Earticle

The extracellular enzyme produced by Bacillus subtilis subsp. subtilis A-53 isolated from sea-water was purified by ammonium sulfate saturation of supernatant of culture broth after removal of cells and column chromatography through HiTrapTM QXL ion exchange column and Mono Q ion exchange column. The molecular weight of purified enzyme was estimated to be about 56 KDa by sodium dodecyl sulfate - polyacrylamide gel electrophoresis (SDS-PAGE). Carboxymethylcellulose (CMC), xylan, cellobiose, Filter paper, cellulose, avicel and p-nitrophenyl-β-D-glucopyranoside (pNPG) as a substrate for the purified enzyme were tested. Among them, carboxymethylcellulose (CMC) was found to be the best substrate. Optimal temperature and pH for the CMCase were determined to be 50℃and 6.5, respectively. The CMCase was found to be stable between pH 6.0 and pH 9.0. The enzyme retained over 40% of its original activity within the pH 6.0 to 9.0 for 8hr. K+, Ni+ and EDTA increased the activity of the CMCase, while Co2+, Hg2+ decreased the activity.

Marine sponges have been known to provide a source of novel bone and cartilage replacements. However, the pharmacological mechanism of action of spongin remains obscure. In this study, it was investigated whether spongin derived from Hymeniacidon sinapium can promote bone mineralization of osteoblast-like MG-63 cells. Our present study provides the first evidence that spongin is effective in activating bone mineralization. Furthermore, spongin increased ALP activity, collagen synthesis and osteocalcin secretion in addition to bone mineralization in osteoblastic cells in vitro. In addition, it was demonstrated that spongin exerted the inhibitory effect on production of inflammatory mediators such as TNF-α, IL-1β and PGE2 in macrophage, RAW264.7 cells. These results suggest that the anti-inflammatory effect of spongin derived from Hymeniacidon sinapium can play a critical role in bone mineralization of osteoblast-like MG-63 cells.

This paper describes the role of nutrients in the production of rifamycin B by Amycolatopsis mediterranei S699 in optimal medium. The central composite design experiments showed that optimal concentrations of glucose and ammonium sulfate were 63 g/L and 7.9 g/L at 10 g/l of corn steep liquor (CSL). A bioreactor culture was performed in optimal medium. The total amino acids derived from CSL were completely depleted and 50% of the ammonium ion was used in lag phase. Cell growth was concomitant with uptake glucose and inorganic phosphate and ceased due to depletion of inorganic phosphate. Rifamycin B production began when the cell growth started, however, its production rate increased after the cell growth stopped. During this period, protein derived from CSL hydrolyzed to free amino acids which were then utilized and simultaneous utilization of ammonium ions was observed. Production stopped at 172 h when the glucose was depleted. The maximum cell concentration and rifamycin B titer were 8.6 g/L and 1260 mg/L, respectively. This result implied that glucose and ammonium sulfate contributed to cell growth and product formation whereas CSL was found to enhance the production rate and increasing rifamycin B titer.

Ghost vaccine production from E.coli / S.iniae 4 different type antigens -enolase, GAPDH, sagA and piaA was maximized α by the optimization of condition parameters such as glucose concentration, agitation speed, aeration, ghost efficiency, and induction temperature point. The glucose concentrations were adjusted to 0g/L, 1g/L, 2g/L and 3g/L in LB broth containing 50㎕/ml ampicillin for carbon source optimization. The highest cell densities in the cultures were 1.87g/l, 1.63g/l, 1.51g/l and 1.60g/l for E.coli / S.iniae antigens α-enolase, GAPDH, sagA and piaA with 1g/l of glucose, respectively. The effect of agitation speed was evaluated ranging from 100 to 400 rpm. The maximum production of cell density was obtained at the agitation speed of 300 rpm with the concentration of 2.5g/l, 2.18g/l, 1.89g/l and 1.95g/l for E.coli / S.iniae antigens α-enolase, GAPDH, sagA and piaA, respectively. The effect of aeration rate on cell density was examined with the different aeration rates of 0.5, 1.0, 1.5 vvm at the agitation speed of 300rpm. Maximum production of cell density was obtained at the aeration rate of 1.0vvm with the concentration of 2.5g/l, 2.18g/l, 1.89g/l and 1.95g/l for E.coli / S.iniae antigens α-enolase, GAPDH, sagA and piaA, respectively. The induction temperature point was determined in three exponential phases, initial exponential phase(OD=1.0), mid exponential phase(OD=2.0), final exponential phase(OD=3.0). At the mid exponential phase(OD=2.0) and the ghost efficiencies was 99.92%. 99.67% and 97.72%, respectively. Therefore the optimal condition parameters of the batch cultures were as follows : glucose concentration of 1g/l, agitation of 300rpm, aeration of 1vvm and induction temperature point of O.D : 2.0. Ghost induction with Streptococcus iniae antigens was analyzed by SDS-PAGE and Western blot

