• KSBB Journal
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• 제19권5호
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• pp.363-367
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• 2004

Leuconostoc mesenteroides NRRL B-1149 produces various glucoseyltransferases for the synthesis of dextran, levan and glucose-1-phosphate using sucrose as a substrate. A sucrose phosphorylase (1149SPase) was purified from L. mesenteroides NRRL B-1149 culture by using hollow fiber filtration (30 kDa cut off), Toyopearl DEAE 650 M column chromatography and following two times of DEAE-Sepharose column chromatographies. The specific activity of the purified 1149SPase was 25.7 (U/mg) with $16\%$ yield. The 1149SPase showed a molecular size of 56 kDa on denatured $10\%$ SDS-PAGE. The N-terminal amino acid sequence of the enzyme was MEIQNKAM. The optimum pH and temperature of this enzyme were 6.2~6.5 and 37^{circ}C, respectively. It had an apparent K_{m} of 6.0 mM and K_{cat} of 1.62/s for sucrose. 1149SPase crystal was formed by hanging drop diffusion technique using 20 mM calcium chloride dihydrate, 100 mM sodium acetate trihydrate pH 4.6 and $30\%$ 2-methyl-2,4-pentanediol as vaporizing and reservation solution. The 1149SPase catalyzes transferring of glucose from isomaltose or sucrose to salicin and salicyl alcohol by disproportionation reaction or acceptor reaction and synthesized two acceptor products, respectively.

• KSBB Journal
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• 제19권5호
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• pp.368-373
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• 2004

The optimal condition for the production of a glucan and a fructan synthesizing enzymes from Leuconostoc mesenteroides NRRL B-1149 were studied based on the different medium compositions. Response surface methodology was applied to find the optimistic condition showing the relationship between the fermentation response (enzyme activities) and the fermentation variable concentrations of yeast extract, peptone concentration, K2HP04 concentration and sucrose. Optimum medium composition for both enzymes production was $0.75\%$ yeast extract, $0.72\%$ peptone, $1\%$ K2HP04 and $2.17\%$ sucrose. Using this medium, the activities produced in culture was 0.90 U/m~ for glucosyltransferase (GTase) and 0.96 U/ml for fructosyltransferase (FTase). After purification of 1149FTase by consecutive chromatographies using Sephadex G-150 and DEAE-Sepharose, a 1149FTase of 210 kDa on $7\%$ polyacrylamide gel was isolated and it synthesized soluble fructan. The 1149GTase showed a band of 180 kDa on $8\%$ polyacrylamide gel after purification using Bio-Gel P-100 gel chromatography and DEAE-Sepharose ion exchange chromatography and it synthesized insoluble glucan. The linkages of polymers were determined by methylation using Hakomori reagent and following NMR analysis. The glucan was composed of a(1~6) and a(1~3) linkages and the fructan was levan.

• Applied Biological Chemistry
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• 제35권3호
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• pp.202-209
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• 1992

청국장 발효시 생산되는 점질물에 대하여 연구하기 위하여 볏짚으로부터 점질물 생성능이 우수하고 좋은 풍미의 청국장 재조균 SB-1를 분리하여 Bacillus sp.로 동정하였다. 이들 균주들은 $42^{\circ}C$에서 48시간때에 가장 많은 점질물을 생산했으며, M.S.G.의 첨가시 점질물 및 아미노태질소와 암모니아태질소의 성분에는 큰 변화가 없었으나, Sucrose 첨가시 점질물과 아미노태질소의 생산량은 증가한 반면 암모니아태질소는 오히려 감소하였다. 점질물의 분자량은 $15,000{\sim}65,000$ 정도로 추정되었다. 점질물은 G.C.와 HPLC의 분석결과 주로 glutamate와 fructose로 구성된 것으로 나타났으며, 배지조성의 차이에 따라 다른 고분자물질이 검출되었다.

• Journal of Microbiology and Biotechnology
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• 제9권2호
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• pp.213-218
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• 1999

Fructans are polyfructose molecules that function as nonstructural storage carbohydrates in several plants. In addition, it has been suggested that, due to their solubility, they can play an important role in helping plants survive periods of osmotic stress. In order to study the effect of levan synthesis on plant growth, the coding region of the levansucrase gene, which was isolated from Zymomonas mobilis, was introduced into tobacco plants using Agrobacterium tumefaciens-mediated transformation. The presence of the levansucrase gene in transgenic plants was verified by genomic DNA gel blot analysis. RNA gel blot and immunoblot analyses showed an accumulation of the corresponding transcript and protein product of the bacterial levansucrase gene in transgenic plants. Furthermore, a thin layer chromatography analysis revealed that fructans were synthesized and deposited in transgenic tobacco plants. When $T_1$ seeds were germinated and grown under polyethylene glycol-mediated drought stress or cold stress, the transgenic seedlings displayed a substantially higher level of growth than that of untransformed plants. These results suggest that fructans may playa significant role in the tolerance of plants under osmotic stress.

