• Journal of Microbiology and Biotechnology
• /
• 제5권1호
• /
• pp.18-21
• /
• 1995

Endoglucanase gene of Pseudomonas sp. LBC505 was previously cloned in pUCl9 to yield plasmid pLC1. overproduction of endoglucanase was attempted by following ways. First, the endoglucanase gene of Pseudomonas sp. LBC505 cloned in pUCl9(pLC1) was tandemly inserted, step by step, into a expression vector pKK223-3 in a directly repeated form to enhance productivity of endoglucanase. Escherichia coli containing pKCC30 among the resulting plasm ids showed the higher yield of the endoglucanase. Ecoli harboring pKCC30 which had three inserted endoglucanase genes expressed about 12.3 times as much CMCase activity as Ecoli harboring pLCl. Second, the endoglucanase gene was subcloned into Bacillus subtilis expression vector pgnt41 for both overproduction and extracellular secretion of the endoglucanase. A resulting plasmid pgntc15 in Bacillus subtilis expressed 4.3-fold higher levels of CMCase activity than that of E.coli harboring pLCl and the endoglucanase produced was entirely secreted into the culture medium.

• 생명과학회지
• /
• 제16권2호
• /
• pp.274-281
• /
• 2006

흑두청국으로부터 분리된 혈전용해력이 우수한 Bacillus subtilis BB-1(KFCC 11344P)으로부터 혈전용해효소 유전자를 PCR법에 의해 크로닝하였고 이를 BCF-1으로 명명하였다. BCF-1의 DNA 염기서열결정 결과 1,145 bp 크기의 혈전용해 효소로, 일본의 natto로부터 분리된 nattokinase 유전자와 99%의 상동성을 보임을 확인하였다. 혈전용해효소 유전자의 발현을 위하여 Bacillus 발현계인 Bacillus-E. coli의 shuttle vector인 pEB vector에 크로닝 하고 host로서 B. subtilis 168에 형질전환시켜 대량 발현시켰다. 생산된 혈전용해효소의 최 적활성 pH와 온도는 7.0과 $35^{\circ}C$로 확인되었다, 기질에 대한 분해양상을 조사한 결과 fibrin에서만 특이적으로 강한 분해가 일어났으며, skim milk에서 아주 약한 분해능을 보였으나 blood agar, gelatin, casein에서는 전혀 분해능을 보이지 않았다. 특히 blood agar plate에서 분해능이 없는 것으로 보아 혈액 내에서의 적혈구 파괴현상과 같은 부작용에 대한 위험을 배제할 수 있을 것으로 사료된다. BCF-1에 의해 생산된 혈전용해효소는 fibrin 특이적으로 활성을 나타냄을 확인할 수 있으며, 이는 임상적이나 산업적으로 적용하였을 때 부작용에 대한 위험적인 문제는 배제될 수 있으리라 생각된다.

• 한국미생물·생명공학회지
• /
• 제40권3호
• /
• pp.186-189
• /
• 2012

A gene coding for mannanase (manA) from Bacillus subtilis was introduced into a shuttle vector pGK12 between Escherichia coli, B. subtilis and Lactobacillus paracasei. As a result of transferring the resultant plasmid, designated pGK12M3, into three different strains, the manA gene was found to be expressed in L. paracasei as well as in B. subtilis and E. coli. In a 4 L fermentor culture, the production of mannanase by recombinant L. paracasei (pGK12M3) reached a maximum level of 5.4 units/ml in an MRS medium with a fixed pH 6.5. Based on the zymogram of mannanase, it is assumed that mannanase produced by recombinant L. paracasei is not maintained stably with proteolytic degradation. The optimal temperature and thermostability of mannanase produced by recombinant L. paracasei were also found to be different from those of enzymes produced by B. subtilis.