Chlamydomonas reinhardtii is a unicellular green microalgae. Normally deriving energy from photosynthesis, with an alternative carbon source, C. reinhardtii can also thrive in total darkness. The relative adaptability and quick generation time has made Chlamydomonas an important model for biological research. Over the years, studies of Chlamydomonas have provided major research contributions in the areas of photosynthesis and molecular biology. C. reinhardtii contains 67% of carbohydrate (54.8% of starch) in the dry weight of algal biomass.1) Most biomass containing starch can be used as a potential substrate for the ethanol fermentation by microbial processes.2) Bioethanol would be an important one of alternative energy sources in post petroleum age. Therefore biomass containing carbohydrates, especially starch would be rise in importance.3) In this study, the pH-stat operation based on acetate/NaOH feeding was established for high cell density culture of starch-rich C. reinhardtii. The biomass concentration was about 3 times higher than that of the control pH-stat operation using HCl and NaOH.

Regulons, sub-parts of functional transcriptional regulatory network(TRN) that are regulated upon a specific stimulus, have been extensively studied and known to consist of transcription factors(TFs) and their target genes. However, most of their dynamic properties – what regulons are really reacting to each stimulus – have not been clearly concluded yet even in microorganisms. In this study, 3 genotoxicants, AgNO3 and Ag-nanoparticles(10nm size) were used to construct the functional TRN in E. coli. First, the significantly perturbed TFs upon each stress were selected from microarray anaylsis referring to DBD and RegulonDB. To check whether those TFs are really responsible with each stress, fitness test was done for each TF deletion strain. For the selected TFs which were shown as essential for cell’s survival upon each stress, their latent target genes were found using microarray with deletion strains. Integrating the results with the prior static TRN from regulonDB, some regulons which have not been expected well were suggested. The result shows that genotoxic stress and metal induced stress have very distinct functional TRNs and some putative TFs are highly confirmative for their relevance on certain stress responses in E. coli.

A bacterial strain producing a high concentration of red pigment isolated from sediment that had been collected East China Sea, which was recorded as CH-34. It was identified as Zooshikella sp.. They produced pigments with maximum absorption at 540 nm, which indicated the presence of prodigiosin1). The radical scavenging activities of the extracts from CH-34 cell and culture broth according to extraction solvents and fractions on hydroxyl, alkyl and DPPH radical were investigated using a ESR spectrophotometer and compared with the ESR signal intensity. The extracts exhibited strong scavenging activity on hydroxyl, alkyl and DPPH radical, and the activity increased with increment of concentration of the extracts. The electron donating abilities (EDAs) of ZC acetone extract (ZCEA) and ZC ether soluble fraction (ZCFE) were 95.74% and 94.51% at 0.5㎎/㎖ respectively. The hydroxyl radical scavenging ability of the ZC hot water extract (ZCE70W) was highest(59.08% at 1㎎/㎖), and alkyl radical scavenging ability was highest in the ZC water extract (84.05% at 1㎎/㎖). From these results, ZC extracts and fractions had strong antioxidant activities.

