• Journal of Microbiology and Biotechnology
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• v.11 no.1
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• pp.131-137
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• 2001

Previous work has identified that only the catabolite responsive element A (creA; previously called cre-2) out of two potential cre sequences (cre-1: nucleotide +160 to +173 and cre-2: +173 to +186), recognized within the coding region of the xylanaseA gene (xynA) of Bacillus stearothermophilus No.236, was actually, was actually involved in the carbon catabolite repression(CCR) of xynA expression in B. subtilis. However, the level of CCR of xynA expression in the original B.stearothermophilus No.236 strain (70-fold repression). Therefore, to search for an additional cre element in the promoter region, the upstream region of the xynA gene was subcloned by chromosome walking, and as a result, another potential cre element (nucleotide -124∼-137; designated creB) was recognized in this region. The cre-like sequence revealed a high homology to the cre consensus sequence. The xylanase activity of B. subtilis MW15 bearing pWPBR14 (containing creA and creB) cultured in a medium containing xylose as the sole carbon source was about 7.7 times higher than that observed for the same culture containing glucose. B. subtilis MW15 bearing pWPBR23 (containing only creA) produced an activity about 2.4 times higher. This pattern of CCR was confirmed using derivatives of xynA::aprA fusion plasmids. Furthermore, a measurement of the amounts of the xynA transcript showed a similar pattern as that for the production of xylanase. In addition, the synthesis of xylanase in B. subtilis QB7115 [a catabolite control protein A (ccpA) mutant strain] carrying pWPBR14 was almost completely relieved from glucose repression. Together, these results lead to a conclusion that the CCR of the expression of the xynA gene is mediated by CcpA binding at creA and creB sites in B. subtilis.

• Microbiology and Biotechnology Letters
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• v.17 no.4
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• pp.334-342
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• 1989

The Bacillus stearothermophilus cdd gene encoding cytidine deaminase (cytidine/2'-deoxycytidine aminohydrolase; EC 3.5.4.5) was isolated through shot gun cloning by oomplementation of an E. coli cdd mutation. Primarily 3.0 kbp of the exogenote was cloned into the Pstl site of pBR322 (pJSC101). By subsequent deletion and subcloning from the insert of pJSC101 with cdd$^+$ and tetracycline resistancy, about 1.35 kbp of the EcoRI$_1$/PstI$_2$ fragment containing the cdd gene was isolated as pJSC201. The minicell experiment revealed a molecular mass of 33,000 dalton for polypeptide from the cloned DNA fragment complementing the cdd gene. From the lacZ fusion of 550 bp fragment of the EcoRI$_1$/AuaI as a putative promoter region, the transcription direction of the cdd gene on pJSC201 is from EcoRI towards the PstI sites, When the cdd gene was expressed in B. subtilis ED4O (cdd$^-$, pyr$^-$) by transformation with the E. coli-B. subtilis shuttle vector, the gene expression occured more efficiently than in E. coli and the gene appears to be stably maintained in B. subtitis as well as in E. coli.

• Microbiology and Biotechnology Letters
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• v.20 no.2
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• pp.136-142
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• 1992

Bacillus stearothemophilus isolated from soil was identified to express multiple extracellular xylanases. Two HindIII restriction fragments of 5.4 and 6.4 kb from B. stearothermophilus genomic DNA were cloned into pBR322 to obtain recombinant plasmids pMG0l and pMG02, respectively, which enabled E. coli HBlOl cells to produce $\beta$-D-xylosidase activity. By subcloning into pUC18 and Southern blotting, the loci of the $\beta$-D-xyiosidase genes were elucidated to be on non-homologous DNA fragments of 2.2 kb from pMGOl(pMG1) and 1.0 kb from pMG02(pMG2), respectively. The two enzymes produced in E. coli cleaved xylobiose, xylotriose, xylotetrose and xylotetrose to produce xylose as a major end product. The gene on pMG1, distinct from that on pMG2 was observed to encode a bifunctional protein that displayed both P-D-xylosidase (EC.3.2.1.37) and a-L-arabinofuranosidase activities (EC.3.2.1.55).

• Applied Chemistry for Engineering
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• v.16 no.1
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• pp.139-143
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• 2005

We evaluated grapefruit seed extract for antibacterial activity at varying time intervals and concentration levels against heat tolerant and spore-forming Bacillus stearothermophilus, mesophilic Bacillus subtilis, and food poisoning Staphylococcus aureus. Grapefruit seed extract showed growth inhibitory activity against B. stearothemophilus and B. subtilis, and S. aureus at the level of 0.01 tp 0.03% in nutrient broth. However, when applied to soymilk in a market, grapefruit seed extract at the level above 0.2% effectively inhibited the growth of B. stearothermophilus, However, it failed to completely sterilize the test organisms. On the other hand, B. subtilis and S. aureus were completely sterilized at the level of 0.2% within 48 hrs and 72 h, respectively. The higher concentration of grapefruit seed extracts showed more effective antibacterial activities against the test organisms, but caused deteriorated organoleptic quality and emulsion stability. We could conclude, by applying grapefruit seed extract (0.015%) with thermal treatment (10 min at $121^{\circ}C$) that the microbial stability of commercial soymilk was enhanced greatly.