• Biotechnology and Bioprocess Engineering:BBE
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• 제9권5호
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• pp.339-344
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• 2004

A method for synthesizing branched fructo-oligosaccharides (BFOS) with a high concentration of sucrose ($1{\~}3$ M) was developed using levansucrase prepared from Leuconortoc mesenteroides B-1355C. The degree of polymerization of oligosaccharides synthesized according to the present method ranged from 2 to over 15. The synthesized BFOS were stable at a pH ranges of 2 to 4 under $120^{\circ}C$. The percentage of BFOS in the reaction digest was $95.7\%$ (excluding monosaccharides; $4.3\%$ was levan). BFOS reduced the insoluble glucan formation by Streptococcus sobrinus on the surfaces of glass vials or stainless steel wires in the presence of sucrose. They also reduced the growth and acid productions of S, sobrinus. Oligosaccharides can be used as sweeteners for foods such as beverages requiring thermo- and acid-stable properties and 3s potential inhibitors of dental caries.

• Food Science and Biotechnology
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• 제16권4호
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• pp.509-514
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• 2007

In this study, we optimized the production of ${\gamma}-polyglutamic$ acid (PGA) in soybean milk cakes (SMC) fermented with Bacillus subtilis GT-D and B. subtilis KU-A, to be utilized as a functional food ingredient. PGA production was dependent upon the glutamate content, fermentation time, and type of Bacillus sp. The consistencies of the SMCs fermented by B. subtilis GT-D and B. subtilis KU-A were highest after 36 hr of fermentation, and then decreased gradually. The SMC fermented by B. subtilis KU-A had a higher consistency than the SMC fermented by B. subtilis GT-D. In the presence of 10% defatted soy flour (DFS), 5% glutamate in the SMC was efficiently converted into polyglutamic acid (PGA) for 24 hr, indicating a conversion yield above 96%, but its conversion then decreased with higher concentrations of glutamate. The soluble solid content (mucilage) of the SMC fermented with B. subtilis KU-A was 9.5%(w/w), and composed of 65.6% PGA (Mw 1,536 kDa) and some polysaccharides. However, the SMC fermented with B. subtilis GT-D had a mucilage content of 7.8%(w/w), and was composed of 66.4% PGA (Mw 1,409 kDa), 11.5% levan, and some polysaccharides. The viscoelastic values of the mucilage obtained using B. subtilis KU-A were much higher than those of mucilage obtained using B. subtilis GT-D. Also, the G'-value (elastic modulus) was higher than the G"-value (viscous modulus).

• Applied Biological Chemistry
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• 제39권3호
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• pp.165-171
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• 1996

볏짚으로부터 분리한 Bacillus sp. K-1과 그 변이주 KM-21과 KM-83이 생산하는 biopolymer들의 물성학적 성질을 비교 조사하였다. 은도에 따른 biopolymer의 겉보기 점도는 온도가 높아짐에 따라 감소하였으며, $20^{\circ}C$일 때에 비해 $80^{\circ}C$일 때 KM-21 균주가 생산하는 biopolymer는 1.7배, K-1균주의 biopolymer는 2.7배, KM-83 균주의 biopolymer는 1.9배 정도로 낮은 점성을 보였다. Biopolymer 용액의 점성은 농도가 증가함에 따라 크게 증가하여 6%의 농도에서 가장 높게 나타났으며, 2%, 4%, 6% 모두 pseudoplastic특성을 나타내어 농도에 대한 의존성이 매우 높았다. 6%의 농도일 때 2%의 농도와 비교해서 전단 속도가 $93\;sec^{-1}$ 일 때 KM-21은 26배, K-1은 56배, KM-83은 28배로 모균주의 biopolymer인 K-1이 가장 높은 점성을 보었다. pH에 따른 점도 변화는 pH7.0에서 최고를 보였으며, 알칼리성 조건일 때에 비해 산성조건에서 상대적으로 낮은 값의 점성을 보였다.

