유가식 배양공정에 의한 Bacillus thurngiensis의 고농도 포자생산

박창열,유연우 한국생물공학회 2000 KSBB Journal Vol.15 No.3

Bacillus thuringiensis를 생물농약으로 이용하기 위해서는 고농도의 포자형성에 의한 높은 살충성의 8 -endotoxin를 생산하는 것이 중요하다. 따라서 본 연구에서는 B. thuringiensis의 고농도 배양에 의한 높은 포자형성 수율을 얻기 위하여 40%의 용존산 소량과 $28^{\circ}C$에서 여랴 가지 방법의 유가식 배양법을 검토하였다. 최종 배양액의 glucose 농도가 50 g/L가 되도록 하는 경우의 유가식 배양에서는 continuos fed-batch culture의 linear gradient f feeding 법에 의하여 $9.37{\times}109$ cell/mL의 최대 생존 세포 수와 8 8.33 X 109 spore/mL의 최대 포자 수를 얻었으며, 이때의 포자 형 성율은 88.9% 이었다 최종 배양액의 glucose 농도가 100 m/L가 되도록 하는 경우의 유가식 배양에서는 intermittent fed-batch culture의 pH-stat 법에 의하여 $1.35{\times}1010$cell/mL의 최대 생존 세포 수와 $1.35{\times}1010$ spore/mL의 최대 포자 수를얻었으며, 이 때의 포자 형성율은 97.8% 이엇다. Both the production of high spore concentration and high insecticidal activity are required in the production of Bacillus thuringiensis to be used for the bacterial insecticide. In the production of high cell and spore concentrations of B. thuringiensis the continuous fed-batch culture(CFBC) and intermittent fed-batch culture(IFBC) were investigated at $28^{\circ}C$ by maintaining 40% dissolved oxygen concentration. When the final glucose concentration was 50 g/L the maximum viable cell number obtained using the CFBC with linear gradient feeding was $9.37{\times}109$ cells/mL and maximum spore concentration was $8.33{\times}109$ spores/mL which was approximately 84.4% yield of spore formation. When the final glucose concentration was 100 g/L the aximum viable cell and spore concentrations obtained using the IFBC with pH-statb were $1.38{\times}$1010 cells/mL and $1.35{\times}1010$ spores/mL respectively and the yield of spore formation was approximately 97.8%.

Bacillus spores: a review of their properties and inactivation processing technologies

조원일,정명수 한국식품과학회 2020 Food Science and Biotechnology Vol.29 No.11

Many factors determine the resistance propertiesof a Bacillus spore to heat, chemical and physical processing,including thick proteinaceous coats, peptidoglycancortex and low water content, high levels of dipicolinicacid (DPA), and divalent cations in the spore core. Recently, attention has been focused on non-thermalinactivation methods based on high pressure, ultrasonic,high voltage electric fields and cold plasmas for inactivatingBacillus spores associated with deterioration inquality and safety. The important chemical sporicides areglutaraldehyde, chorine-releasing agents, peroxygens, andethylene oxide. Some food-grade antimicrobial agentsexhibit sporostatic and sporicidal activities, such as protamine,polylysine, sodium lactate, essential oils. Surfactantswith hydrophilic and hydrophobic properties havebeen reported to have inactivation activity against spores. The combined treatment of physical and chemical treatmentsuch as heating, UHP (ultra high pressure), PEF(pulsed electric field), UV (ultraviolet), IPL (intense pulsedlight) and natural antimicrobial agents can act synergisticallyand effectively to kill Bacillus spores in the foodindustry.

Crystal structure of the inactive state of the receiver domain of Spo0A from Paenisporosarcina sp. TG-14, a psychrophilic bacterium isolated from an Antarctic glacier

이창우,박선하,이성구,신승철,한세종,김한우,박현호,김성환,김학준,박현,박하정,이준혁 한국미생물학회 2017 The journal of microbiology Vol.55 No.6

