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Bacillus sp. SH-8과 Bacillus sp. SH-8M의 Protease 생산 및 특성에 미치는 pH의 영향
심창환,정광선,신원철,유주현 한국산업미생물학회 1994 한국미생물·생명공학회지 Vol.22 No.1
Bacillus sp. SH-8과 Bacillus sp. SH-8M을 이용하여 생육 pH 변화에 따른 protease 생산과 효소학적 특성을 검토하였다. 알칼리성 배지(pH 10.2)에서는 Bacillus sp. SH-8과 Bacillus sp. SH-8M 균주 모두 protease를 생산하였으며 60시간, 96시간에서 각각 70 units/㎖과 50 units/㎖로 최고의 활성을 보였다. 중성 배지(pH 6.9, 7.7)에서 Bacillus sp. SH-8은 protease 활성을 나타내지 않았으나 Bacillus sp. SH-8M은 pH 6.9와 7.7에서 각각 8일과 6일에 최고 활성을 나타내었다. 또한 배양도중 배지의 pH를 알칼리에서 중성으로 변화시킬 경우 Bacillus sp. SH-8M은 protease를 생산하였으나 Bacillus sp. SH-8은 생산하지 않았다. 알칼리 및 중성 배지에서 생산된 protease는 배양 pH를 변화시켜도 효소의 물리 화학적 특성이 비슷하였다. The production and the characteristics of protease produced by Bacillus sp. SH-8 and Bacillus sp. SH-8M were investigated under the different pH conditions. Bacillus sp. SH-8 and Bacillus sp. SH-8M showed the maximum activity of protease at 60 hours(70 units/㎖) and 96 hours(50 units/㎖) cultivation, respectively, under the alkaline condition(pH 10.2). However, Bacillus sp. SH-8M exhibited the maximum activities in 8 days cultivation at pH 6.9 and in 6 days cultivation at pH 7.7. Bacillus sp. SH-8M showed the protease activity at the pH change from alkaline to neutral condition, whereas Bacillus sp. SH-8 did not. In addition, all the enzymatic characteristics of protease produced by Bacillus sp. SH-8 and Bacillus sp. SH-8M were similar with the regardless of different pH conditions.
극한 환경하에서 생육하는 호알카리성 Bacillus sp. 의 생리적 특성
심창환,정용준 ( Chang Whan Shim,Yong Joon Chung ) 한국환경생물학회 1997 환경생물 : 환경생물학회지 Vol.15 No.1
To understand the differences in the physiological characteristics between neutrophile and alkalophile, an absolute alkalophilic Bacillus sp. SH-8 which was able to grow only at alkaline pH range up to pH 11.0 was isolated from the soil, and its mutant strain, SH-8M which was able to grow both at neutral and alkaline pH was obtained by UV mutagenesis. The changes of ΔpH and Na^+ ion concentration in the wild type and the mutant strain were then measured under various growth conditions. The ΔpH value of the two strains were calculated to be within the range of 70∼82㎷ in alkaline condition (pH 10.2). In neutral condition (pH 7.7) the ΔpH value of SH-8 strain was decreased to 23㎷ but that of SH-8M was maintained at the level of 62-83㎷. The circulation of Na^+ ions through cell membranes of the both strains at pH 10.2 and pH 7.7 was detected. When the two strains were cultured in the alkaline condition, high fluctuation in the external Na^+ ion concentration was observed to be 870∼900㎍/㎖, but when the mutant strain SH-8M was cultured in the neutral condition, the fluctuation was reduced and became to be increased to the same level in the alkaline condition by addition of NaCl to the medium. This may indicate that functional Na^+/H^+ antiporter and Na^+/solute symporter are present in the membrane of two strains.
심창환,정용준 한국식품영양학회 1999 韓國食品營養學會誌 Vol.12 No.3
새로운 Bacillus sp.의 숙주균주로서 가능성을 조사하기 위해 알칼리 내성 Bacillus sp. YA-14의 형질전화최적조건을 검토한 결과 다음의 결과를 얻었다. 형질전화배지로서 0.4% MgSO_4, 0.5mM CaCl_2가 함유된 modified SPI배지 (pH 8.0)를 사용하였으며, 말기 대수증식기의 균체가 최적이었다. 또한 competent cell 1ml에 plasmid DNA 0.5g을 첨가하여 40분간 진탕배양하는 것이 형질전환에 가장 좋았으며, 이러한 최적조건하에서 형질전환율은 4.53×10 exp (-6)CFU/ml/㎍ DNA이었다. 전기영동실험을 통하여 transformant 내에 분자량 3.0×10 exp (6) daltons의 pUB110 plasmid DNA가 도입되었음을 확인하였고, 20세대 계대배양 후에도 안정하였다. 이로부터 알카리내성 Bacillus sp. YA-14는 Bacillus속 미생물의 host-vector system에서 새로운 숙주균주로서 이용가능성을 보여 주었다. To develop the potential use as new host strain for gene cloning, the optimal conditions for transformation of alkali-tolerant Bacillus sp. were examined. The Bacillus sp. YA-14 was cultured to late logarithmic growth phase at 37℃ in modified SPI medium (pH8.0) containing 0.4% MgSO_4, 0.5mM CaCl_2, 1 ml of competent cell was mixed with 0.5㎍ of plasmid DNA and incubated with shaking at 37℃ for 40 min. The transformation frequency under the optimal condition was 4.53×10 exp (-6)CFU/ml/g plasmid DNA. The presence of 3.0×10 exp (6) daltons molecular weight pUB110 plasmid DNA in trasformants was confirmed by electrophresis and stably maintained in the new host.
