Bacillus subtilis와 Bacillus megaterium에서의 $\beta$-1,3-glucanase 유전자의 발현

김기훈,김지연,김한복,이동석 한국미생물학회 2001 미생물학회지 Vol.37 No.4

A Bacillus circulans KCTC3004 $\beta$-1,3-glucanase gene contained in a recombinant plasmid pLM460 derived from subcloning the original recombinant plasmid pLM530 was trasferred into a new shuttle vector plasmid pLMS1180 by ligating linearized DNAs of pLM460 and pUB110. B. subtilis RM125 and B. megaterium ATCC14945 transformed with pLMS1180 produced the $\beta$-1,3-glucanase substantially. Most of the enzyme was produced during the exponential growth period. The maxium activities of the $\beta$-1,3-glucanase produced by the Bacillus transformants were compared with that of the B. circulans gene donor strain. The B. subtilis RM125 (pLM1180) enzyme showed the activity 14 times higher than that of the gene donor cells, followed by the B. megaterium ATCC14945 (pLMS 1180) enzyme with activity 5 times higher than that of the gene donor cells. While E. coli secreted about 7% of the produced enzyme, B. subtilis excreted the enzyme into the medium wholly and B. megaterium about 97% of the total product. The SDS-PAGE of this enzyme produced in E. coli (pLMS1180), B subtilis (pLMS1180) or B. megaterium (pLMS1180) indicated a molecular weight of 38,000. The enzymes overproduced in three different host cells hydrolyzed laminarin to produce mainly laminaribiose, laminaritriose, and laminarioligosaccharides. The plasmid pLMS1180 was stable in B. megaterium, E. coli, but was unstable in B. subtilis. Bacillus circulans KCT3004 기원의 $\beta$-1,3-glucanase 유전자를 함유한 재조합 플라스미드 pLM460과 pUB110을 이용하여 shuttle 플라스미드 pMLS1180을 제작하고 Bacillus 세포에 이동.발현시켰다. pLMS1180으로 형질전환된 B. subtilis와 B. megaterium은 효율적으로 $\beta$-1,3-glucanase를 생산하였고, 이 효소들은 세포의 증식과 비례하여 생산되었다. 형질전환체가 생산하는 $\beta$-1,3-glucanase의 최대 활성을 유전자 공여 균주인 B. circulans와 비교하여 보니, B. subtilis는 14배, B. megaterium은 5배 정도의 높은 활성을 나타내었다. 그리고 대장균 형질전환체는 분비율이 7% 정도인데 반하여 B. subtilis 형질전환체는 생산된 효소를 전부, B. megaterium 형질전환체는 약 97%를 세포 외로 분비하는 것을 알 수 있었다. SDS-PAGE를 통해 대장균과 B. subtilis, B. megaterium에서 발현된 효소의 분자량을 분석해 보니 약 38,000으로 추정되었다. 또한, 이들 형질전환체가 생산하는 $\beta$-1,3-glucanase는 laminarin에 작용하여 주된 산물로서 laminaribiose (G2), laminaritriose (G3) 이상의 다양한 laminarioligosaccharide들을 생산함이 확인되었다. pLMS1180의 각 숙주 내에서이 안정성을 살펴본 결과 B.megaterium에서는 88%, 대장균에서는 75%, B. subtilis에서는 48%로 나타났다.

High prevalence of <i>Bacillus subtilis</i>-infecting bacteriophages in soybean-based fermented foods and its detrimental effects on the process and quality of <i>Cheonggukjang</i>

Ghosh, Kuntal,Kang, Hai Seong,Hyun, Woo Bin,Kim, Kwang-Pyo Elsevier 2018 FOOD MICROBIOLOGY Vol.76 No.-

