• Journal of Microbiology and Biotechnology
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• 제16권3호
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• pp.368-373
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• 2006

Bacillus subtilis and related Bacillus species are frequently used as hosts for the mass production of recombinant proteins. Accordingly, this study examined the cellular response of B. subtilis to the overexpression of a soluble secretory protein. As such, the lichenase derived from B. cereus was overexpressed in B. subtilis, initially localized in the cytoplasm as a mature form and then secreted into the medium. Thereafter, the proteome of B. subtilis was analyzed using 2D electrophoresis and MALDI-TOF mass spectrometry. The expression of several heat-shock proteins, such as dnaK and groEL, was increased under this condition. In addition, manganese superoxide dismutase and NADH dehydrogenase were also upregulated in the lichenase-secreting B. subtilis. Therefore, it was concluded that the transient accumulation of a secreted protein in B. subtilis before secretion acted as a stress on the cell, which in turn induced the expression of various protective proteins.

• Journal of Microbiology and Biotechnology
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• 제15권3호
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• pp.537-543
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• 2005

Subtilisin (EC 3.4.21.14) is the major extracellular alkaline serine protease of Bacillus species. Previously, we found that subtilisins did not migrate in the electrophoretic field in the Laemmili buffer system due to their high pI values (over 8.8); however, it formed a 'binding mode' at the top of the separating gel [5]. Utilizing this characteristic, four subtilisins from Bacillus sp. strains (e.g., B. subtilis 168, B. subtilis KCTC 1021, B. amyloliquefaciens KCTC 3002, and Bacillus sp. DJ-1 and DJ-4) were easily and quickly identified by an over-running electrophoretic technique with a miniscale culture supernatant (less than 20 ml) without any column chromatographic steps. Two subtilisins (DJ-l and a recombinant version) from Bacillus sp. DJ-l were characterized, and the enzymatic properties were determined by SDS-fibrin zymography and densitometric analysis. Based on this observation, the recombinant pro-subtilisin DJ-l showed the same 'binding mode,' similar to native subtilisin DJ-l. On the other hand, mature subtilisin DJ -1 without pro-peptide showed no enzymatic activity.

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

• 한국미생물·생명공학회지
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• 제21권2호
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• pp.113-118
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• 1993

Fro the purpose of producing glouse from cellobiose or oligo saccharide and obtaining genetic information of beta-1,4-glucosidase gene, alpha beta-1,4-glucosidase gene of Pseudomonas sp. LBC505, potent cellulase complex and xylanase producing strain, was cloned in Esherichia coli and Bacillus subtilis into pUC19 and pBD64, respectively. Recombinant plasmid pGL1 contained 1.2kb EcoRI fragment was isolated from transformants forming blue color around colony on LB agar plate containing 20 ng/ml of 5-bromo-4-chloro-3-indolyl-beta-D-glucopyranoside(X-glu) and ampicillin.

• Journal of Microbiology and Biotechnology
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• 제14권6호
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• pp.1295-1302
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• 2004

A gene encoding the mannanase of Bacillus subtilis WL-7, which had been isolated from Korean soybean paste, was cloned into Escherichia coli, and the gene product was purified from the culture filtrate of the recombinant E. coli. This mannanase gene, designated manA, consisted of 1,086 nucleotides, encoding a polypeptide of 362 amino acid residues. The deduced amino acid sequence was highly homologous to those of mannanases belonging to the glycosyl hydrolase family 26. The molecular mass of the purified mannanase was 38 kDa as estimated by SDS-PAGE. The enzyme had a pH optimum at 6.0 and a temperature optimum at $55^{\circ}C$. The enzyme was active on locust bean gum, konjak, guar gum, and lichenan, while it did not exhibit activity towards yeast mannan, laminarin, carboxymethylcellulose, $\beta$­glucan, xylan, and para-nitrophenyl-$\beta$-mannopyranoside.

• Journal of Microbiology and Biotechnology
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• 제19권12호
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• pp.1514-1519
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• 2009

A gene encoding the xylanase of Bacillus subtilis AMX-4 isolated from soil was cloned into Escherichia coli and the gene product was purified from the cell-free extract of the recombinant strain. The gene, designated xylA, consisted of 639 nucleotides encoding a polypeptide of 213 residues. The deduced amino acid sequence was highly homologous to those of xylanases belonging to glycosyl hydrolase family 11. The molecular mass of the purified xylanase was 23 kDa as estimated by SDS-PAGE. The enzyme had a pH optimum of 6.0-7.0 and a temperature optimum of $50-55^{\circ}C$. Xylanase activity was significantly inhibited by 5 mM $Cu^{2+}$ and 5 mM $Mn^{2+}$, and noticeably enhanced by 5 mM $Fe^{2+}$. The enzyme was active on xylans including arabinoxylan, birchwood xylan, and oat spelt xylan, but it did not exhibit activity toward carboxymethylcellulose or p-nitrophenyl-$\beta$-xylopyranoside. The predominant products resulting from xylan and xylooligosaccharide hydrolysis were xylobiose and xylotriose. The enzyme could hydrolyze xylooligosaccharides larger than xylotriose.

