• Journal of Microbiology and Biotechnology
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• 제5권1호
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• pp.26-30
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• 1995

The cytoplasmic endo-$\beta$-l, 4-glucanase (endoglucanase) was purified from cell extracts of Escherichia coli (pBS1) transformant carrying the Bacillus subtilis endo-$\beta$-l, 4-glucanase gene after full growth, and its molecular weight was found to be 52 kilodaltons (kDa). The endo-$\beta$-l, 4-glucanase isolated from the periplasmic space was smaller than 52-kDa cytoplasmic enzyme. The 52-kDa endoglucanase was found to be cleaved in the periplasm and finally converted to 34.5-kDa protein. Small amounts of both 52-kDa and 34.5-kDa proteins were secreted into the culture broth. The cleavage took place in the C-terminal portion of the enzyme. The N-terminal amino acid residues of both 52-kDa and 34.5-kDa enzymes were determined to be the same, Ala, the 30th residue of the primary translation product. Cleavage of the C-terminal portion showed to have no significant effect on the basic enzyme properties.

• 한국미생물·생명공학회지
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• 제13권3호
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• pp.297-302
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• 1985

미생물중 urease생성능이 아주 강한 B. pasteurii의 Hind III partial digest 된 chromosomal DNA를 E. coli-B. subtilis bifunctional plasmid vector pGR 71으로 E. coli RR1 균주에 cloning 하므로써 그 urease gene을 expression시킬 수 있었다. 그러나 B. subtilis에서는 insertion DNA fragment의 deletion으로 expression되지 않았다. Cloning된 E.coli RR1 균주로부터 분리 정제한 urease gene함유 Plasmid(pGU66)의 restriction map을 작성하여 본 결과 7.1 Mdal의 insertion fragment가 삽입된 12.6Mdal의 plasmid에 Hind III, Bgl II, Xba I, Sal I등 몇 개의 cleavage site 위치를 찾을 수 있었다. Cloning된 E. coli의 urease는 periplasmic space에 많은 비율로 축적되며, 그 효소학적 성질은 donor인 B.pasteurii의 그것과 매우 유사하였다.

• Journal of Microbiology and Biotechnology
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• 제27권1호
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• pp.9-18
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• 2017

Nine bacilli with fibrinolytic activities were isolated from doenjang, a traditional Korean fermented soy food. Among them, RSB34 showed the strongest activity and was identified as Bacillus amyloliquefaciens by 16S rRNA and recA gene sequencing. During growth on LB up to 96 h, RSB34 showed the highest fibrinolytic activity ($83.23mU/{\mu}l$) at 48 h. Three bands of 23, 27, and 42 kDa in size were observed when the culture supernatant was analyzed by SDS-PAGE and 27 and 42 kDa bands by fibrin zymography. The gene encoding the 27 kDa fibrinolytic enzyme AprE34 was cloned by PCR. BLAST analyses confirmed that the gene was a homolog to genes encoding AprE-type proteases. aprE34 was overexpressed in Escherichia coli BL21(DE3) using pET26b(+). Recombinant AprE34 was purified and examined for its properties. The $K_m$ and $V_{max}$ values of recombinant AprE34 were $0.131{\pm}0.026mM$ and $16.551{\pm}0.316{\mu}M/l/min$, respectively, when measured using an artificial substrate, N-succinyl-ala-ala-pro-phe-p-nitroanilide. aprE34 was overexpressed in B. subtilis WB600 using pHY300PLK. B. subtilis transformants harboring pHYRSB34 (pHY300PLK with aprE34) showed higher fibrinolytic activity than B. amyloliquefaciens RSB34.

• KSBB Journal
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• 제9권1호
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• pp.16-25
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• 1994

Bacillus subtilis를 재조합 이종 단백질 생산 균주로 만들기 위하여 포자형성 변이주를 만들었다. 균주는 두 개의 프로테아제가 제거된 균주인 DB104로부터 spoOJ와 spoIIG 변이주를 유전자 조작법에 의해 만들고 두 개의 유전자가 모두 제거된 균주도 만들었다. 이에 목적 aprE 유전자를 삽입 벡터 형태로 만들어 integration시킨 뒤 변이주 각각의 형태적인 변화를 투과성 전자현미경으로 살펴 보았다. 각각 변이주의 모습은 이전에 보고된 것과 거의 일치하였으며 spoOJ spoIIG 이중포자변이주의 경우는 spoIIG 변이주와 더욱 닮은 것을 알 수 있었으며, 훨씬 주름진 것과 같은 투박한 세포벽 및 막을 가지고 있음을 관찰하였다. spoOJ 변이는 포자형성 빈도를 낮추고 aprE 활성을 감소시키는 반면, spoIIG 변이는 포자형성을 거의 하지 않으면서 aprE 활성에 상승효과를 가져왔다. spoOJ와 spoIIG 이중포자변이주는 spoOJ 변이의 효과는 거의 나타나지 않은채, spoIIG와 비슷한 aprE 활성을 보였다.

