김치에서 분리한 Lactobacillus sp. JC-7과 Lactobacillus acidophilus 88간의 Electrofusion 최적조건 설정

조영배,최현정,백형석,전홍기 부산대학교 유전공학연구소 1997 분자생물학 연구보 Vol.13 No.-

김치 발효 숙성기간을 연장하고 신선도를 오랫동안 유지 할 수 있는 김치발효 starter의 개발을 목적으로 최적숙성기에 있는 김치로부터 bacteriocin생성능이 없는 유산균을 분리하여 동정하였으며, bacteriocin생성능이 우수한 L.acidophilus 88을 융합시키기 위해 electrofusion에 대한 최적 조건을 검토하였다. 분리균주는 Lactobacillus속으로 동정되어 Lactobacillus sp. JC-7이라 명명하였다. Electrofusion에 의해 생성된 융합주를 식별하기 위해 streptomycin(2.5㎎/ml)에 내성을 나타내는 Lactobacillus sp. JC-7변이주와 kanamycin(600㎍/ml)에 내성을 나타내는 L.acidophilus 88변이주를 분리하였다. Electrofusion을 100V/㎝, 120msec(72ohms,1670 capacitance) 에서 수행했을 때 융합효율이 가장 양호하였으며 전기장의 세기와 시간이 중가할수록 융합 효율이 현저히 감소하였다. 2가 양이온은 농도가 중가할수록 대체적으로 융합효율을 감소시키는 경향을 나타내었으나 1mM MGCl_2에서는 대조군에 비해 융합효율이 약간 증가하였다. PEG매개에 의한 융합법의 융합효율을 비교한 결과, 융합효율은 chemical fusion<electrofusion<electrofusion+20% PEG순을 나타내었다. Evaluation of Optimum Conditions for the Electrofusion between Lactobacillus sp. JC-7 Isolated from Kimchi and Lactobacillus acidophilus 88. Young-Bae Jo, Hyun-Jung Choi, Hyung-Suk Baik and Hong-Ki Jun*. Department of Microbiology, Pusan National University, Pusan 609-735,Korea-A lactic acid bacterium was isolated from kimchi. The isolated strain was identified as the genus Lactobacillus thrugh its morphological characteristics and named as Lactobacillus sp. JC-7. The optimum conditions for the electrofusion between streptomycin(2.5㎎/ml)resistant mutant of Lactobacillus acidophilus 88 and kanamycin(600㎍/ml) resistant mutant Lactobacillus sp. JC-7 were evaluated. The highest number of fusants were obtained at a capacitance value of 120msec(1670㎌), a field strength of 100V/㎝,and a pulse controller setting of 72Ω. The potimum pH of elecroporation buffer was 7.5 and the concentration of divalent cation was 1mM MG^2+. Electrofusants were efficiently obtained by addition 20% polyethylene glycol to electroporation buffer. The yield of fusion was better than that of using polyethylene glycol mediated chemical induction.