Reactive oxygen species (ROS) are constantly generated in aerobic organisms during normal metabolism in response to both internal and external stimuli. Imbalances in the production and removal of ROS have been hypothesized to play a causative role in numerous disease such as cancer, ischemia/reperfusion injury and degenerative diseases such as photoaging, atherosclerosis, arthritis, and neurodegeneration. A feature often associated with these diseases is a malfunctioning of the connective tissue remodeling process due to increased activity of extracellular matrix metallo proteinases (MMPs). Algae extracts have been studied as potential natural antioxidants in recent years. Coralline red alga, Amphiroa dilatata is distributed widely coastal area of east asia. In the present study, the free radical scavenging potency and inhibitory effects methanolic extract Amphiroa dilatata(AD) on ROS production and MMP-2 and 9 activities were investigated It's scavenging activity of ROS such as 1,1-diphenyl-2-picrylhydrazyl(DPPH), superoxide and alkyl radical was determined using electron spin resonance(ESR) spectroscopy. The levels of intracellular ROS was measured using 2,7- chlorofluorescindiacetate. Their levels significantly decreased in the presence AD extracts, and the DPPH radical scavenging effects were higher than superoxide and alkyl radicals. Furthermore, MMP-2 and -9 expression levels were significantly decreased in a dose dependent manner by treatment of AD extracts. Therefore, these results suggest that AD extract could have a therapeutic potential for prevention and treatment of several diseases such as tumor metastasis, inflammation and neurodegeneration.

The effects of dietary β-glucan administration on non-specific immune parameters in Oliver flounder, Paralichthys olivaceus were evaluated. In this study investigated the effects of mushroom mycelium mixed solution extract (MMSE) on the immune responses of olive flounder, Paralichthys olivaceus. The culture extracts were evaluated for the growth, hematology, lysozyme activity, leukocyte phagocytic activity, and disease resistance against Vibrio anguillarum. In the effect of the growth, the body weight and length gain in the group, which fed with mushroom mycelium cultural extract, were the body weight 52 g and length gain 2.1cm higher than that in the control. For the hematology, the administration of MMSE resulted in increase of glucose. However, there was no distinct differences in GOT (glutamic oxaloacetic transaminase), GPT (glutamlc pyruvic transaml- nase), TG, TP, and LDH (lactate dehydronase) among each group. The activities of Lysozyme were 80% higher in the experimental groups than in the control. The activities of Leucocyte were 66% higher in the experimental groups than in the control. Although lysozyme activity and leucocyte activity showed somewhat decrease after 12 weeks, these activities were still higher than in the control. The relative percent survival rate (RPS) after an artificial challenge with 7˟108 CFU of Vibrio anguillarum per fish was 25% higher in the experimental groups than the control.

Flow cytometry offers quick and statistically reliable information about the physiological state of cells. In this study, the inactivation of Salmonella typhimurium treated at the various temperature (35-55°C at 100 bar) and pressure (80-250 bar at 35°C) by supercritical carbon dioxide (SC-CO2) was analyzed by the flow cytometry. Using fluorescent dyes such as propidium iodide (PI) and ethidium bromide (EB), which distinguish the physiological state by specific dyes indicating the membrane integrity and the functionality of efflux pump, respectively, the state of S. typhimurium treated with SC-CO2 was analyzed. By the combined staining with SYTO 9 and PI, there was a remarkable decrease in the of membrane integrity as increasing the SC-CO2 treatment. The staining with SYTO 9 and EB staining revealed that the efflux pump system was damaged even at a less severe condition compared to the membrane integrity.

