β. Taken together, these results suggest that O-GlcNAcylation of MAVS may be a master regulatory event that promotes host defense against RNA viruses.

XIAP (X-linked inhibitor of apoptosis) is an inhibitor of apoptotic cell death. It consists of a RING domain and functions as an E3 ligase. In this study, we identify OGT (O-linked N-acetylglucosamine (O-GlcNAc) Transferase) as a substrate of XIAP. In addition, we show that OGT catalyses the O-GlcNAcylation XIAP at serine 406, and that this modification in turn regulates the E3 ligase activity of XIAP towards OGT. Substitution of XIAP serine 406 by alanine decreased its E3 ligase activity toward OGT but not toward other substrates. Stable overexpression of XIAP in HCT116 human colorectal carcinoma cells decreased OGT protein levels and inhibited cancer cell growth and invasion. These results suggest that O-GlcNAcylation of XIAP is required for its E3 ligase activity toward OGT, which leads to OGT degradation and ultimately the inhibition of cancer cell growth.

Rehmannia glutinosa (RG) is one of the major medicinal crops in Korea. The problem with using it is that it contains many indigestible sugars such as stachyose and raffinose. According to our previous experiments about aging technique, the best conditions for making fermented liquor with RG is adding 6% alcohol and aging at 55℃. First, we counted yeast cell number and found that the most yeast was produced when RG was aged for 8 days and fermented for 2 days. Second, we measured soluble solid content (°Brix) of aged RG with different aging periods during alcohol fermentation using a refractive sugar meter. Unlike the difference in the content of fermentable sugar, there is no difference in sugar content depending on the aging period because there is a factor in the suppression of fermentation in the RG, so that fermentable sugar cannot be consumed more than a certain amount. Third, we compared alcohol content (%) of aged RG with different aging periods after alcohol fermentation using an alcoholometer. When aged for more than 5 days, ethanol content increases dependently in aging periods, representing 7.3% alcohol production. What should be considered in future research is that RG has factors that hinder fermentation.

Eukaryotic cells have evolved a N-glycan-dependent endoplasmic reticulum -mediated glycoprotein quality control (ER-QC) system. The ER-QC facilitates folding and modification of secretory and membrane proteins and eliminates terminally misfolded glycoproteins through ER-associated degradation (1). UDP-glucose: glycoprotein glucosyltransferase (UGGT), a key player in ER-QC, selectively reglucosylates misfolded glycoproteins for binding to CNX or CRT lectin/chaperones (2). The human pathogenic yeast Cryptococcus neoformans has unique N-linked glycosylation pathway, in which the mature precursor N-oligosaccharides without glucose residues are linked to asparagine on proteins (3). To investigate the physiological importance of ER-QC in C. neoformans, we constructed a mutant strain lacking UGGT by deleting the gene encoding UGGT. Notably, the C. neoformans uggtΔ stain showed a delayed growth rate even at normal growth condition and severe growth defects under several stress conditions, including high temperature, ER stress, and cell wall stress. Furthermore, the C. neoformans uggtΔ strain displayed defect in the capsule formation and the melanin biosynthesis, which are representative virulence-associated factors. Our results demonstrated that the UGGT-mediated ER-QC system plays a significant role in stress resistance, virulence, and even normal cell growth of Cryptococcus neoformans.

Eukaryotes evolved a N-glycan-dependent endoplasmic reticulum (ER)-associated degradation (ERAD) pathway to monitor protein folding (1). Specific mannose trimming of the protein-bound oligosaccharide is essential for degradation of misfolded proteins (2). The human-pathogen Cryptococcus neoformans has a unique N-linked glycosylation pathway lacking glucosyltransferases but carrying multiple mannosidases (3). To investigate the molecular assembly of C. neoformans specific-ERAD pathway and its impact on pathogenicity, we disrupted the homologue genes to MNS1 (MNS1A and MNS1B), MNL1 and MNL2. The N-glycan profiles indicated MNS1A and MNS1B as a major and minor α-mannosidases, respectively. Whereas the double deletion of MNS1A and MNS1B generated mild growth retardation under several stress conditions, the simultaneous deletion of MNL1 and MNL2, encoding putative ERAD-associated mannosidases, resulted in hypersensitivity to ER, cell wall and osmostress. Notably, it also showed rather increased resistance to high temperature, caffeine and fluconazole. In vitro analysis of virulence factors revealed reduced capsule mass in all the constructed mutants, suggesting attenuation of virulence. Interestingly, mnl1Δmnl2Δ cells did not survive in capsule-inducing media, implying an essential role of the ERAD pathway under specific growth conditions. Overall, our results show the importance of ERAD pathway in protein quality control system for C. neoformans survival and virulence.