Lactobacillus casei와 Lactobacillus delbrueckii간의 Protoplast 융합에 관한 연구

전홍기,김미경,백형석 부산대학교 유전공학연구소 1992 분자생물학 연구보 Vol.8 No.-

유산균주의 균주개량방법의 일환으로 protoplast fusion 방범을 사용하여 linconmycin에 내성을 나타내는 Lactobacillus casei KCTC 1121과 rifampicin에 내성을 나타내는 Lactobacillus delbrueckii JK-414의 protoplast 형성과 재생, 융합에 대한 조건 및 융합주의 생리학적 성질 등을 검토하였다. Lactobacillus casei와 Lactobacillus delbrueckii JK-414는 삼투압 안정제로 sucrose가 함유된 protoplast forming buffer에서 5μg/ml의 mutanolysin으로 42˚C, 15분간 처리했을 때 Protoplast 형성율이 높게 나타났다. L. casei와 L. delbrueckii는 대수 증식기 중반에서 protoplast가 가장 잘 형성되었으며, MRS 배지에 삼투압 안정제로 sucrose 10%, MgCl_2 6mM, CaCl_2 6mM, gelatin 2.5%를 첨가하여 만든 재생배지에서 재생효율이 가장 좋았다. 한편, L. casei와 L. delbrueckii 사이의 protoplast 융합은 40%의 PEG 4,000을 처리하였을 때 가장 양호하였으며, 그 융합 효율은 3.2×10^-4이었다. L. casei가 L. delbrueckii보다 높은 산 생성능을 보였으며 융합주 중 특히 F23, F35의 산 생성능이 우수하였다. 융합주 중 F23, F24의 protease 활성이 모균주의 protease 활성보다 높았으며, 이들 융합주의 DNA 함량은 모균주의 2배였다. Protoplast fusion between lincomycin resistant Lactobacillus casei KCTC 1121 and rifampicin resistant Lactobacillus delbrueckii JK-414 was attempted to obtain the improved strains. Protoplasts of L. casei and L. delbrueckii were produced by mutanolysin digestion at 42˚C for 15 min. L. casei cells were converted to protoplasts by treating with 5 μg/ml of mutanolysin in 20 mM HEPES buffer (pH 7.0) containing 0.75 M sucrose at the middle logarithmic growth phase. In case of L. delbrueckii 1.0M sucrose was used osmotic stabilizer. Regeneration of protoplast in both strains was efficiently accomplished on the regeneration medium containing 10% sucrose, 6mM MgCl_2, 6mM CaCl_2, and 2.5% gelatin. Protoplast fusion between L. casei and L. delbrueckii was carried out in the presence of 40% of PEG 4,000. The frequency of protoplast fusion was found to be about 3.2 × 10^_4. Acid production of L. casei was better than that of L. delbrueckii. Among fusants, F23 and F35 exhibited excellent lactic acid production. F23 and F24 exhibited the improved proteolysis compared to that of the parent strains and they had twice as much as DNA content of the parents.

Lactobacillus casei와 Lactobacillus delbrueckii간의 Protoplast 융합에 관한 연구

전홍기,김미경,백형석 한국산업미생물학회 1992 한국미생물·생명공학회지 Vol.20 No.1

유산균주의 균주개량방법의 일환으로 protoplast fusion 방법을 사용하여 lincomycin에 내성을 나타내는 Lactobacillus casei KCTC 1121과 rifampicin에 내성을 나타내는 Lactobacillus delbrueckii JK-414의 protoplast 형성과 재생, 융합에 대한 조건 및 융합주의 생리학적 성질 등을 검토하였다. Lactobacillus casei와 Lactobacillus delbrueckii JK-414는 삼투압 안정제로 sucrose가 함유된 protoplast forming buffer에서 5㎍/㎖의 mutanolysin으로 42℃, 15분간 처리했을 때 protoplast 형성율이 높게 나타났다. L. casei와 L. delbrueckii는 대수 증식기 중반에서 protoplast가 가장 잘 형성되었으며, MRS 배지에 삼투압 안정제로 sucrose 10%, MgCl_2 6 mM, CaCl_2 6 mM, gelatin 2.5%를 첨가하여 만든 재생배지에서 재생효율이 가장 좋았다. 한편, L. casei와 L. delbrueckii 사이의 protoplast 융합은 40%의 PEG 4,000을 처리하였을 때 가장 양호하였으며, 그 융합 효율은 3.2×10^-4이었다. L. casei가 L. delbrueckii보다 높은 산 생성능을 보였으며 융합주 중 특히 F23, F35의 산 생성능이 우수하였다. 융합주 중 F23, F24의 protease 활성이 모균주의 protense 활성보다 높았으며, 이들 융합주의 DNA 함량은 모균주의 2배였다. Protoplast fusion between lincomycin resistant Lactobacillus cαsei KCTC 1121 and rifampicin resistant Lactobacillus delbrueckii JK-414 was attempted to obtain the improved strains. Protoplasts of L. casei and L. delbrueckii were produced by mutanolysin digestion at 42℃ for 15 min. L. casei cells were converted to protoplasts by treating with 5㎍/㎖ of mutanolysin in 20 mM HEPES buffer (pH 7.0) containing 0.75 M sucrose at the middle logarithmic growth phase. In case of L. delbrueckii 1.0 M sucrose was used osmotic stabilizer. Regeneration of protoplast in both strains was efficiently accomplished on the regeneration medium containing 10% sucrose, 6 mM MgCl_2, 6 mM CaCl_2, and 2.5% gelatin. Protoplast fusion between L. casei and L. delbrueckii was carried out in the presence of 40% of PEG 4,000. The frequency of protoplast fusion was found to be about 3.2×10_-4. Acid production of L. casei was better than that of L. delbrueckii. Among fusants, F23 and F35 exhibited excellent lactic acid production. F23 and F24 exhibited the improved proteolysis compared to that of the parent strains and they had twice as much as DNA content of the parents.