Carotenoids are a structurally diverse group of natural pigmented chemicals of emerging importance as food supplements or colorants and in nutraceutical and pharmaceutical applications1). Carotenoids are structurally classified based on the number of backbone carbon molecules, usually C30, C40 or C50. Carotenoid biosynthesis occurs via a head to head condensation reaction of isoprenoid precursors followed by a desaturation reaction to increase the number of conjugated double bonds generating the distinctive carotenoid chromophore. Non-carotenogenic Escherichia coli cells can be transformed with heterologous carotenoid biosynthetic pathway genes of microbial or plant sources for the production of structurally diverse of carotenoids1). A new carotenoid desaturase homologue from S. aureus (CrtOx) was identified2). When expressed in engineered E. coli cells synthesizing linear C30 carotenoids3), novel polar carotenoid products were generated, identified as aldehyde and carboxylic acid C30 carotenoid derivatives. The major product in this engineered pathway is the fully desaturated C30 dialdehyde carotenoid 4,4’-diapolycopen-4,4’-dial. Very low carotenoid yields were observed when CrtOx was complemented with the C40 carotenoid lycopene pathway. But extension of an in vitro evolved pathway of the fully desaturated 2,4,2’,4’-tetradehydrolycopene produced the structurally novel fully desaturated C40 dialdehyde carotenoid 2,4,2’,4’-tetradehydrolycopendial. Directed evolution of CrtOx by error-prone PCR resulted in a number of variants with higher activity on C40 carotenoid substrates and improved product profiles. These findings may provide new biosynthetic routes to highly polar carotenoids with unique spectral properties desirable for a number of industrial and pharmaceutical applications. These results also demonstrate the utility of extending an in vitro evolved central metabolic pathway with catalytically promiscuous downstream enzymes in order to generate structurally novel compounds that are inaccessible without directed evolution4).

Coenzyme Q10을 생산하는 광합성세균을 분리하기 위해 부산 낙동강하구, 양어장, 광합성세균 생산 공장에서 각각 광합성 균을 샘플링하여 총 3종을 분리하였고, 16s-rDNA sequencing을 통해 동정한 결과 Rhodobacter sphaeroids, Rhodobacter capsulatus 및 Rhodopseudomonas palustris로 밝혀졌다. 기초배지에서 배양한 R.sphaeroids균의 비증식속도는 0.13 h-1이였고, 1L 반응기에서의 배양한 결과 반응6시간 이내에 용존산소는 소모되었고, 최종 pH는 증가하고 ORP는 감소함을 알 수 있었다. HPLC 분석결과 R. sphaeroids균이 다른 균에 비해 가장 높은 Q10값(59.2mg/L)을 가짐을 알 수 있었다.

For screening of Q10-producing photosynthetic bacteria (PSB), we took samples from three different sites: silt at Nakdonggang, aquaculture farm and PSB production plant. Three PSB were isolated and found to be Rhodobacter sphaeroids, Rhodobacter capsulatus and Rhodopseudomonas palustris, respectively by the 16s-rDNA sequence analysis. The specific growth rate of R. sphaeroids cultivated on the basal medium was calculated to be 0.13 h-1. The result of experiment in 1 L bioreactor showed that dissolve oxygen in the broth was depleted within 6 h, pH increased and ORP decreased finally. From the HPLC analysis, it was found that R. sphaeroids had the highest Q10 content, 59.2 mg/L among the three PSB.

Lipases are used as efficient biocatalysts in many industrial processes for food, detergent, fine chemical, and biodiesel. Recently, Candida antarctica lipase B (CalB) draws attention because it can be used for stereoselective transformations and polymer synthesis and the potential process for biodiesel production using lipases instead of chemical catalysts is under development. Since E. coli expression systems are extensively studied and have advantages of ease to use and low cost for screening of gene libraries as compared with the other expression systems, E. coli strains, Rosettagami with enhanced disulfide bond formation, Novablue, and DH5α, were exploited in this study. CalB was cloned into the plasmid pCold, and the constructed pCold/CalB was used to transform the E. coli strains. In addition, the host cells having the pCold construct were transformed with chaperone plasmids containing groES/groEL, groES/groEL/tig, tig, dnaK/dnaJ/grpE, and dnaK/dnaJ/grpE/groES/groEL for coexpression of CalB and chaperones. The colonies expressing functional lipase were selected for each E. coli strain, and the CalB expression levels were examined.

Lactic acid bacteria (LAB) have long been used in fermentations to preserve the nutritive qualities of various foods. Recently, a number of antimicrobial metabolites, e.g. cyclic dipeptides, phenyllactic acid, proteinaceous compounds, and 3-hydroxylated fatty acids have been isolated from lactic acid bacteria.1) The antioxidative effect of lactic acid bacteria was also reported.2) This paper investigated antimicrobial activities and antioxidative effect of silk peptide transformed by lactic acid bacteria. Among the 38 lactic acid bacteria as lyophilized stock purchased, 25 of them showed good growth. I indicator strain were used for screening of antimicrobial activity and 19 strains of lactic acid bacteria showing antimicrobial activity. The antimicrobial compound from the 3 strains of lactic acid bacteria showing strong antimicrobial activity are under investigation. Free radical scavenging activities the culture supernatants were measured according to the modified method of Bilos.3) The highest radical scavenging activity was obtained in 3 lactic acid bacteria cultured under aerobic condition. Tyrosinase inhibition activites were also measured.