<P><B>Abstract</B></P> <P>While the detrimental effect of bacteriophages on lactic acid bacterial fermentation is well documented, the importance of <I>Bacillus subtilis</I> phages in soybean-based fermented foods is not. In this study, we show for the first time that 100% of Korean soybean-based fermented foods (<I>Doenjang, Gochujang, and Cheonggukjang</I>) and 70% of raw materials (<I>Meju</I> and rice straw) were contaminated with <I>B. subtilis</I>-infecting phages (as high as 3.7 × 10<SUP>4</SUP> PFU g<SUP>−1</SUP>). Among 15 isolated <I>B. subtilis</I>-infecting phages, BSP18 was selected for further studies due to its specificity to and relatively broad host infectivity (34%) against <I>B. subtilis</I>. This <I>Myoviridae</I> family phage, BSP18 could infect all of the tested wild-type and commercially-used strains for soybean-based fermented food preparation. Furthermore, artificial contamination of as low as 10<SUP>2</SUP> PFU g<SUP>−1</SUP> of BSP18 significantly inhibited <I>B. subtilis</I> growth during <I>Cheonggukjang</I> fermentation. Moreover, phage-treated samples contained considerably more degraded γ-PGA which could negatively affect the functional property of <I>Cheonggukjang</I>. We also present the data, strongly suggesting BSP18-encoded, not bacterial, γ-PGA hydrolase was responsible for γ-PGA degradation. In conclusion, <I>B. subtilis</I> phages are widespread in Korean soybean-based fermented foods and it should be of great concern as phages may hamper the bacterial growth during fermentation and yield poor quality products.</P> <P><B>Highlights</B></P> <P> <UL> <LI> High prevalence of <I>B. subtilis</I> phages in Korean soybean-based fermented foods. </LI> <LI> Inhibition of <I>B. subtilis</I> growth in BSP18-applied <I>Cheonggukjang</I> samples. </LI> <LI> Fragmented γ-PGA was detected in BSP18-applied <I>Cheonggukjang</I> samples. </LI> <LI> <I>B. subtilis</I> phage contamination decreases the quality of soybean fermented foods. </LI> </UL> </P>

Edwardsiella tarda의 특이 Bacteriophage와 Bacillus subtilis KM-1혼합액이 Edwardsiella tarda 에 미치는 항균효과

백민석 ( Min Suk Baek ),황요셉 ( Yo Sep Hwang ),최상훈 ( Sang Hoon Choi ) 한국어병학회 2013 한국어병학회지 Vol.26 No.3

본 연구는 E. tarda에 대한 특이박테리오phage와 생균제의 일종인 B. subtilis KM-1의 혼합제가 E. tarda에 대한 항균활성에 미치는 효과를 알아보고자 수행되었다. 가장 효과적인 혼합제의 항균활성을 보인 phage의 적정 수는 B. subtilis 가 2×105 CFU/㎖의 농도로 존재할 때 배양 36시간째 2×105 PFU/㎖로 나타났다. B. subtilis의 경우 2×105 PFU/㎖의 phage가 존재할때 첨가량에 비례하는 항균활성을 보여주었다. phage와 B. subtilis의 혼합투여는 phage나 B. subtilis 각각을 투여한 그룹보다 훨씬 높은 항균활성을 나타냈다. 본 연구의 결과는 수산양식에서 대두되고 있는 양식어류의 E. tarda 감염성 질병 치료를 위한 항생제 대체 제로서의 사료첨가제 개발 가능성을 제시하고 있다. The present study was performed to investigate an antibacterial activity of specific bacteriophage (phage) and Bacillus subtilis KM-1 (B. subtilis) mixture against Edwardsiella tarda (E. tarda). An appropriate number of phage showing the most effective antibacterial activity was 2×105 PFU/㎖ with 1×10 CFU/㎖ of B. subtilis 36 h post incubation. On the other hand, B. subtilis showed a dose dependant manner in inducing antibacterial activity in the presence of phage (2×105 PFU/㎖). The phage and B. subtilis mixture showed higher antibacterial activity against E. tarda than phage or B. subtilis only. These results suggest that the phage and B. subtilis mixture could be utilized as an alternative to antibiotics in the control of fish diseases caused by E. tarda.