Glycosylation, which is highly sensitive to the external environment, has been considered as a window reflecting physiological state of cell. Since abnormal glycans are a hallmark of diseases, the characterization of glycans is gaining attention in biomarker research for treatment and diagnosis. In particular, gastric cancer (GC) with various subtypes has highly different clinical results for each patient. Although gastric cancer patients are classified into different subgroups according to genetic profiling and clinical symptoms, targeted treatment is difficult. In this study, we have characterized the glycans obtained from representative GC cell lines including SNU-1, SNU-16, SNU-5, NCI-N87, AGS, KATO-Ⅲ, MNK-45 and MKN-74. In order to selectively separate the glycans located on the cell surface, the cell membrane extraction was first performed. Subsequently, glycans were enzymatically released, enriched and profiled via PGC nanoLC/MS and LC/MS/MS. Using normalized glycan intensity, a correlation among GC cell lines was examined through hierarchical analysis. The heterogeneity of each cell line was distinguished by moietise of glycans such as fucosylation, sialylation, LacdiNAc and bisecting structures. These results suggested that the glycan signatures of each cell line can differentiate GC cell lines. A deeper understanding of glycan profiling in different subtypes of gastric cancer may provide important clues for precision medicine and tumor targeted treatment.

From the genome of Streptococcus equinus, the putative gene encoding an intracellular α -L-arabinofuranosidase (EC 3.2.1.55, SteqAF43C) has been found, cloned, and constitutively expressed in Escherichia coli. Its open reading frame consists of 1,002 nucleotides encoding 334 amino acids (~38.4 kDa). SteqAF43C with C-terminal six histidines showed the highest activity on debranched arabinan at 30℃ and pH 5.0 in 50 mM sodium acetate buffer. SteqAF43C could exclusively produce L-arabinose from the linear arabinan and linear arabinooligo- saccharides, not from the branched arabinosyl and arabinoxylosyl substrates. These modes of action proposed that SteqAF43C specifically catalyzes the hydrolysis of the α-(1,5)-L-arabinofuranosyl linkages of arabinan backbone, not α-(1,2)/(1,3)-linkages of arabinan branches. Accordingly, SteqAF43C is an arabinan-specific α -(1,5)-L-arabinofuranosidase belonging to GH43.

From the genome of Lactobacillus shenzhenensis, the probable genes encoding five α -L-arabinofuranosidases (AF, EC3.2.1.55) and an β-L-arabinopyranosidase (AP, EC3.2.1.88) were found and cloned in Escherichia coli. Except LbshAF-B1, five genes were functionally expressed and characterized in this study. The comparative studies on the hydrolytic modes of action revealed that these exo-hydrolases can play the important roles in the enzymatic saccharification of arabinan polymers into L-arabinose. LbshAF-A GH51 could hydrolyze both α-(1,5)- and α-(1,2)/(1,3)-linkages in arabinan. LbshAF-B2 GH51 showed only α-(1,2)/(1,3)-specific debranching activity to generate linear products, whereas LbshAF-C1 and C2 GH43 could exclusively hydrolyze α-(1,5)-linked arabinan backbone. LbshAP showed the activity against p-nitrophenyl arabinopyranoside. It was proposed that the synergistic actions of these exo-hydrolases can facilitate the complete degradation of sugar beet arabinan to L-arabinose.