• Journal of Microbiology and Biotechnology
• /
• 제17권6호
• /
• pp.1018-1023
• /
• 2007

Bacillus subtilis CH3-5 was isolated from cheonggukjang prepared according to traditional methods. CH3-5 secreted at least four different fibrinolytic proteases (63, 47, 29, and 20 kDa) into the culture medium. A fibrinolytic enzyme gene, aprE2, encoding a 29kDa enzyme was cloned from the genomic DNA of CH3-5, and the DNA sequence determined. aprE2 was overexpressed in heterologous B. subtilis strains deficient in extracellular proteases using a E. coli-Bacillus shuttle vector. A 29 kDa AprE2 band was observed and AprE2 seemed to exhibit higher activities towards fibrin rather than casein.

• 미생물학회지
• /
• 제42권4호
• /
• pp.313-318
• /
• 2006

가정에서 제조된 된장으로부터 cellulase 생산균으로 분리된 고온성 WL-12는 형태적 특성, 생화학적 성질 및 16S rRNA의 염기서열에 근거하여 Bacillus licheniformis로 동정되었다. B. licheniformis WL-12의 cellulase 유전자를 클로닝하여 그 염기서열을 결정한 결과 cellulase 유전자(celA)는 517 아미노산으로 구성된 단백질을 코드하며 1,551 뉴클레오티드로 이루어졌다. 아미노산 잔기배열을 분석한 결과 WL-12의 cellulase는 활성영역과 cellulose 결합영역으로 구성되어 있었으며, glycosyl hydrolase (GH) family 5에 속하는 B. licheniformis, B. subtilis와 B. amyloliquefaciens의 cellulase와 높은 상동성을 보였다. 클론된 celA를 발현용 vector에 도입하여 B. subtilis에서 발현시켜 cellulase 최대생산성이 7.0 units/ml에 이르렀다.

• Asian-Australasian Journal of Animal Sciences
• /
• 제30권4호
• /
• pp.576-584
• /
• 2017

Objective: To generate recombinant Bacillus subtilis (B. subtilis) engineered for expression of porcine ${\beta}-defensin-2$ (pBD-2) and cecropin P1 (CP1) fusion antimicrobial peptide and investigate their anti-bacterial activity in vitro and their growth-promoting and disease resisting activity in vivo. Methods: The pBD-2 and CP1 fused gene was synthesized using the main codons of B. subtilis and inserted into plasmid pMK4 vector to construct their expression vector. The fusion peptide-expressing B. subtilis was constructed by transformation with the vector. The expressed fusion peptide was detected with Western blot. The antimicrobial activity of the expressed fusion peptide and the recovered pBD-2 and CP1 by enterokinase digestion in vitro was analyzed by the bacterial growth-inhibitory activity assay. To analyze the engineered B. subtilis on growth promotion and disease resistance, the weaned piglets were fed with basic diet supplemented with the recombinant B. subtilis. Then the piglets were challenged by enteropathogenic Escherichia coli (E. coli). The weight gain and diarrhea incidence of piglets were measured after challenge. Results: The recombinant B. subtilis engineered for expression of pBD-2/CP1 fusion peptide was successfully constructed using the main codons of the B. subtilis. Both expressed pBD-2/CP1 fusion peptide and their individual peptides recovered from parental fusion peptide by enterokinase digestion possessed the antimicrobial activities to a variety of the bacteria, including gram-negative bacteria (E. coli, Salmonella typhimurium, and Haemophilus parasuis) and grampositive bacteria (Staphylococcus aureus). Supplementing the engineered B. subtilis to the pig feed could significantly promote the piglet growth and reduced diarrhea incidence of the piglets. Conclusion: The generated B. subtilis strain can efficiently express pBD-2/CP1 fusion antimicrobial peptide, the recovered pBD-2 and CP1 peptides possess potent antimicrobial activities to a variety of bacterial species in vitro. Supplementation of the engineered B. subtilis in pig feed obviously promote piglet growth and resistance to the colibacillosis.