Bacillus subtilis와 Listeria monocytogenes의 일반 스트레스반응의 비교

신지현,Shin, Ji-Hyun 한국미생물학회 2009 미생물학회지 Vol.45 No.1

A diverse range of stresses such as heat, cold, salt, ethanol, oxygen starvation or nutrient starvation induces same stress-responsive proteins. This general stress response enhances bacterial survival significantly. In Bacillus subtilis and closely related Gram-positive bacteria Listeria monocytogenes, the general stress response is controlled by the alternative transcription factor ${\sigma}^B$. The activity of ${\sigma}^B$ is regulated post-translationally by a signal transduction network that has been extensively studied in B. subtilis, and serve as a model for L. monocytogenes. The proposed model of L. monocytogenes signal transduction network is similar to that of B. subtilis, but the energy stress pathway is missing. More than 150 general stress proteins belong to ${\sigma}^B$ regulon of B. subtilis and L. monocytogenes. In both bacteria, ${\sigma}^B$ function is primarily important for resistance to diverse stresses. In addition, ${\sigma}^B$ function contributes to the control of important virulence genes in food-borne pathogen L. monocytogenes. Therefore, understanding of the general stress response is important not only for bacterial physiology, but also for pathogenicity. 일부 그람양성세균들은 고온, 저온, 염, 에탄올, 산소와 영양분 고갈과 같은 다양한 스트레스 상태에 노출되면, 일반 스트레스반응(general stress response)에 의해서 일련의 스트레스 단백질군을 발현시켜 외부 스트레스를 극복하고 세균의 생존력을 증가시킨다. 비병원성균인 Bacillus subtilis의 일반 스트레스반응에 관해서는 많은 연구가 이루어져 있으므로 다른 균의 연구모델로 이용이 가능하다. 본 총설에서는 B. subtilis와 병원성균인 Listeria monocytogenes의 일반 스트레스반응의 유사성과 차이점을 B. subtilis를 모델로 하여 비교하였다. 두 균의 일반 스트레스반응은 대체 전사 인자인 ${\sigma}^B$ (alternative transcription factor sigma B)에 의해서 조절되고 신호전달 네트워크 또한 매우 유사하며, ${\sigma}^B$ 의존성 유전자들에 의해 150여 개의 스트레스 단백질들이 발현된다. 그러나 L. monocytogenes는 B. subtilis의 에너지 스트레스 신호 경로를 가지고 있지 않은 점과, 일반 스트레스반응에 의해 병독 유전자들(virulence genes)이 조절되는 것이 가장 큰 차이점이다. 그러므로 L. monocytogenes의 생리 및 병원성 규명을 위해서는 일반 스트레스반응에 관한 이해가 매우 중요하다.

Bacillus circulans $\alpha$-amylase 유전자의 Basillus subtilis와 Bacillus megaterium에서의 클로닝 및 발현