Ntirogen source dependent growth and antibiotics production regulator (NdgR) was initially found from S. peucetius by DNA affinity capture assay method. NdgR was well conserved not only through the completely sequenced Streptomcyes species such as S. coelicolor and S. avermitilis, but also through many other bacteria such as Mycobacteria and Corynebacteria. Furthermore, it is quite different from known IclR-type regulators, which are known to be involved in glycerol operon, acetate production and sporulationspecific cell division and morphogenesis. In S. coelicolor, ndgR deletion mutant showed overall late growth and promoted actinorhodin (ACT) production in minimal media with various amino acids, even where wild type strain could not produce ACT. According to RT-PCR results, it was observed that NdgR affects glutamine/glutamate and proline. Moreover, surprisingly, it controls S. coelicolor quorum sensing system such as scbR/scbA by binding to its promoter region and autoregulate itself.

Effects of pH shock on the secretion system of S. coelicolor A3(2) have been investigated at a transcriptional level by using DNA microarrays. Actinorhodin secretion was observed to be highly enhanced when an acidic pH-shock was applied to surface grown cultures of S. coelicolor A3(2). In this culture, a gene of actVA-orf1 coding a putative efflux pump or transporter protein for actinorhodin was strongly upregulated. A major number of efflux pumps for other metabolites, and a major number of secretion proteins for protein secretion were also observed to be upregulated with pH shock. The secretion of actinorhodin was observed to be remarkably enhanced in liquid culture also.

The efficient soluble expression of human epidermal growth factor (hEGF) was achieved by using functional fusion partners in cytoplasm and periplasm of Escherichia coli (E. coli). hEGF was over-expressed in inactive inclusion body form in cytoplasm of E. coli due to improper disulfide bond formation and hydrophobic properties, resulting in about 5.9 mg/L in flask culture. Six functional fusion partners were introduced by linking to N-terminal part of hEGF gene for the high-level expression of soluble and active hEGF in cytoplasm and periplasm region. Three fusion partners such as ATS, thioredoxin and lipase for cytoplasmic expression, and three fusion partners such as periplasmic cystein oxidoreductases (DsbA and DsbC) and maltose binding protein for periplasmic expression were investigated. hEGF fused with ATS and DsbA showed over 90% of solubility in cytoplasm and periplasm, respectively. Especially DsbA was found to be an efficient fusion partner for soluble and high-level expression of hEGF, resulting in about 18.1 mg/L, that is three-fold higher concentration compared to that of insoluble non-fusion hEGF.

Minimization of a genome provides many advantages over conventional approaches. Targeted deletion of large blocks of unnecessary genes can reduce production of unwanted by-products, and increase genome stability while simultaneously streamlining or simplifiing metabolism without physiological compromise. Recently, a reduced-genome Escherichia coli strain, MDS42 (Science, 312, 1044-1046) lacking 14.3% of the chromosome has been constructed. The reduced-genome E. coli has been reengineered to increase the productivity of an essential amino acid L-threonine, by over-expressing a feedback resistant threonine operon (thrA*BC), deleting the genes encoding threonine dehydrogenase (tdh) and threonine transporter (tdcC and sstT), and introducing a mutant threonine exporter (rhtA23). The resulting strain, MDS42-thr, shows a 20% increase in threonine production relative to the wildtype E. coli strain engineered with the same threonine specific modifications. This result clearly demonstrates that elimination of large numbers of genes unnecessary for cell growth from the genome can increase the productivity of the strain significantly, most likely by reducing the metabolic burden on the cells.