endo-(1,5)-α-L-Arabinanase (AN, EC 3.2.1.99) is an endo-acting hydrolase which can randomly hydrolyze the internal α-(1,5)-L-arabinofuranosidic linkages of arabinan polymers. The arabinan-utilization gene cluster was found from the genome of Streptococcus equinus, and a probable extracellular endo-AN GH43 (SteqAN43B) gene was cloned and heterologously expressed in Escherichia coli. The corresponding open reading frame consists of 2,283 nucleotides encoding 761 amino acids. This enzyme possesses the longer N- and C-terminal domains, compared with common bacterial ANs. Although the debranched (linear) arabinan is the most preferred substrate, SteqAN43B showed the significant hydrolytic activity on sugar beet (branched) arabinan as well. Its modes of action are suitable for the production of various branched arabinooligosaccharides from sugar beet arabinan, and the synergistic hydrolysis of arabinans in combination with the other exo-AFs.

Sugar beet arabinan is a hemicellulosic polymer which consists of L-arabino- furanoside. Sugar beet pulp (SBP) is a waste from the sugar industry. In this study, the efficient arabinan extraction process is examined from SBP by using hot water or autoclaving treatments. Before the extraction of sugar beet arabinan, the pellets were 2-3 times repeatedly washed with distilled water to remove the residual sucrose. The autoclaving treatment showed the highest extraction yield. As the longer extraction time, the more arabinan was extracted, while its purity was lowered. Thin layer chromatography analyses revealed that the optimal extraction time for 5% (w/v) SBP was determined as 1 hr and the yield (w/w%) of the arabinan extract was approximately 6%. The purity of arabinan extracted from SBP was 23.7%. After the ethanol precipitation, the purified arabinan was obtained from the supernatant and its purity improved to 66.5%.

Resistant maltodextrin (RMD) is a soluble dietary fiber derived from starch that known to promote the growth of beneficial intestinal bacteria. The aim of this study is to investigate the potential prebiotic activity of RMD on the growth of Bacteroides xylanisolvens and 15 species of probiotic strains. For this, individual culture and co-culture were performed by measuring the growth rate of 15 types of probiotics and Bac. xylanisolvens . and checking symbiosis relationship between them. As results, in case of Bac. xylanisolvens , optical density reached 1.1 and pH decreased to 5.3. However, none of 15 probiotics made a growth using RMD as a sole carbon source in the medium. When co-cultures were conducted using transwell system cultivating Bac. xylanisolvens and probiotics by separating 0.2μm membrane, only Bac. xylanisolvens made a growth revealing no cross-feeding exist in between those bacteria. In conclusion, RMD showed the prebiotics effect on Bac. xylanisolvens , but not on 15 species of probiotics.

Hepatocellular carcinoma (HCC) ranks as the most common and the second deadliest cancer worldwide and its development is related with changes in several important cellular signaling pathways. Quercetin is considered to possess anticancer activities. Meanwhile, ganglioside, which is a sialic acidcontaining glycosphingolipid, is significantly involved in cell adherence, cell growth, proliferation, differentiation, cellcell communication and cacinogenesis in many types of carcinomas including HCC cells. However, the apoptotic effect of quercetin in HCC Huh7 cells and PRC/PRF/5 cells via regulation of cell cycle and cell growth, and the change of ganglioside synthesis has not been studied. Here, we demonstrate that quercetin decreased cell growth and induced cell cycle apoptosis in both cell lines using several experimental techniques such as 3( 4,5dimethyl2yl) 5( 3carboxymethoxyphenyl) 2( 4sulfophenyl )2Htetrazolium, inner salt (MTS) assay, immunoblot analysis, Annexin V and highperformance thinlayer chromatography (HPTLC) analysis through doseand timedependent manner. Treatment of HCC cells with quercetin reduced cell growth and induced apoptosis, followed by regulation of cell cycle and growth protein and also changed ST3GAL2, which is a ganglioside GD1a synthase. Taken together, our findings indicated that quercetin suppressed the expression of ganglioside GD1a and thereby inhibited cell growth and cell cyclerelated apoptosis.