Bacillus sp. SH-8과 Bacillus sp. SH-8M의 세포벽과 세포막에 미치는 pH의 영향
심창환,정용준,신원철 한국산업미생물학회 1995 한국미생물·생명공학회지 Vol.23 No.1
토양에서 분리한 호알칼리성 Bacillus sp. SH-8과 중성에서 생육이 가능한 변이주 Bacillus sp. SH-8M을 중성(pH 7.7) 및 알칼리성(pH 10.2) 배지에서 생육시키면서 각각의 세포벽과 세포막을 분석, 비교하였다. 그 결과 두 균주 모두 배지의 초기 pH에 관계없이 세포벽을 구성하는 아미노산이 alanine, glutamic acid, lysine, aspartic acid 및 meso-diaminopimelic acid로 성분이나 함량에 큰 차이가 없었으며, 산성아미노산인 glutamic acid와 aspartic acid가 약 50%정도 존재하였다. Crude cell membrane을 SDS polyacrylamide gel 1차원 전기영동을 행한 결과, 알칼리성 배지에서 생육시켰을 때 두 균주의 전기영동 pattern에 차이가 있음을 확인하였다. Crude cell membrane을 acetone으로 분획 후 2차원 전기영동을 행한 결과, 두 균주 모두 배지의 초기 pH에 따라 세포막의 전기영동 pattern에 큰 차이를 나타내어 세포막을 구성하는 단백질 성분은 배지의 pH에 영향을 받는 것을 알 수 있었다. 한편, 알칼리성 배지에서 생육시켰을 때 두 균주의 전기영동 pattern에는 차이점이 없었으나, 중성 배지에서 생육시키면 여러 부분에 차이가 있음을 확인하였다. Using the alkalophillic Bacillus sp. SH-8 and its mutant Bacillus sp. SH-8M capable of growing at the neutral pH, the amino acid compositions of the cell wall and cell membrane were studied at varying cultivation pH's. The pattern of protein electrophoresis was also tested. It was elucidated that the amino acids consisting of the cell wall were alanine, glutamic acid, lysine, aspartic acid, and meso-diaminopimelic acid. There was not any significant difference in the amino acid composition between two strains regardless of the culture pH. As the results of HPLC assay, glutamic acid and aspartic acid accounted for more than 50% in the amino acid composition of the cell wall. By the isolation of the crude cell membrane and the SDS-PAGE analysis, it was found that there was a considerable difference in the protein pattern when the strains were cultured at the neutral pH. In addition, by the two dimensional gel electrophoresis, it was confirmed that there was a difference in the protein pattern between two strains cultivated at the neutral pH medium, but no difference at the alkaline medium.
호알칼리성 미생물의 분리, 동정 및 중성에서 생육 가능한 변이주의 분리
심창환,신원철,유주현 한국산업미생물학회 1991 한국미생물·생명공학회지 Vol.19 No.6
토양으로부터 호알칼리성 미생물을 분리하여 동정한 결과, Bacillus속의 특성을 나타내어 Bacillus sp. SH-8로 명명하였다. Bacillus sp. SH-8은 초기 pH 9.0이상의 알칼리에서만 생육이 가능하였으며 중성 pH에서는 불가능하였다. Bacillus sp. SH-8로부터 U.V.조사에 의해 중성배지에서도 생육이 가능한 변이주를 분리하여 Bacillus sp. SH-8M으로 명명하였으며, 초기 pH 6.9부터 11.0까지 생육이 가능하였다. 따라서 이들 두 균주는 pH 변화에 따른 생육과 효소생산을 비교, 검토하는데 적합한 균주로 생각되었다. An alkalophilic microorganism, SH-8, was isolated from soil samples. It was a Grampositive, catalase-positive, spore-forming and motile rod which was capable of growth in aerobic condition at the initial pH 9.0 or above up to 11.0 and between 15 and 42℃. The characteristics of this strain resembled those of the Bacillus group of bacteria. The mutant, Bacillus sp. SH-8M, was selected from Bacillus sp. SH-8 by U.V. mutagenesis and was able to grow at pH 6.9 up to 11.0. These two strains will be suitable for the comparative study on growth and enzyme production at various pH conditions.