이동석,김지연,김한복 한국미생물학회 2000 미생물학회지 Vol.36 No.3

재조합 플라스미드 pAL850에 함유된 Bacillus circulans KCTC3004 $\alpha$-amylase 유 전자를 pUB110을 이용하여 shuttle 플라스미드 pALS111을 만들어 Bacillus 세포에 이동. 발현시켰다. Bacillus subtilis(고초균)와 Bacillus megaterium(거대균)으로 형질전환된 pALS111로부터 $\alpha$-amylase는 세포증식과 비례하여 생산되었다. 형질전화주가 생산하는 $\alpha$ -amylase의 최대활성을 유전자 공여 균주인 B. circulans와 비교했을 때 고초균은 약 95배, 거대균은 약 34배 정도의 높은 활성을 나타내었다. 그리고 대장균 형질전환주는 분비율이 10% 정도인데 반하여 고초균 형질전화주는 생산된 효소전부를 , 거대균 형질전환주는 약 98%를 세포외로 분비함을 보임으로써 고초균과 거대균은 실용적인 면에서 대장균보다 우월 함을 나타내었다, pALS111의 각 숙주 내에서의 안정성을 살펴본 결과 거대균에서는 92%, 고초균에서는 76%, 대장균에서는 38% 로 나타났다. SDS-PAGE와 zymogram을 통해 추정 된 대장균과 고초균, 거대균에서 발현된 효소의 분자량은 약 55,000으로 확인되었다. 이들 형질전환주가 생산하는 $\alpha$-amylase는 starch 에 작용하여 주된산물로서 maltotriose 이상의 다양한 maltooligosaccharide들을 생산함이 확인 되었다. A Baczllus circdans KCTC3004 $\alpha$-amylase gene contained in a recombinant plasmid pAL850 was transferred into a new shuttle vector plasmid pALSIlI by ligating linearlzed DNAs of pUC19 and pUB110. B. subtilis RM125 and B. megatenurn ATCC14945 transfonned with pALS111 produced the $\alpha$-amylase substantially Most of the enzyme was produced during the exponential growth period. The maxiinurn activities of the $\alpha$-amylase produced by the Bucillus transformants were compared with that of the B. circulans gene donor strain. The B. subtilis RM125(pALS111) enzyme showed the actlvicy 95 times higher than that of the gene donor cells, followed by the B, nzegaterium ATCC14945(pALSlll) enzyme with activity 34 limes higher than that of the gene donor cells. While E coli secreted about 10% of the produced enzyme, B. subtilis excreted the enzyme inlo the medium wholly and B. megaterirun about 98% ofthe total product. The plasmid pALSI11 was quite stable inB. nzegaterium (92%), inoderately stable in B. subtilis (76%), but was unstable in E. coli (38%). The SDS-PAGE and zymogram of this enzyme produced in E. coli(pALS111), B. subtilis( pALS111) or B. megateril~m (pALS111) indicated a molecular weight of 55,000. The enzymes overproduced in three different host cells hydrolyzed starch to produce mainly maltoaiose and mallooligosaccharides.

유용식용 균주에 의한 발효 누에분말의 이화학적 특성과 생리활성

Jae-Young Cha(차재영),Yong-Soon Kim(김용순),Hee-Young Ahn(안희영),Min-Jung Kang(김민정),Su-Jin Heo(허수진),Young-Su Cho(조영수) 한국생명과학회 2011 생명과학회지 Vol.21 No.1

누에 분말을 이용하여 B. subtilis 및 A. kawachii 균주에 의한 고체 발효를 통해 얻어진 발효 누에분말의 발효시간에 따른 이화학적 특성(단백질 함량 및 단백질 분해 패턴) 및 생리활성 효과(항산화 작용, 환원력 및 혈전용해 활성)를 평가하였다. 누에 단백질 함량과 항산화 활성은 B. subtilis 및 A. kawachii 두 균주 발효 모두 12일째에 가장 높았으며, 발효시간 증가와 함께 증가하는 경향을 보였다. 발효 누에분말의 환원력은 B. subtilis 균주 발효에 의해서는 6일째에 A. kawachii 균주 발효에 의해서는 12일째 가장 높은 것으로 나타나 균주간에 차이가 있었다. 혈전 분해 활성은 B. subtilis 및 A. kawachii 두 균주 발효 모두 6일째에 가장 높았으며, 이러한 효과는 B. subtilis 발효 누에분말보다는 A. kawachii 발효 누에분말에서 더 높았다. SDS-PAGE상의 단백질 패턴 분석에서는 66-97 kDa 크기의 단백질 밴드가 B. subtilis 발효에 의해서는 발효 초기인 3일째 대부분 분해가 일어났으나, A. kawachii 발효 누에분말에서는 발효 3일째부터 차츰 분해되기 시작하여 12일째 분해가 대부분 이루어졌다. 따라서 미생물을 이용한 발효 누에에서 특정의 생리활성 작용을 기대하기 위해서는 사용하는 균종에 따라 발효 기간이 달라져야 한다는 것을 시사하였다. Biological activities (α,α'-diphenyl-β-picrylhydrazyl (DPPH) free radical scavenging activity, fibrinolytic activity and reducing power) and biochemical properties (protein content and electrophoretical protein patterns) were examined in solid state fermentation with Bacillus subtilis and Aspergillus kawachii using silkworm powder (SP) as substrate. The highest protein contents and free radical scavenging activities were seen in the SP fermented for 12 days with B. subtilis and A. kawachii, and these were in a time-dependent manner. The highest reducing power was seen in the SP fermented for 6 days with B. subtilis and for 12 days with A. kawachii, respectively. The highest fibrinolytic activities were seen in silkworm fermented for 6 days with B. subtilis and A. kawachii, but this activity was higher in the A. kawachii fermented SP than that of B. subtilis. When total protein patterns were analyzed by SDS-polyacrylamide gel electrophoresis (PAGE), the proteins of the SP fermented with B. subtilis for 3 days were completely degraded, while the protein degradation in the SP fermented with A. kawachii occurred after 12 days and this degradation increased proportionally to culture time. As a result, the SP fermented with both B. subtilis and A. kawachii showed higher fibrinolytic activities after 6 days of fermentation and antioxidative activity after 12 days, indicating that physiological activities of the fermented SP using these strains were highly improved compared to the unfermented SP, and that this compound could be a candidate material as a dietary supplement of healthy functional foods.