The two-component phosphorelay system is the most pre-valent mechanism for sensing and transducing environ-mental signals in bacteria. Spore formation, which relies on the two-component phosphorelay system, enables the long- term survival of the glacial bacterium Paenisporosarcina sp. TG-14 in the extreme cold environment. Spo0A is a key re-sponse regulator of the phosphorelay system in the early stage of spore formation. The protein is composed of a regu-latory N-terminal phospho-receiver domain and a DNA- binding C-terminal activator domain. We solved the three- dimensional structure of the unphosphorylated (inactive) form of the receiver domain of Spo0A (PaSpo0A-R) from Paenisporosarcina sp. TG-14. A structural comparison with phosphorylated (active form) Spo0A from Bacillus stearo-thermophilus (BsSpo0A) showed minor notable differences. A molecular dynamics study of a model of the active form and the crystal structures revealed significant differences in the α4 helix and the preceding loop region where phosphorylation occurs. Although an oligomerization study of PaSpo0A-R by analytical ultracentrifugation (AUC) has shown that the protein is in a monomeric state in solution, both crosslinking

Flusilazole의 훈증 효과에 의한 양파검은곰팡이병균(Aspergillus niger)의 포자 형성 억제

김흥태,박세원,최경자,김진철,조광연,Kim, Heung-Tae,Park, Se-Won,Choi, Gyung-Ja,Kim, Jin-Cheol,Cho, Kwang-Yun 한국식물병리학회 2002 식물병연구 Vol.8 No.2

1998년 전남 신안군의 양파 상온 저장고에서 발생한 검은색의 곰팡이는 검은곰팡이병을 일으키는 Aspergillusniger로 동정되었다. A. niger AnYD-1은 3$0^{\circ}C$에서 균사생장, 포자발아 그리고 양파에 대한 병원성이 가장 양호하였다. PDA 배지 상에서 실시한 in vitro의 실험에서 flusilazole과 hexaconazole은 A. niger AnYD-1의 균사 생장 억제 효과는 적었지만, 병원균에 살균제를 직접 처리하지 않고 증기 상태로 처리하였을 때 포자의 형성을 강하게 억제하는 특이적 인 훈증효과를 보였다. Flusilazole과 hexaconazole의 훈증의 효과는 사용하는 용매, 처리 농도와 배양 온도에 따라서 차이가 있었다. Dimethylsulfoxide와 dimethylformamide를 용매로 사용하였을 때 가장 높은 효과가 나타났으며, 고온에서 배양할수록 포자형성 억제 효과가 크게 나타났다. In 1998, a pathogen isolated from infected onions was identified as Aspergillus niger. At 3$0^{\circ}C$, A. niger AnYD-1 showed the best mycelial growth, spore germination, and high pathogenicity to onions. In spite of the weak inhibitory effect of flusilazole and hexaconazole on the mycelial growth on PDA, they showed the specific inhibitory activity against the formation of spores in the vapour phase. With flusilazole and hexaconazole, the effects of the solvent, the applied concentration and the incubating temperature on the activities inhibiting the spore formation were confirmed. Their inhibitory effect on the spore formation in vapour phase was excellent by solving them with dimethylsulfoxide and dimethylformamide among tested solvents, and applying them at high temperature such as 30~35$^{\circ}C$.

Effects of Oxygen Volumetric Mass Transfer Coefficient on Transglutaminase Production by Bacillus circulans BL32

Claucia Fernanda Volken de Souza,Rafael Costa Rodrigues,Marco Antônio Záchia Ayub 한국생물공학회 2009 Biotechnology and Bioprocess Engineering Vol.14 No.5