• Journal of Microbiology and Biotechnology
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• 제31권10호
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• pp.1446-1454
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• 2021

Herein, we cloned and expressed an endo-β-1,4-glucanase gene (celA1805) from Bacillus subtilis B111 in Escherichia coli. The recombinant celA1805 contains a glycosyl hydrolase (GH) family 8 domain and shared 76.8% identity with endo-1,4-β-glucanase from Bacillus sp. KSM-330. Results showed that the optimal pH and temperature of celA1805 were 6.0 and 50℃, respectively, and it was stable at pH 3-9 and temperature ≤50℃. Metal ions slightly affected enzyme activity, but chemical agents generally inhibited enzyme activity. Moreover, celA1805 showed a wide substrate specificity to CMC, barley β-glucan, lichenin, chitosan, PASC and avicel. The K_m and V_max values of celA1805 were 1.78 mg/ml and 50.09 µmol/min/mg. When incubated with cellooligosaccharides ranging from cellotriose to cellopentose, celA1805 mainly hydrolyzed cellotetrose (G4) and cellopentose (G5) to cellose (G2) and cellotriose (G3), but hardly hydrolyzed cellotriose. The concentrations of reducing sugars saccharified by celA1805 from wheat straw, rape straw, rice straw, peanut straw, and corn straw were increased by 0.21, 0.51, 0.26, 0.36, and 0.66 mg/ml, respectively. The results obtained in this study suggest potential applications of celA1805 in biomass saccharification.

• 한국미생물·생명공학회지
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• 제32권4호
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• pp.322-327
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• 2004

구성적 promoter인 $P_{JH}$ promoter 하류에 연결된 Bacillus sp. 유래의 endoxylanase를 B. subtilis에서 과발현 하였다. 발현된 plasmid, pJHKJ4는 자체 promoter($P_{B}$)와 Bacillus의 강력한 promoter($P_{JH}$)가 tandem으로 위치하여 transcription되었다. B.subtilis DB431에 형질전환된 균주를 glucose가 함유된 LB 배지에서 배양 후 총활성 및 분비효율은 140 U/ml 으로 나타났다. 배양상등액은 ultrafiltration(MW cut off 10 kDa and 30 kDa)과 염석법으로 농축하였다. 효소의 농축에서 ultrafiltration이 70% ammonium sulfate precipitation 보다 효과적이었다. $50^{\circ}C$ 에서 농축된 효소액의 안정화는 NaCl, glycerol, sorbitol, and $CaCl_{2}$ 등의 다양한 안정제를 농도별로 첨가하여 안정제의 효과를 검토한 결과 가장 효과적인 안정제는 NaCl과 $CaCl_{2}$ 이었다.

• Journal of Microbiology and Biotechnology
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• 제28권11호
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• pp.1850-1858
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• 2018

Glucosamine (GlcN) is widely used in the nutraceutical and pharmaceutical industries. Currently, GlcN is mainly produced by traditional multistep chemical synthesis and acid hydrolysis, which can cause severe environmental pollution, require a long prodution period but a lower yield. The aim of this work was to develop a whole-cell biocatalytic process for the environment-friendly synthesis of glucosamine (GlcN) from N-acetylglucosamine (GlcNAc). We constructed a recombinant Escherichia coli and Bacillus subtilis strains as efficient whole-cell biocatalysts via expression of diacetylchitobiose deacetylase ($Dac_{ph}$) from Pyrococcus furiosus. Although both strains were biocatalytically active, the performance of B. subtilis was better. To enhance GlcN production, optimal reaction conditions were found: B. subtilis whole-cell biocatalyst 18.6 g/l, temperature $40^{\circ}C$, pH 7.5, GlcNAc concentration 50 g/l and reaction time 3 h. Under the above conditions, the maximal titer of GlcN was 35.3 g/l, the molar conversion ratio was 86.8% in 3-L bioreactor. This paper shows an efficient biotransformation process for the biotechnological production of GlcN in B. subtilis that is more environmentally friendly than the traditional multistep chemical synthesis approach. The biocatalytic process described here has the advantage of less environmental pollution and thus has great potential for large-scale production of GlcN in an environment-friendly manner.