오염된 양식 해수의 수질정화능력이 뛰어난 광합성세균을 고순도 대량배양하기 위하여 광합성세균의 최적 배양 배지를 탐색해 보고, 실험실 단위에서의 최적 반응조건을 알아보는 실험을 수행하였다. 다양한 배지에서 광합성세균을 배양해 본 결과, 기초배지가 광합성세균의 최적배지임을 알 수 있었다. Lab-scale에서의 반응 최적화를 위하여 기초배지를 사용하여 5L bioreactor에서 광합성세균을 배양한 결과, 반응 12시간 이후 pH를 6.8로, air의 양을 0.32 vvm으로, 교반속도를 340 rpm으로 유지시켜 주었을 때 광합성세균의 성장( μ=0.15h-1 ) 및 pigment(maroon)생산이 가장 뛰어난 것으로 나타났다. 이때, DO값은 0 ppm, ORP 값은 -150 mV, 최대 cell수는 1.0*1010 CFU/ml로 나타났다.

For high-density mass culture of photosynthetic bacteria, optimal media and culture conditions for its successful cultivation were investigated in lab-scale. The best results of cell growth and pigment production were achieved on the basal medium. Optimization of the culture was carried out in 5 L bioreactor using the basal medium. When cultivation was maintained at pH 6.8, 340 rpm and 0.32 vvm of aeration, maximum cell growth ( μ=0.15h-1) and pigment production (color: maroon) were achieved. At this cultivation, the values of DO, ORP and maximum cell number were measured to be 0 ppm, -150 mV and 1.0*1010 CFU/ml, respectively.

Various methods (OECD 301B, ISO 9439 and ASTM 5864) for biodegradability test of lubricants were reviewed, and a standard procedure was developed. Most lubrication products are released in rivers or sea then is degraded by microbial action in aerobic condition. Most international method are based on CO2 evolution test. Inoculum obtained from a sewage disposal plant and test compound are cultivated in a mineral medium. Organic carbon of the test compound is degraded and oxidized through the enzymatic actions of inoculum, and ultimately mineralized to carbon dioxide. Biodegradability test conditions of lubricant oils were optimized. The highest biodegradability was achieved when the same medium as in ASTM 5864 and inoculum concentration of 105 cell/L were used. The optimum standard materials were selected as aniline and sodium acetate. Additionally the effects of inoculum type on microbial growth and biodegradability were examined. Finally the standard operating procedure (SOP) for biodegradability test method was proposed.

Shikimic acid (SA) is a hydroaromatic intermediate in the commom pathway of aromatic amino acid biosynthesis. Due to its highly functionalized chiral characteristics, it has been recognized as an essential starting material for synthesizing neuramidase in hibitors (marketed as Tamiflu) which are effective in the treatment of influenza. The current state-of-the art for isolation of shikimic acid from the fruit of Illicium plants is cumbersome and costly, which eventually motivates the microbial production shikimic acid from renewable resource. (1) In this study, effects of various industrially important carbon sources (glucose, sucrose, xylose, gluconate and glycerol) on the SA biosynthesis in Escherichia coli were investigated to gain new insight into the metabolic capability for producing SA (2). Constraints-based flux analysis using the genome-scale in silico model of E. coli allows us to quantify the threoretical maximum SA yield. The corresponding flux distributions fueled by different carbon sources under investigation were compared with respect to theoretical yields and energy utilization. For the experimental observation, E coli in which the accumulation of SA can be facilitated by blocking the aromatic amino acid pathway were developed, and production of SA in this strain under various carbon sources was investigated through 5L fermentation.

The extraction and separation method of protein-bound polysaccharides from fruiting body and mycelium of Inonotus obliquus were investigated. For fruiting body and mycelium of Inonotus obliquus, the good extraction conditions were achieved for 1 hour at 180℃ and 2 hours at 150℃, respectively. Two step extraction was much better than one step extraction to separate the protein-bound polysaccharides. For fruiting body of Inonotus obliquus, the protein-bound polysaccharides were separated by microwave extraction for 1 hour at 150℃ after the first extraction was done by hot water for 4 hours at 100℃. For mycelium of Inonotus obliquus, the protein-bound polysaccharides were separated by microwave extraction for 1 hour at 150℃ after the first extraction was achieved by hot water for 2 hours at 80℃.