• 대한화장품학회:학술대회논문집
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• 대한화장품학회 2003년도 IFSCC Conference Proceeding Book I
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• pp.700-718
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• 2003

Fructan, a polysaccharide existing in plants or produced by microorganisms, is a sugar polymer of fructose with $\beta$-2,6 linkages. In this study, we investigated some cosmeceutical properties of Fructan such as moisturizing effect, cell proliferation effect, anti-inflammation effect and cell cytotoxicity. Zymomonas mobilis, a microorganism producing Fructan, was cultured in a medium containing 10％ sucrose and 2％ yeast extract as main components for 24 hours at 37$^{\circ}C$ and pH 7. Fructan was obtained by precipitation from the cultured medium by adding alcohol (alcohol ratio of 1:3) after removing the enzyme by centrifuging. Fructan exhibited almost same moisturizing effect as hyaluronic acid and cell proliferation effect on human fibroblast and keratinocyte as well. Moreover, on cell proliferation test on bio-artificial skin constructed by 3-dimensional(3-D) culture after inducing primary skin inflammation with 0.5％ sodium lauryl sulfate (SLS), the 3-D artificial skin treated with 0.01 mg/ml, 0.05mg/ml of Fructan exhibited higher cell proliferation than the 3-D artificial skin treated with SLS only. On anti-inflammation test on 3-D artificial skin evaluated by measuring secreted quantity of interleukin-1$\alpha$ (IL-1$\alpha$) which is a pre-inflammatory mediator induced by SLS, the quantity of IL-1$\alpha$on the 3-D artificial skin treated with 0.01 mg/ml, 0.05mg/ml of Fructan was less than the one on the 3-D artificial skin treated with SLS only. As a result of these studies, Fructan has anti-inflammation effect against inflammatory reaction by a skin irritant as well as cell proliferation effect in bio-artificial skin. Fructan was also evaluated as a safe material without any toxicity in safety tests using fibroblasts and animals.

• The Plant Pathology Journal
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• 제39권3호
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• pp.275-289
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• 2023

Fungal isolates from infected Chinese quince trees were found to cause black rot in Yeongcheon, Gyeongsangbuk Province, Korea. The quince leaves withered and turned reddish-brown and fruits underwent black mummification. To elucidate the cause of these symptoms, the pathogen was isolated from infected leaf and fruit tissues on potato dextrose agar and Levan media. Several fungal colonies forming a fluffy white or dark gray mycelium and two types of fungi forming an aerial white mycelium, growing widely at the edges, were isolated. Microscopic observations, investigation of fungal growth characteristics on various media, and molecular identification using an internal transcribed spacer, β-tubulin, and translation elongation factor 1-α genes were performed. The fungal pathogens were identified as Diplodia parva and Diplodia crataegicola. Pathogenicity tests revealed that the pathogen-inoculated fruits exhibited a layered pattern, turning brown rotting; leaves showed circular brown necrotic lesions. The developed symptoms were similar to those observed in the field. Fungal pathogens were reisolated to fulfill Koch's postulates. Apples were inoculated with fungal pathogens to investigate the host range. Strong pathogenicity was evident in the fruits, with browning and rotting symptoms 3 days after inoculation. To determine pathogen control, a fungicidal sensitivity test was conducted using four registered fungicides. Thiophanate-methyl, propineb, and tebuconazole inhibited the mycelial growth of pathogens. To the best of our knowledge, this is the first report on the isolation and identification of the fungal pathogens D. parva and D. crataegicola from infected fruits and leaves of Chinese quince, causing black rot disease in Korea.

• 농업과학연구
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• 제34권2호
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• pp.161-170
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• 2007

Poly($\gamma$-glutamic acid) 및 levan의 생성균주로 알려진 Bacillus subtilis BS 62의 $\gamma$-GTP(ggt) 유전자를 해석하기 위하여 PCR 반응에 의해 BS 62의 염색체 DNA로부터 약 2.5 kb의 $\gamma$-GTP(ggt) 유전자 분획을 얻어 그 PCR 산물의 염기서열을 분석하여 기왕에 보고된 기타의 ggt 유전자와 비교 분석한 결과, B. subtilis $\gamma$-GTP 유전자(BSU49358)와 98%의 높은 상동성을 보였으며, Pseudomonas sp. A14(S63255)와는 37%, 방선균인 Streptomyces avermitils(AP005028)의 게놈 DNA와는 38%의 상동성을 나타냈다. BS 62의 $\gamma$-GTP 유전자의 open reading frame은 587개의 amino acid로 구성된 polypeptide의 것으로 해석되었으며, N-terminal의 28개 아미노산은 B. subtilis 펩타이드의 전형적인 형태를 보였고, 전형적인 리보솜의 부착부위는 개시코돈 ATG의 위쪽 7번에서 12번 염기(AGGAGG)에 위치하였고, 그리고 종지코돈 다음에서는 stem-loop 구조, ORF의 위쪽 약 50 bp 지점에서는 catabolite-responsive element가 발견되었다. 또한 B. subtilis 효소의 촉매자리로 추정되는 467번 잔기는 threonine으로서, 다른 박테리아의 serine, 포유동물의 cysteine과는 구별되는 것이었다.