Antimicrobial Gageomacrolactins Characterized from the Fermentation of the Marine-Derived Bacterium <i>Bacillus subtilis</i> under Optimum Growth Conditions

Tareq, Fakir Shahidullah,Kim, Ji Hye,Lee, Min Ah,Lee, Hyi-Seung,Lee, Jong-Seok,Lee, Yeon-Ju,Shin, Hee Jae American Chemical Society 2013 Journal of agricultural and food chemistry Vol.61 No.14

<P>Marine bacteria are a potential source of structurally diversified bioactive secondary metabolites that are not found in terrestrial sources. In our continuous effort to search for new antimicrobial agents from marine-derived bacteria, we isolated bacterial strain 109GGC020 from a marine sediment sample collected from Gageocho, Republic of Korea. The strain was identified as <I>Bacillus subtilis</I> based on a 16s rRNA sequence analysis. After a 7-day fermentation of the <I>B. subtilis</I> strain under optimum growth conditions three new and four known secondary metabolites were discovered using chromatographic procedures, and their biological activities were evaluated against both bacteria and crop-devastating fungi. The discovered metabolites were confirmed by extensive 2D NMR and high-resolution ESI-MS data analyses to have the structures of new macrolactin derivatives gageomacrolactins <B>1</B>–<B>3</B> and known macrolactins A (<B>4</B>), B (<B>5</B>), F (<B>6</B>), and W (<B>7</B>). The stereoconfigurations of <B>1</B>–<B>3</B> were assigned based on coupling constant values, chemical derivatization studies, and a literature review. The coupling constants were very crucial to determine the relative geometries of olefins in <B>1</B>–<B>3</B> because of overlap of the <SUP>1</SUP>H NMR signals. The NMR data of these compounds were recorded in different solvents to overcome this problem and obtain accurate coupling constant values. The new macrolactin derivatives <B>1</B>–<B>3</B> displayed good antibiotic properties against both Gram-positive (<I>S. aureus</I>,<I> B. subtilis</I>, and <I>B. cereus</I>) and Gram-negative (<I>E. coli</I>,<I> S. typhi</I>, and <I>P. aeruginosa</I>) bacteria with minimum inhibitory concentration (MIC) values of 0.02–0.05 μM. Additionally, the antifungal activities of <B>1</B>–<B>7</B> were evaluated against pathogenic fungi and found to inhibit mycelial growth of <I>A. niger</I>,<I> B. cinerea</I>,<I> C. acutatum</I>,<I> C. albicans</I>, and <I>R. solani</I> with MIC values of 0.04–0.3 μM, demonstrating that these compounds were good fungicides.</P><P><B>Graphic Abstract</B> <IMG SRC='http://pubs.acs.org/appl/literatum/publisher/achs/journals/content/jafcau/2013/jafcau.2013.61.issue-14/jf4009229/production/images/medium/jf-2013-009229_0008.gif'></P><P><A href='http://pubs.acs.org/doi/suppl/10.1021/jf4009229'>ACS Electronic Supporting Info</A></P>