The effects of oxygenation in cultures of Bacillus circulans BL32 on transglutaminase (TGase) production and cell sporulation were studied by varying the agitation speed and the volume of aeration. Kinetics of cultivations has been studied in batch systems using a 2 L bioreactor, and the efficiency of agitation and aeration was evaluated through the oxygen volumetric mass transfer coefficient (kLa). It was adopted a two-stage aeration rate control strategy: first stage to induce biomass formation, followed by a second stage, in which cell sporulation was stimulated. A correlation of TGase production, spores formation, and oxygen concentration was established. Under the best conditions (500 rpm; 2 vvm air flow, followed by no air supply during stationary phase; kLa of 33.7 h –1 ), TGase production reached a volumetric production of 589 U/L after 50 h of cultivation and the enzyme yield was 906 U/g cells. These values are 61% higher than that obtained in shaker cultures and TGase productivity increased 82%, when kLa varied from 4.4 to 33.7 h –1 . The maximal cell concentration increased four times in relation to shaker cultures and the cultivation time for the highest TGase activity was reduced from 192 h to just 50 h. These results show the importance of bioprocess design for the production of microbial TGase, especially concerning the oxygen supply of cultures and the induction of cell sporulation The effects of oxygenation in cultures of Bacillus circulans BL32 on transglutaminase (TGase) production and cell sporulation were studied by varying the agitation speed and the volume of aeration. Kinetics of cultivations has been studied in batch systems using a 2 L bioreactor, and the efficiency of agitation and aeration was evaluated through the oxygen volumetric mass transfer coefficient (kLa). It was adopted a two-stage aeration rate control strategy: first stage to induce biomass formation, followed by a second stage, in which cell sporulation was stimulated. A correlation of TGase production, spores formation, and oxygen concentration was established. Under the best conditions (500 rpm; 2 vvm air flow, followed by no air supply during stationary phase; kLa of 33.7 h –1 ), TGase production reached a volumetric production of 589 U/L after 50 h of cultivation and the enzyme yield was 906 U/g cells. These values are 61% higher than that obtained in shaker cultures and TGase productivity increased 82%, when kLa varied from 4.4 to 33.7 h –1 . The maximal cell concentration increased four times in relation to shaker cultures and the cultivation time for the highest TGase activity was reduced from 192 h to just 50 h. These results show the importance of bioprocess design for the production of microbial TGase, especially concerning the oxygen supply of cultures and the induction of cell sporulation

한국산 및 중국산 김치의 Bacteria 군집 분석 및 발효과정 중 Bacilli 포자 형성 규명

안두현 ( Doo Hyun An ),김혜림 ( Hye Rim Kim ),정도원 ( Do Won Jeong ),( Jane M. Caldwell ),이종훈 ( Jong Hoon Lee ) 한국미생물생명공학회(구 한국산업미생물학회) 2014 한국미생물·생명공학회지 Vol.42 No.2

Bacteria 군집 차이를 이용한 신속한 한국산 및 중국산 김치 원산지 판별 가능성의 검토를 위하여 Terminal Restriction Fragment Length Polymorphism (T-RFLP) 분석법을 적용하였다. T-RFLP 분석은 김치발효에 관여하는 주요 유산균의 빠르고, 재현성 있는 검출에는 효과적이었지만, 종(species) 수준에서의 미생물 확인에는 한계를 가지고 있어 한국산 및 중국산 김치에 특이적으로 존재하는 bacteria의 검출에는 부적합한 것으로 평가되었다. T-RFLP를 적용한 발효과정 중의 한국산 및 중국산 김치에 존재하는 bacteria 군집 천이 분석은 비슷한 양상으로 나타났고, Bacillus 속이 발효 후기까지 검출되었다. 또한 Bacillus 속은 발효 후기에 포자를 형성하는 것으로 확인되었다. Terminal Restriction Fragment Length Polymorphism (T-RFLP) analysis was adopted to explore rapid differentiation in the diversity and dynamics of bacteria in kimchi made in Korea and China for future application in kimchi origin discrimination. TRFLP analysis supported the reproducible and rapid detection of major lactic acid bacteria known to be involved in kimchi fermentation. The taxonomic resolution level of this T-RFLP analysis was between the species and genus level, but was not specific enough for the detection of a bacterium found only in one origin, either Korea or China. The bacterial community structure successions in kimchi samples from Korea and China analyzed by T-RFLP analysis occurred with a similar pattern. Bacillus spp. which were not detected in the early microbial studies of kimchi were constantly detected until the late fermentation stage of kimchi in our T-RFLP analysis and their existence was proved by culture-based identification. Additionally, sporulation of Bacillus spp. during kimchi fermentation was discovered.