김치 분리균인 Bacillus sp. JK-43이 생산하는 Cyclodextrin Glucanotransferase의 생산 및 특성

전홍기,배경미,김영희,백형석 부산대학교 유전공학연구소 2000 분자생물학 연구보 Vol.16 No.-

김치 시료로부터 자동산화되지 않으며 열 및 중성 pH에서 안정한 AA 유도체인 AA-2G를 생산할 수 있는 당전이활성을 가진 CGTase 생산균주를 분리하였고, 분리균주의 형태학적, 배양학적, 생리학적 성질 및 16s-rDNA sequences를 조사한 결과 그람 양성의 간균으로 호기성이며 내생포자를 형성하는 전형적인 중온성 Bacillus sp. JK-43으로 동정되었다. Bacillus sp. JK-43의 CGTase는 AA-2G 뿐만 아니라 AA-6G로 추정되는 물질을 함께 생산하였으며, 효소 최적생산조건은 1.0% soluble starch, 1.0% yeast extract, 1.0% Na_2CO_3, 0.1% K_2HPO_4 그리고 0.02% MgSO_4·7H_2O가 함유된 배지에서 pH 7.0, 37℃에서 26시간 동안 진탕배양하였을 때였다. 각종 당공여체에 따른 Bacillus sp. JK-43의 AA-2G 생산성을 조사한 결과 β-CD에서 가장 높은 AA-2G 생산성을 보였으며, 식혜제조폐액인 엿기름 및 밥당화액에서도 비교적 높은 AA-2G 생산성을 보였다. 또한 여러 가지 당수용체에 대한 JK-43의 CGTase의 당전이 반응을 검토한 결과 sucrose, mannitol 및 inositol에서 높은 당전이 수율인 70∼90%를 나타내었다. A bacterial strain, designated as JK-43, producing extracellular cyclodextrin hlucanotransferase (CGTase)[EC 2.4.1.19] was isolated from kimchi. The CGTase from isolated strain JK-43 showed the transglucosylation activity from soluble starch to L-ascorbic acid (AA) compared to those obtained from other strains. A main product formed by this reaction was identified as 2-O-α-glucopyranosyl L-ascorbic acid (AA-2G) by testing its susceptibility to α-glucosidase hydrolysis, the HPLC prodiles, and through the elementary analysis. The β-CD, γ-CD, potato starch and corn starch were identified to be suitable glucosyl donor for transglucosylation reaction on AA by CGTase. Acceptor specificity on AA-2G production was examined by use of AA, Iso-AA and AA-2P. Transglucosylation was observed toward AA-2P as well as AA. The microorganism isolated from kimchi was identified as a strain of Bacillus sp. JK-43 based on the maximal CGTase production was observed in a medium containing 1.0% soluble starch, 1.0% yeast extract, 1.0% Na_2CO_3, 0.1% K_2HPO_4, and 0.02% MgSO_4·7H_2O with initial pH 7.0. The strain was cultured at 37℃ for 26 hrs with reciprocal shaking.