신균주 Bacillus amyloliquefaciens NBF11-1을 이용한 청국장의 항균활성과 열 및 pH 안정성

김한수(Han Soo Kim) 한국산학기술학회 2016 한국산학기술학회논문지 Vol.17 No.7

본 연구의 목적은 대나무 줄기 마디 부분의 표면에서 처음 발견 되었으나, 아직까지는 연구가 미흡한 신 균주 Bacillus amyloliquefaciens NBF11-1로 발효한 청국장 추출물의 항균 활성을 알아보려고 한다. 청국장 전통 발효 균주인 Bacillus subtilis NG24를 대조 군으로 하고, 비교 분석을 실시하였다. 실험 방법은 항균 활성, 최소 억제 농도(MIC)와 열 및 pH 안정성 을 측정하였다. 항균 활성 실험 결과, 병원성 6종 균주에서 그람 양성 균주 C.perfringens, S.aureus와 그람 음성 균주 A.faecalis, E.coli에서 B.amyloliquefaciens NBF11-1이 B.subtilis NG24에 비해 강한 항균 활성을 나타내었다. 최소 억제 농도 실험 결과, C.perfringens, S.aureus, A.faecalis, E.coli에서 B.amyloliquefaciens NBF11-1이 B.subtilis NG24에 비해 0.01 %, 0.21 %, 0.45 %, 0.29 %의 농도로 억제 효과를 나타내었다. 열과 pH 안정성 실험 결과, B.subtilis NG24와 더불어 B.amyloliquefaciens NBF11-1 청국장 추출물은 121℃, 15분 간 열처리와 pH 2-10 처리에 의한 안정성에서 활성이 감소하지 않았고, 열과 pH 실험에서 비교적 안정하였다. This study aims to determine the antimicrobial activity of fermented Cheonggukjang extract using the new Strain, Bacillus amyloliquefaciens NBF11-1, which was first found on the surface of the node part of bamboo stems, but has been studied very little so far. Bacillus subtilis NG24, which is the traditional fermented strain of Cheonggukjang, was selected as the control group and a comparative analysis was performed. The experimental method included measurements of the antimicrobial activity, minimum inhibitory concentration (MIC) and heat and pH stability. B. amyloliquefaciens NBF11-1 had stronger antimicrobial activity than B. subtilis NG24 against the gram-positive bacteria, C. perfringens and S. aureus and the gram-negative bacteria, A. faecalis and E. coli among the six species of pathogenic bacteria studied. When the minimum inhibitory concentration was measured, B. amyloliquefaciens NBF11-1 in C. perfringens, S. aureus, A. faecalis, and E. coli had an inhibitory effect at concentrations of 0.01 %, 0.21 %, 0.45 % and 0.29 %, respectively, compared to B. subtilis NG24. When the heat and pH stability was measured, B. subtilis NG24 and B. amyloliquefaciens NBF11-1 Cheonggukjang extract did not show any decrease in activity when held at a temperature of 121℃ for 15 minutes and at pH values ranging from 2 to 10 and were therefore considered to be relatively stable against heat and pH changes