• Journal of Microbiology and Biotechnology
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• 제30권7호
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• pp.1082-1091
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• 2020

Microbial transglutaminases (MTGs) are widely used in the food industry. In this study, the MTG gene of Streptomyces sp. TYQ1024 was cloned and expressed in a food-grade bacterial strain, Bacillus subtilis SCK6. Extracellular activity of the MTG after codon and signal peptide (SP Ync M) optimization was 20 times that of the pre-optimized enzyme. After purification, the molecular weight of the MTG was 38 kDa and the specific activity was 63.75 U/mg. The optimal temperature and pH for the recombinant MTG activity were 50℃ and 8.0, respectively. MTG activity increased 1.42-fold in the presence of β-ME and 1.6-fold in the presence of DTT. Moreover, 18% sodium chloride still resulted in 83% enzyme activity, which showed good salt tolerance. Cross-linking gelatin with the MTG increased the strength of gelatin 1.67 times and increased the thermal denaturation temperature from 61.8 to 75.8℃. The MTG also significantly increased the strength and thermal stability of gelatin. These characteristics demonstrated the huge commercial potential of MTG, such as for applications in salted protein foods.

• Food Science and Biotechnology
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• 제16권4호
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• pp.655-658
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• 2007

Bacillus polyfermenticus SCD was transformed by the recombinant shuttle vector for Bacillus and Escherichia coli containing 3 antibiotic resistant genes and an ${\alpha}$-amylase gene from Bifidobacterium adolescentis Int57. The ${\alpha}$-amylase gene fused to a secretion sequences was expressed under the control of the promoter of amylase gene from B. subtilis var. natto. The recombinant plasmid was maintained stably in the transformants producing the ${\alpha}$-amylase. The enzyme was secreted to outside of the cell and showed the similar enzyme activity as that of Bacillus subtilis BD170 under the same conditions of pH and growth temperature. Because of the relatively easy transformation and the secretion of the enzyme, the transformants of B. polyfermenticus SCD may give a new strategy in the production of foreign genes.

• 생명과학회지
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• 제14권3호
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• pp.391-395
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• 2004

B. stearothermophilus NO2의 CGTase 유전자 (cgtS)를 구성적 $P_{JH}$ promoter 하류에 subcloning 하여 재조합 plasmid pIH-CGT1 (8.14 kb)을 구축하고 B. subtilis DB431에 형질 전환하였다. B. subtilis DB431/pJH-CGT1를 5가지 배지(LB, 2${\times}$LB, 5% molasses+2% CSL, CS, LBG)로 flask 배양하여 균체증식과 CGTase발현량 및 분비국재성을 조사하여 최적 배지를 결정하였다. 그 중 〔5% molasses+2% CSL〕 배지에서 9시간에 1.8 unit/$m\ell$의 CGTase가 발현$.$생산되었다. 이 결과를 토대로 3. subtilis DB431/pJH-CGT1를 〔10% molasses + 5% corn steep liquor〕 배지에서 발효조 회분 배양한 결과, 30시간 배양시 CGTase의 최대 발현량은 4.2 unit/$m\ell$, 90%의 분비 효율, 90% 이상의 plasmid 안정성을 나타내었다. 저렴한 산업용 molasses 배지로 발효조 회분배양시 플라스크 배양보다 균체증식과 CGTase 발현량이 2배 이상의 증가된 값을 얻었다.

• Applied Biological Chemistry
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• 제45권4호
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• pp.190-194
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• 2002

Bacillus subtilis의 glutamyl-tRNA synthetase(GluRS)는 대장균에서 발현될 때 숙주세포의 $tRNA_1^{Gln}$에 glutamate를 잘못 아실화하여 독성을 나타내는 것으로 추정되고 있다. 이러한 B. subtilis GluRS를 대장균에서 과발현 시키기 위하여 B. subtilis 168 균주의 chromosomal DNA에서 GluRS의 유전자(gltX)를 PCR을 이용하여 증폭하고 T7 promoter에 의해 발현이 조절되는 pET11a expression vector에 클로닝하였다. 이 재조합된 pEBER plasmid DNA로 T7 RNA polymerase를 갖는 대장균 NovaBlue(DE3)에 형질전환하였다. 형질전환된 대장균에 IPTG를 처리하여 과량 생성된 GluRS 단백질은 ammonium sulfate 분별침전 후 EPLC를 이용한 Source Q column anion exchange chromatography, Superdex 200 column gel filtration, Mono Q column anion exchange chromatography로 정제하였다. 정제된 B. subtilis의 GluRS 분자량은 약 55 kDa이었으며 효소의 활성도는 조효소액에 비해 18배로 증가하였다.