Lactobacillus acidophilus 88과 Lactobacillus bulgaricus IFO 13953간의 세포융합주의 특성에 관한 연구

조영배,김혜정,김성구,백형석,전홍기 부산대학교 유전공학연구소 1996 분자생물학 연구보 Vol.12 No.-

유산균의 균주개량방법의 일환으로 protoplast fusion 기법과 electrofusion법을 이용하여 protease 활성, lipase 활성, 내열성, 내산성 등이 우수한 L. bulgaricus 와 bacteriocin을 생산하는 L. acidophilus를 융합시켜 얻은 융합주들의 생리학적 성질을 검토하였다. 산 생성능, 내열성, 내산성, protease, lipase 활성 등은 L. bulgaricus가 L. acidophilus 보다 우수하였다. L. bulgaricus는 lactose와 sorbose를 이용하였으나 maltose와 sorbitol을 이용하지 못하는 반면, L. acidophilus는 maltose를 이용하고 lactose와 sorbose를 이용하지 못하였다. 융합주 가운데서는 3, 6, 7, 8, 10번이 모균주의 발효능 특성을 함께 지님으로서 재조합체임을 확인 할 수 있었으며, sorbitol의 경우 모균주에서는 발효능이 전혀 나타나지 않았음에도 불구하고 융합주 4, 7번이 발효능을 나타내어 융합과정에서 새로운 형질을 획득하기도 한다는 사실을 알 수 있었다. Lactase 활성은 모균주 모두 높은 β-galactosidase 활성을 보였으나, phospho-β-galactosidase 활성은 거의 없었으며 융합주들도 다소 차이는 있었지만 모균주와 유사한 효소 활성을 나타내었다. 발효에 있어서 key enzyme으로 작용하는 protease, lipase 등의 효소 활성도 모균주의 활성 보다 우수한 융합주도 존재하였다. Aninterspecific fusant was made from the protoplasts of two strains of lactobacillus genus (streptomycin and lincomycin resistant L. bulgaricus and kanamycin resistant L. acidophilus 88). The functional properties of the fusant were examined by determining bacteriocin productivity, acid producing activity, ability of carbohydrates fermentation and three important enzyme activities. The recombinant strain revealed bacteriocin productivity. Acid production and β galactosidase, phospho-β-galactosidase, lipase and protease activity of L. bulgaricus were better than those of L. acidophilus 88. Among fusants, β-galactosidase activity of two strains were better than that of the parent strains but phospho-β-galactosidase activity remarkably lower. One fusant revealed the improved proteolysis compared to that of the parent strains- Lipase activity of L. bulgaricus was better than that of L. acidophilus but another fusant exhibited the highest lipase activity.