In batch cultures, after 25 h, the maximum cell mass of Bifidobacterium bifidum BGN4 was 4.5 g/L and the maximum cell count was and 3.0 × 109 cfu/mL at pH 6.0 and 50 g/L sucrose. To increase the viable counts of bifidobacteria, cell retentive culture was applied using a submerged membrane bioreactor with suction and gas sparging. The maximum mass, count, and productivity of the cells after 36 h were 12.0 g/L, 2.2 × 1010 cfu/mL, and 6.1 × 108 cfu/mL·h, respectively, at the feeding (dilution) rate of 120 mL/h (0.06 h-1) in the feeding medium. The accumulated levels of organic acids and ammonium ions at the end of the cultivation were 1.5 and 1.0 g/L, respectively. The viable counts and volumetric productivity of the cells after the cell retentive culture were 7.3- and 5.1-fold higher, respectively, than the values obtained during batch culture. These high viable counts and volumetric productivity were obtained by maintaining lower concentrations of organic acids and ammonium ions so that the growth of B. bifidum BGN4 was not inhibited. The submerged membrane bioreactor produced the highest viable counts of B. bifidum without membrane fouling and cell damage.

The extraction method of protein-bound polysaccharides from fruiting body of Sparasis crispa were investigated. For fruiting body of Sparasis crispa, the good extraction conditions were achieved for 2 hours at 150℃ by microwave. Then, sugar and protein content were 20 and 8.7 percents, respectively. Also, two step extraction was much better than one step extraction to separate the protein-bound polysaccharides. For fruiting body of Sparasis crispa, the protein-bound polysaccharides were separated by microwave extraction for 30 minutes at 150℃ after the first extraction was done by hot water for 1 hour at 100℃.

Hepatitis B virus causing the chronic diseases of the liver is surrounded by envelop proteins. Their major component is the small hepatitis B surface antigen (sHBsAg).1) A plasmid containing the sHBsAg gene under the Gal promoter and MFα signal sequence was constructed for the chromosomal integration and expression of the sHBsAg gene in recombinant S. cerevisiae. Batch fermentation of S. cerevisiae 2805/Mδα _sHBsAg with 20 g/L glucose and 30 g/L galactose resulted in 2.21 mg/L of sHBsAg concentration. Appearance of three different sizes of sHBsAg with 25, 35 and 40 kDa indicated that sHBsAg was as three types of authentic, MFα containing and N-glycosylated MFα containing sHBsAg. To improve the expression level of sHBsAg, the PDI1 gene was episomally co-expressed. Batch fermentation of S. cerevisiae 2805/Mδα_sHBsAg_Pdi1 resulted in 9.75 g/L of dry cell mass and 13.3 mg/L of sHBsAg concentration. Modulation of galactose addition yielded the optimal initial galactose concentration of 15 g/L. Finally, 14.2 mg/L of sHBsAg concentration was obtained in batch fermentation using 20 g/L glucose and 15 g/L galactose, which was 6.5 times higher than that for S. cerevisiae 2805/Mδα_sHBsAg.

Succinic acid is a useful chemical in many field of industry : making foods, pharmaceuticals and cosmetics [2, 3]. It is a valuable 4-carbon intermediate and is useful for the production of 1,4-butanediol, tetrahydrofuran, and gammabutyrolactone [1]. Recently, fermentation of microbe for production of succinic acid has become of interest [4]. At previous study, lab scale batch fermentation of Mannheimia succiniciproducens LPK7 was performed at optimal conditions. In order to test the industrial applications, bench scale batch fermentation was carried out at optimal conditions in lab scale fermentation. After extensive fermentation experiments, final succinic acid concentration and yield were obtained as 14.5 g/L and 0.61 mol/mol. This result shows a similar amount of succinic acid concentration and yield with the lab scale batch fermentation experiment. With these optimal conditions it will be possible to apply succinic acid production industrially.

Swollenin, a fungal protein, is presumed to disrupt hydrogen bonding between cell wall polysaccharides. Despite of the disruption of hydrogen bonding, the swollenin is known not to possess the hydrolytic activity against cellulose. Therefore, the swollenin is thought to play some roles other than hydrolysis of cellulose when the fungi attack the lignocellulose which is the main body of plant biomass. In this study, the gene encoding swollenin was cloned from Trichoderma reesei into Saccharomyces cerevisiae. The activity of the swollenin was then evaluated by quantifying how much the swollenin preparation assist the action of cellulose on the cellulose when the protein was incubated with cellulose and cellulase.