Saccharomyces cerevisiae의 감수분열 특이적 Protein Kinase인 Ime2의 역할

임선희,탁연수,선우양일,Leem, Sun-Hee,Tak, Yon-Soo,Sunwoo, Yang-Il 한국미생물학회 1999 미생물학회지 Vol.35 No.4

출아효모에서는 질소원의 고갈과 MATa/MAT${\alpha}$ 이배체 세포의 감수분열기 특이적인 유전자 발현에 의해 체세포분열기의 G1기에서 감수분열기로의 진행이 결정된다. 이러한 두 경로는 감수분열기 특이적인 IME 유전자군에 의한 전사조절에 의해 활성화되어 감수분열기가 시작된다. 본 연구는 IME2 유전자가 protein kinase 로서 감수분열기의 어떤 과정에 직접 관여하는가를 조사하기 위하여 먼저 PCR mutagenesis를 통하여 온도감수성 ime2 변이주를 분리하였다. 전체 62개의 온도감수성 변이주 중에서 온도에 따른 포자형성능과 감수분열기 재조합 빈도에 대하여 명확한 차이를 나타내는 3종류의 변이주들(ts ${\cdot}$ ime2-11, ts ${\cdot}$ ime2-12와 ts ${\cdot}$ ime2-13)을 선택하였다. 이러한 3종류의 온도감수성 변이주를 이용하여 제한온도에서 감수분열기 초기과정 중 결손을 조사하기 위해, FACScan analysis를 한 결과 IME2유전자가 감수분열기의 DNA 복제과정의 개시 및 완료에 관여함을 알 수 있었고, his4 혹은 arg4 locus에서 감수분열기 재조함 빈도의 측정으로 재조합 과정에 중요한 역할을 한다는 것을 알 수 있었다. 더욱이${\Delta}$mre4 파괴주에 IME2유전자를 과다발현시켜 그 영향을 조사한 결과, 감수분열기 특이적인 protein kinase 인 IME2와 MRE4가 감수분열기 초기과정인 재조합 과정에서는 동일한 경로에 작용한다는 것이 제시되었다. Entry into meiosis in the yeast Saccharomyces cerevisiae is regulated by two major factors: the cell type MATa/MAT${\alpha}$ and the nutriational state (starvation) of the cell. The two independent regulations act through IME1and IME2 expression to initiate meiosis. IME2 encodes a meiosis-specific protein kinase, and it enabled MATa/MAT${\alpha}$ diploid cells to undergo meiosis and sporulation. The PCR mutagenesis method was applied for the isolation of thermosensitive ime2 mutants. Among sixty two mutants isolated from the phenotype of defective spore formation under the restrictive temperature, three with the most easily observed temperature-sensitive phenotype (ts ${\cdot}$ime2-11, ts ${\cdot}$ime2-12 and ts ${\cdot}$ime2-13) were selected for further study. To understand the detailed functions of IME2, we examined the defects of these mutants in the early meiotic pathway including the premeiotic DNA replication and exhibited decreased level in meiotic recombination. These results suggest that the IME2 gene plays essential role in meiotic recombination pathway as well as premeiotic DNA replication. As the result of the IME2 overexpression in ${\Delta}$mre4. moreover, it was suggested that the IME2 and MRE4 genes act on the same pathway of initiation step in meiotic recombination.

오이 절편과 곡류 배지를 이용한 Botryosphaeria dothidea 분생포자의 생성과 분출

박은우 한국식물병리학회 1998 Plant Pathology Journal Vol.14 No.1

Proteomic Reference Map and Comparative Analysis between Streptomyces griseus S4-7 and wbiE2 Transcription Factor-Mutant Strain

Jisu Kim,Young Sang Kwon,Dong-Won Bae,Youn-Sig Kwak 한국식물병리학회 2020 Plant Pathology Journal Vol.36 No.2

Streptomyces griseus S4-7, a well-characterized key- stone taxon among strawberry microbial communi- ties, shows exceptional disease-preventing ability. The whole-genome sequence, functional genes, and bioactive secondary metabolites of the strain have been described in previous studies. However, proteomics studies of not only the S4-7 strain, but also the Streptomyces genus as a whole, remain limited to date. Therefore, in the pres- ent study, we created a proteomics reference map for S. griseus S4-7. Additionally, analysis of differentially expressed proteins was performed against a wblE2 mu- tant, which was deficient in spore chain development and did not express an antifungal activity-regulatory transcription factor. We believe that our data provide a foundation for further in-depth studies of functional keystone taxa of the phytobiome and elucidation of the mechanisms underlying plant-microbe interactions, es- pecially those involving the Streptomyces genus.