Interaction of Plasmid pKM101 Mutator Function with uvrA and uvrB Genes

Baik, Hyung Suk,Park, Hyun Jeong 부산대학교 1986 자연과학논문집 Vol.42 No.-

DNA 회복 기능이 결여된 Escherichia colir 균주에 nurA^(-)와 nurB^(-) mutator 기능을 가진 plasmid pKM101 또는 pSL4를 도입시키고, 돌연변이유발에 DNA 회복에 미치는 pKM101 mutator의 기능 그리고 pKM101과 DNA 회복 기능에 관여하는 유전자와의 상호작용을 조사하였다. 또한 UV와 4-NQO가 유발하는 DNA손상회복과 돌연변이에 있어서의 이들 DNA 회복 유전자의 역할을 조사하였다. 본 연구에서 얻어진 결과는 다음과 같았다. 1. DNA 회복 기능이 결여되어 있는 E. coli B/r 균주에 pKM101 또는 pSL4를 도입시킬 때의 빈도는 10^(-4)-10^(-6)이었으며 균주내에서 안정하였다. 2. E. coli B/r 균주에 pKM101 또는 pSL4를 도입시키면 자발적 인돌연변이율은 증가되었는데 pSL4가 도입되었을 때가 pKM101이 도입되었을 때보다 전반적으로 자발적 돌연변이율이 더 증가되었다. 3. uvrA^(-) 균주에서 pKM101과 pSL4는 UV나 4-NQO에 의한 돌연변이에는 아무 영향도 미치지 못하였다. 4. uvrB^(-) 균주는 UV와 4-NQO에 대해 민감하였으나 uvr+에 대해서는 민감하지 않았다. uvrB^(-) 균주에서 pKM101과 pSL4 모두 UV에 대한 보호효과를 나타내었다. uvrB-돌연변이는 4-NQO에 의한 돌연변이를 강하게 증가시켰으며 UV에 의한 돌연변이에는 아무런 영향을 미치지 않았다. uvrB^(-) 균주에 pKM101이나 pSL4가 도입되면 4-NQO에 의한 돌연변이에는 강하게 증가시켰으며 UV에 의한 돌연변이에는 아무런 영향을 미치지 않았다. uvrB^(-) 균주에 pKM101이나 pSL4가 도입되면 4-NQO에 의한 돌연변이에 대해서는 억제 효과를 나타내었으나 UV에 의한 돌연변이에 대해서는 아무런 영향을 미치지 못하였다. 5. UV나 4-NQO에 의한 DNA 상해의 회복은 uvrB 유전자의 기능이 관여하는 같은 DNA에 의하여 진행된다고 생각되지만 이들 돌연변이원에 의한 돌연변이 유발은 서로 다른 기작에 의하여 진행되는 것 같았다. 6. 본 연구의 결과를 근거로 해서 다음과 같은 가설을 세울 수 있다. 즉, pKM101의 mutator 효과와 돌연변이원에 대한 보호 효과는 플라스 미드의 한 유전자의 기능에 의한 것이며 이 유전자 산물은 error-free DNA 회복과 error-prone DNA 회복 system 양쪽 다에 상호작용을 가지며 어떤 돌연변이원에 의해서 uvrA-와 uvrB^(-)군주에서 나타난 pKM101의 anti-mutator 효과는 이 pKM101 유전자 산물이 error-prone DNA 회복 기능을 억제함으로써 더 많은 error-free DNA회복을 유발하여 나타나는 것이라고 생각할 수 있다. A plasmid with mutator effect, pKM101, and its mutant pSL4 were introduced into Escherichia coli B/r strains possesing DNA repair capacities (uvr A^(-) and uvr B^(-)) and determined the function of pKM101 mutator in the mutagenesis and DNA repair and their interactions with DNA damage and the mutagenesis induced by mutagens (UV and 4-NQO). The following results were obtained. 1. The transfer frequencies of pKM101 and pSL4 to E. coli B/r repair deficient mutants were in the range of 10^(-4) to 10^(-6) and the plasmids were maintained stably in the cells. 2. Whenplasmid pKM101 or pSL4 was introduced into the E. coli B/r strains, the spontaneous mutation rate was increased. The effect of pSL4 was generally more pronounced than that of pKM101. 3. The uvrA^(-) strain was sensitive to UV and 4-NQO. Both plasmid pKM101 and pSL4 had a protection effect against UV and 4-NQO. The uvrA^(-) mutation increased the mustagenesis induced by 4-NQO and had no effect on the UV-mutagenesis. Both plasmid pKM101 and pSL4 had no effect on the UV- and 4-NQO mutagenesis in the uvrA^(-) strain. 4. The uvrB strain was sensitive to UV and 4-NQO. Both plasmid pKM101 and pSL4 had a protection effect against UV but only pSL4 showed the protection effect against 4-NQO in the uvrB- strain. The uvrB^(-) mutation sharply increased the 4-NQO mutagenesis and had no effect on the UV-mutagenesis. 5. The DNA lesions caused by the UV and 4-NQO treatment seems to be repaired by the same repair processes involng the functions of the uvrB gene. However, different processes seems to be involved in the mutagenesis induced by these two mutagens. 6. On the basis of results obtained in this work, it was postulated that the mutator effect and the mutagen protection effect of pKM101 may be the results of the plasmid codes one gene function and that the gene product may interact with both the error-free DNA repair and error-prone DNA repair system. The anti-mutator effect of pKM101 seen in the uvrA- and uvrB^(-) strain for certain type of mutagenes may be due to the pKM101 gene product interferring the error-prone repair processes by inducing more error-free DNA repair.