신 균주 Bacillus amyloliquefaciens NBF11-1을 이용하여 발효한 청국장의 항산화효과

김한수(Kim, Han Soo),윤현(Yun, Hyun) 한국산학기술학회 2015 한국산학기술학회논문지 Vol.16 No.8

본 연구의 목적은 대나무 줄기표면에서 처음 발견 하였으나, 아직까지는 연구가 미흡한 신 균주 Bacillus amyloliquefaciens NBF11-1의 생리활성 효과를 알아보기 위하여 청국장 전통 발효 균주인 Bacillus subtilis NG24를 대조군으로 하여 항산화효과에 대한 비교분석을 실시하였다. 청국장 추출물의 항산화 실험 결과에서 Total Polyphenol 추출물 함량의 측정 결과는 B.subtilis NG24에 비해 B.amyloliquefaciens NBF11-1 시료에서 유의하게 증가하였다(p=0.032). SOD 유사활성과 DPPH radical 소거능 및 NO radical 소거능에서 B.subtilis NG24에 비해 B.amyloliquefaciens NBF11-1을 포함한 시료에서 농도가 증가함에 따라 유의하게 증가하였다(p<.05). 추가적으로, 각 항산화 실험의 IC50은 B.subtilis NG24에 비해 B.amyloliquefaciens NBF11-1 시료에서 SOD 유사활성(p=0.045), DPPH radical 소거능(p=0.041), NO radical 소거능(p=0.019)과 같이 유의한 차이로 감소하였다. This study aims to compare and analyze the antioxidant effect of Cheonggukjang's traditional fermentation strain, Bacillus subtilis NG24 which was a control of the study, in order to see the biological activity effect of the new strain, Bacillus amyloliquefaciens NBF11-1 that was first found in the surface of the bamboo stem, but hasn't been insufficiently researched. In the antioxidant activity experiment of the Cheonggukjang extract, the B.amyloliquefaciens NBF11-1 sample showed a significant increase in the total polyphenol extract content, compared to B.subtilis NG24(p=0.032). Also, compared to B.subtilis NG24, the sample containing B.amyloliquefaciens NBF11-1 showed a significant increase in SOD-like Activity, DPPH radical scavenging, and NO radical scavenging, as the concentration rose(p<.05). Additionally, IC50 in each antioxidant activity experiment significantly decreased in the B.amyloliquefaciens NBF11-1 sample like SOD-like Activity(p=0.045), DPPH radical scavenging(p=0.041), and NO radical scavenging(p=0.019), compared to B.subtilis NG24.

Bacillus circulans β-1, 3-1, 4-glucanase 유전자의 고초균과 거대균에서의 클로닝 및 발현

김지연,이동석 인제대학교 1998 仁濟論叢 Vol.14 No.2

재조합 플라스미드 pLL200K에 함유된 Bacillus circluans ATCC21367 β-1,3-1,4-glucanase 유전자를 Pub110을 이용하여 shuttle 플라스미드 pLLS920을 만들면서 Bacillus 세포로 이동·발현시켰다. 고초균과 거대균으로 형질전환된 pLLS920은 β-1,3-1,4-glucanase를 실질적으로 생산하였는데 효소들은 세포증식과 비례하여 생산되었다. 형질전환주가 생산하는 β-1,3-1,4-glucanase의 최대활성을 유전자 공여균주인 B. circulans와 비교하여 보니, 고초균은 약 83배, 거대균은 약 7배 정도의 높은 활성을 나타내었다. 고초균이나 거대균 형질전환주가 생산하는 효소들은 대부분 성장배지로 분비되는 반면에 대장균 형질전환주의 경우에는 생산하는 전체효소의 10% 정도만이 분비되는 것을 알 수 있었다. 한편, activity staining을 통해 대장균과 고초균, 거대균에서 발현된 효소를 분석해 본 결과 분자량은 약 28,000으로 추정되었으며, 특히 고초균에서는 효소 단백질의 processing이 일어남을 관찰할 수 있었다. A Bacillus circulans ATCC21367 β-1,3-1,4-glucanase gene contained in a recombinant plasmid pLL200K was transferred into a new shuttle vector plasmid pLLS920 by ligating linearized DNAs of pLL200K and pUB110. B. subtilis RM125 and B. megaterium ATCC14945 transformed with pLLS920 produced the β-1,3-1,4-glucanase substantially. Most of the enzyme was produced during the exponential growth period. The maximum activities of the β-1,3-1,4-glucanase produced by the Bacillus transformants were compared with that of the B. circulans gene donor strain. The B. subtilis RM125(pLLS920) enzyme showed the activity 83 times higher than that of the gene donor cells, followed by the B. megaterium ATCC14945(pLLS920) enzyme with activity 7 times higher than that of the gene donor cells. Although all or most of the enzyme produced by the B. subtilis or B. megaterium was secreted into the culture broth, only about 10% of the total enzyme produced by the Escherichia coli transformant was detected in the culture supernatant. The activity staining of the enzyme produced in E. coli(pLLS920) or B. megaterium(pLLS920) indicated a molecular weight of about 28,000. The zymogram of the enzyme from B. subtilis(pLLS920), however, showed shortened band, suggesting proteolytic processing of the enzyme.