• Food Science and Biotechnology
• /
• 제17권6호
• /
• pp.1372-1375
• /
• 2008

The aprE2 gene from Bacillus subtilis CH3-5 was expressed in Bacillus licheniformis ATCC 10716 using a Bacillus-Escherichai coli shuttle vector, pHY300PLK. The fibrinolytic activity of transformant (TF) increased significantly compared to B. licheniformis 10716 control cell. During the 100 hr incubation in Luria-Bertaini broth at $37^{\circ}C$, fibrinolytic activity of B. licheniformis TF increased rapidly at the late growth stage, after 52 hr of incubation, which was confirmed by zymography using a fibrin gel. pHY3-5 was stably maintained in B. licheniformis without tetracycline (Tc) in the media, 60.9% of cells still maintained pHY3-5 after 100 hr of cultivation.

• Journal of Microbiology and Biotechnology
• /
• 제19권2호
• /
• pp.194-203
• /
• 2009

$HpaG_{Xooc}$, from rice pathogenic bacterium Xanthomonas oryzae pv. oryzicola, is a member of the harpin group of proteins, eliciting hypersensitive cell death in non-host plants, inducing disease and insect resistance in plants, and enhancing plant growth. To express and secret the $HpaG_{Xooc}$ protein in Bacillus subtilis, we constructed a recombinant expression vector pM43HF with stronger promoter P43 and signal peptide element nprB. The SDS-PAGE and Western blot analysis demonstrated the expression of the protein $HpaG_{Xooc}$ in B. subtilis. The ELISA analysis determined the optimum condition for $HpaG_{Xooc}$ expression in B. subtilis WBHF. The biological function analysis indicated that the protein $HpaG_{Xooc}$ from B. subtilis WBHF elicits hypersensitive response(HR) and enhances the growth of tobacco. The results of RT-PCR analysis revealed that $HpaG_{Xooc}$ induces expression of the pathogenesis-related genes PR-1a and PR-1b in plant defense response.

• 미생물과산업
• /
• 제14권1호
• /
• pp.17-20
• /
• 1988

A genetic analysis of the complex Bacillus subtilis transcriptional apparatus is essential to understand the function, regulation, and interaction of the transcriptase components during growth and sporulation. This approach in Escherichia coli has uncovered fundamental mechanisms regulating gene expression Cole and Nomura, 1986; Lindahl and Zengel, 1986) and an analysis of the B. subtilis transcriptase will allow comoparison of the E.coli system to another bacterium that has evolved under different selective pressures. To this end we used antibody probes to isolate the alpha, beta, and beta' core subunit genes from a .lambda.gtill expression vector library. To address the question of function ans regulation of the minor sigma factors that confer promoter specifity on the polymerase core (Losick et al., 1986), we used the same approach to isolate the gene for the 37,000 dalton sigma factor, sigma-37.

• 한국미생물·생명공학회지
• /
• 제24권4호
• /
• pp.408-414
• /
• 1996

The SecY is a central component of the protein export machinery that mediate the translocation of secretory proteins across the plasma membrane, and has been known to be rate-limiting factor of secretion in Escherichia coli. In order to study the extracellular protein secretion in Gram-positive microorganism, we have, constructed strains harboring more than one copy of the gene for SecY. Firstly, the gene, for B. subtilis SecY and its promoter region was subcloned into pDH32 and the chimeric vector was inserted into amyE locus by homologous recombination. Secondly, low copy number vector, pCED6, was also used for subcloning the secY gene and for constructing a strain which harbors several copies of secY. The KH1 cell which harbor two copies of secY on the chromosome excreted more extracellular proteins than the wild type PB2. Moreover, the KH2 cells which harbor several copies of secY in pCED6 vector excreted more extracellular proteins than the KH1 cells. Here, we found that the capacity of protein secretion is partly controlled by the number of secY and it is suggested that SecY has also an important role in protein secretion in B. subtilis, a gram positive microorganism, as like in E. coli. This will promote the use of B. subtilis as a host for the expression of useful foreign gene and excretion of precious proteins.