In a previous study we have isolated Lactobacillus sp. MS79 showing high resistance to various environmental stresses. MS79 was identified as L. fermentum using PCR-RFLP and 16S rDNA sequencing. In order to determine the mechanism of stress resistance at the molecular level, we attempted to clone rpoE gene from MS79. Initially we cloned partial rpoE (directed RNA polymerase, delta subunit) using degenerate primers. It has been shown that rpoE is induced and required during survival at extreme osmotic stress. Using Northern blot analysis two transcripts were observed from the stressed cells and displayed similar levels of induction. These results suggest that the extreme osmotic stress response of MS79 is under transcriptional regulation of rpoE and possibly cotranscription of two promoters like other bacteria. rpoE fragment of MS79 was cloned into nonreplicative delivery vector pRV300. The recombinant plasmid was successfully inserted into MS79 chromosome by single-crossover integration. Currently we are comparing the stress resistance response between rpoE knock-out mutant and MS79 to determine the direct involvement of rpoE in stress response of MS79.

Acanthopanax koreanum has been used in Korea to cure rheumatism, neuralgia, edema, dermatopathy, and impotence.1) Especially, this is cultivated only in Jeju. In this study, we heve investigated the antimicrobial and antioxidant activities on herb extract from Acanthopanax koreanum. Preparation of Acanthopnax koreanum used were a water extract of stems of the plant. The extracts of Acanthopnax koreanum originated from Jeju Island were provided from a stock farm product company(Jewoo Bio, Korea). Ferment strain was by four mixed strain(Lactobacillus acidophilus, Lactobacillus brevis, Lactobacillus plantrum, Bacillus subtilis) from 4.5brix concentration. The extract activity was compared at 7.5 brix, 4.5 brix and 4 mixed strains being concentrated liquid. We have tested the antioxidant activity by ESR(Electron Spin Resonance)2), DPPH(1,1-diphenyl-2-picrylhydrazyl) and hydroxyl radical activity. Four mixed strains culture extracts showed 40-89% of the DPPH radical scavenger activity and 25-78% of the hydroxyl radical scanvenger activity. The sensitivity of extracts was determined against different bacterial strains by disc diffusion method using 24h-old bacterial culture suspension (105CFU/ml) in agar media. The extracts exhibited antimicrobial activity against Vibrio alginolyticus KCCM 40513, Vibrio campbellii KCCM 41986, Vibrio harveyi KCCM 40866, Vibrio salmonicida KCCM 41663, Vibrio furnissii KCCM 41679, Vibrio anguillarum KCTC 2711, Streptococcus parauberis KCTC 3651, Streptococcus iniae KCTC 3657, Edwardsiella tarda KCTC 12267. The higher antimicrobial activity was observed on 4 mixed strains containing the 4.5 brix extract.

A Multi-fermentor is an efficient equipment th investigate the effect of operating conditions. In order to exploit the advantage, maintaining the same stable conditions throughout a mult-fermentor is essential except for changing parameter. The oxygen transfer is one of the most important factor in all aerobic fermentations.1,2) Especially, the oxygen mass transfer coefficient (kLɑ) can play a primary role in the scale-up and economical process.3) Since coefficient depends on operational conditions (power input, stirrer speed and gas flow), geometry of the vessel, stirrer, it can serve as a determinate factor on evaluating fermentor performance. In this study, the impact factor of measurement of the kLɑ value were sparger type, impeller size and baffle. In order to obtain the constant value of kLɑ each fermentor has modified sparger design, impeller size and baffle. The initial average estimated kLɑ values of each six fermentors were 0.032 s-1 with 0.011 standard deviation at 400rpm. The optimal kLɑ average values of each fermentor was 0.030 s-1 and standard deviation was 0.001, which values were obtained by changing sparger and baffle design, position of impeller. The each fermentor also have maintained stable conditions.