• 생명과학회지
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• 제26권10호
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• pp.1113-1120
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• 2016

우리나라의 전통 발효 된장으로부터 균체외 효소로 mannanase를 생산하는 세균 2주가 분리되었다. 분리균 WL-6과 WL-11은 형태적 특성, 생화학적 성질 및 16S rDNA의 염기서열에 따라 Bacillus subtilis로 확인되었다. 이들 두 균주로부터 각각 mannanase 유전자를 대장균에 클로닝하여 염기서열을 결정한 결과 mannanase 유전자는 362 아미노산으로 구성된 단백질을 코드하며 1,086 뉴클레오티드로 동일하게 이루어졌다. WL-6과 WL-11 mannanase (Man6, Man11)의 아미노산 잔기 배열은 서로 8개 잔기가 다르며 GH family 26에 속하는 B. subtilis의 mannanases와 매우 상동성이 높았다. Man6과 Man11의 아미노 말단의 26개 아미노 잔기가 signal peptide로 예측되었다. 재조합 대장균로부터 각각 생산된 Man6과 Man11은 94~95% 정도가 균체내에 존재하였고, mannotriose, mannotetraose, mannopentaose, mannohexaose와 같은 만노올리고당과 locust bean gum을 유사하게 분해하여 주된 반응산물로 mannobiose와 mannotriose를 생성하였다. Man6는 55℃와 pH 6.0, Man11은 60℃와 pH 5.5에서 각각 최대 반응활성을 보였으며, Man11이 Man6에 비해 열안정성이 높았다.

• Journal of Microbiology and Biotechnology
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• 제8권1호
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• pp.28-33
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• 1998

A gene coding for ${\beta}$-xylosidase from thermophilic xylanolytic Bacillus sp. KK-1 was cloned into Escherichia coli using plasmid pBR322. Recombinant plasmid DNAs were isloated from E. coli clones which were capable of hydrolyzing 4-methylumbelliferyl-${\beta}$-D xylopyranoside. Restriction analysis showed the DNAs to share a common insert DNA. Xylo-oligosaccharides, including xylotriose, xylotetraose, xylopentaose, and xylobiose were hydrolyzed to form xylose as an end product by cell-free extracts of the E. coli clones, confirming that the cloned gene from strain KK-1 is ${\beta}$-xylosidase gene. The ${\beta}$-xylosidase gene of strain KK-1 designated as xylB was completely sequenced. The xylB gene consisted of an open reading frame of 1,602 nucleotides encoding a polypeptide of 533 amino acid residues, and a TGA stop codon. The 3' flanking region contained one stem-loop structure which may be involved in transcriptional termination. The deduced amino acid sequence of the KK-1 ${\beta}$-xylosidase was highly homologous to the ${\beta}$-xylosidases of Bacillus subtilis and Bacillus pumilus, but it showed no similarity to a thermostable ${\beta}$-xylosidase from Bacillus stearothermophilus.

• BMB Reports
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• 제44권3호
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• pp.193-198
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• 2011

The chitinase-producing strain SC081 was isolated from Korean traditional soy sauce and identified as Bacillus atrophaeus based on a phylogenetic analysis of the 16S rDNA sequence and a phenotypic analysis. A gene encoding chitinase from B. atrophaeus SC081 was cloned in Escherichia coli and was named SCChi-1 (GQ360078). The SCChi-1 nucleotide sequences were composed of 1788 base pairs and 596 amino acids, which were 92.6, 89.6, 89.3, and 78.9% identical to those of Bacillus subtilis (ABG57262), Bacillus pumilus (ABI15082), Bacillus amyloliquefaciens (ABO15008), and Bacillus licheniformis (ACF40833), respectively. A recombinant SCChi-1 containing a hexahistidine tag at the amino-terminus was constructed, overexpressed, and purified in E. coli to characterize SCChi-1. $H_6SCChi$-1 revealed a hydrolytic band on zymograms containing 0.1% glycol chitin and showed the highest lytic activity on colloidal chitin and acidic chitosan. The optimal temperature and pH for chitinolytic activity were $50^{\circ}C$ and pH 8.0, respectively.