Mutational Analysis of Cucumber Mosaic Virus Movement Protein Gene

BAIK, HYUNG SUK,Paek, Kyung Hee,You, Jin Sam 생화학분자생물학회 1997 BMB Reports Vol.32 No.1

The movement protein of cucumber mosaic virus (CMV) is required for cell-to-cell movement of viral RNA. The movement of viral RNA occurs through the plant intercellular connection, the plasmodesmata. The viral movement protein was known to be multifunctional. In this work, a series of deletion mutants of CMV movement protein gene were created to identify the functional domains. The mutated movement proteins were produced as inclusion body in E. coli, and purified and renatured. A polyclonal antibody was raised against the CMV-Kor strain (Korean isolate) movement protein expressed in E. coli. The ability of the truncated proteins to bind to ssRNA was assayed by UV cross-linking and gel retardation analyses. The results indicate that the domain between amino acids 118 and 160 of CMV movement protein is essential for ssRNA binding.

Salmonella System 에 있어서의 농약의 돌연변이 유발성

백형석(Hyung - Suk Baik),변우현(Woo - Hyun Byeon),전문진(Moon - Jin Chun),이세영(Se - Young Lee) 한국미생물생명공학회 1977 한국미생물·생명공학회지 Vol.5 No.4

합성세제를 분해하는 미생물의 분리

백형석,안영희,전홍기 부산대학교 1988 자연과학논문집 Vol.45 No.-

This study was carried out to obtain microorganisms having strong synthetic detergent-degrading plasmid. For that purpose, the medium containg sodium dodecylbenzenesulfonate(soft type) as sole source of carbon was employed to isolate synthetic detergent-degrading bacteria in sewages, river waters and soils. Isolated bacteria(152 strains) were examined for primary biodegradation rate of synthetic detergent in the medium by methylene blue-active subtance(MBAS) method and tested for resistance to several metal compounds by metal compound gradient method. Eventually, 13 strains were selected, which having both relatively efficient degradation rate of synthetic detergent and higher resistance to metal compounds.

원통모양의 유리 전박 피판으로 경부 식도의 재건

백봉수,류형호,이정형,조병채,박준식,변진석 大韓成形外科學會 1996 Archives of Plastic Surgery Vol.23 No.4

Various reconstruction of pharyngoesophageal defects after ablative surgery have been made to restore functions of the pharyngoesophagus. A tubed free radial forearm flap was used to reconstruct the pharyngoesophagus is 23 patients after resection of neoplasms from May 1989 to October 1994. 19 were male and 4 were female. Average patient age 62.2 years. Follow-up period ranged 10 to 64 months with mean 18 months. Oral intake within 3 weeks was possible in 18 patients(78%). 19patients(82%) were discharged within 4 weeks. The immediate postoperative complications were hematoma (n=1), bleeding(n=2), infection(n=3), fistula(m=4), and venous thrombosis(n=1). The late complication was stricture of lower anastomosing site (n=3). The tubed free radial forearm flap had advantages over free jejunal transfer : Larger caliber of the vascular pedicle, longer ischemic time, no laparotomy with less morbidity of the donor site and better toleration to the radiotherapy. Troublesome disadvantages were stricture and fistula formation at suture sites. We modified conventional free radial forearm flap to reduce the complications. 1. A small monitoring flap supplied by the septocutaneous branch of the radial artery was elevated to check the survival of the flap. 2. During tubing, the longitudinal suture site was overlapped with a deepithelialized skin flap and double layer sutures were done to prevent the fistula. 3. Two small triangular flaps were designed and inserted in the distal anastomotic site to prevent circular contracture. The outer layer sutures were anchored to surrounding rigid structure to withstand shrinkage and circular contraction. With this modification, the incidence of stricture and fistula formation was reduced to 13.0% 17.4% respectively and these complication could be treated conservatively.