• Journal of Microbiology and Biotechnology
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• v.8 no.1
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• pp.21-27
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• 1998

The xylA gene of Bacillus stearothermophilus encoding the major ${\beta}$-xylosidase was previously cloned and sequenced. In the present study we examined the regulation of the cloned xylA gene expression in Bauillus subtilis MW15 carrying the xylA::aprA fusion plasmids. The induction of the fused xylA gene expression remained uninfluenced by any of the carbon sources tested but the gene expression was repressed about 2-3 fold in the presence of glucose. Two CRE-like sequences (CRE-1: nucleotides ＋ 124 to ＋136 and CRE-2: ＋247 to ＋259) were recognized within the reading frame region of the xylA gene. The deletion experiments showed that the CRE-2 sequence had a role in catabolite repression (CR) as a true CRE of the xylA gene, but the CRE-1 had no effect on CR of the xylA gene expression. Surprisingly, the deletion of the CRE- 1 sequence reduced about 2~3 fold of the expression of the xylA fused gene. The repression ratios of the xylA gene expression were estimated to be about 0.4 from the assay of subtilisin activity, and about 0.3 at the level of transcription by determining the amounts of xylA transcripts in B. subtilis. While, the level of CR of the xylA gene was assessed to be about l0-fold in previous work when the relative amounts of the xylA transcripts were measured in B. stearothermophilus.

• Journal of Microbiology and Biotechnology
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• v.2 no.2
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• pp.115-121
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• 1992

In order to broaden the spectrum of substrate utilization of a Gram negative bacterium Zymomonas mobilis which has a great potential as an industrial ethanol producing microorganism, cloning of $\alpha$-amylase gene into Z. mobilis ZM4 was tried. The $\alpha$-amylase gene was isolated from Bacillus stearothermophilus. By Southern blot analysis, it was proven that the $\alpha$-amylase gene fragment was originated from a naturally occuring plasmid of B. stearothermophilus ATCC 31195. To place $\alpha$-amylase gene under the control of Z. mobilis promoter, two different Z. mobilis expression vectors, pZA26 and pLOI204, were used. The truncated $\alpha$-amylase gene was then introduced into these vectors. Both qualitative and quantitative activities of $\alpha$-amylase were observed in Z. mobilis cells harboring these plasmids with the $\alpha$-amylase gene inserted. Gas chromatographic analysis of ethanol showed that one of the Z. mobilis transconjugants was capable of producing 67 mM ethanol from rich medium(RM) containing 5% soluble starch as a sole carbon source.

• Journal of Life Science
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• v.8 no.3
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• pp.326-332
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• 1998

Cyclodextrin glucanotransferase from B. stearothermophilus KJ16 that can produce both cyclodextrin glucanotransferase and cyclodextrinase was purified by ammonium sulfate precipitation, DEAE-cellulose chromatography, Sephadex G-100 chromatography, and FPLC. The molecular weight of the purifice enzyme was about 65,000 dalton by SDS-PAGE. The optimal pH and temperature were 6.0 and $60^{\circ}C$, respectively. The enzyme was stable at $50^{\circ}C$ for 1 hr and in the pH range of 5.5 and 8.5. Mercaptoethanol and dithiothreitol inhibited the enzyme activity strongly. The enzyme produced 60% cyclodextrin(CD) from 5% soluble starch with the $^{\alpha}$, $^{\beta}$, $^{\gamma}$-CD ratio of 42:46:12. Amylopectin was the most suitable substrate with 67% conversion to CD.

• Food Science and Biotechnology
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• v.16 no.6
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• pp.1082-1084
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• 2007

Ultra-high temperature (UHT) processed full fat milk inoculated with Escherichia coli, Pseudomonas fluorescens, and Bacillus stearothermophilus was exposed to 30-60 kV/cm square wave pulsed electric field (PEF) with $1\;{\mu}sec$ pulse width, and $26-210\;{\mu}sec$ treatment time in a continuous PEF treatment system. Eight log reduction was obtained for E. coli and P. fluorescens and 3 logs reduced for B. stearothermophilus under PEF treatment conditions of $210\;{\mu}sec$ treatment time, 60 kV/cm pulse intensity at $50^{\circ}$. There was no significant change in pH and titration acidity of milk after PEF treatment. The electrical energy required to achieve 8 log reduction for E. coli and P. fluorescens was estimated to be about 0.74 kJ/L.

• Journal of Life Science
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• v.14 no.3
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• pp.391-395
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• 2004

To overproduce the cyclodextrin glucanotransferase(CGTase) of Bacillus stearothermophilus NO2 in B. subtilis, the pJH-CGTl plasmid (8.14 kb) was constructed, in which the ORF of CGTase gene could be transcribed by strong constitutive promoter, P$\_$JH/. To overproduce CGTase from a recombinant B. subtilis, the effect of media on the cell growth and expression level of CGTase were investigated with various media (LB, 2${\times}$LB, 5% molasses+2% CSL, CS, LBG) in the flask culture. Among them, [5% molasses+2% CSL] medium revealed the maximum expression level of CGTase with 1.8 unit/$m\ell$ at 9 hr culture. In the batch culture on [10% molasses+5% corn steep liquor] medium the expression level of CGTase, the secretion efficiency, and plasmid stability were about 4.2 unit/$m\ell$, 90% and 90%, respectively, at 30 hr culture. The cell growth and expression level in the fermenter culture with the industrial molasses medium were increased by 2-folds over the flask culture.

• Microbiology and Biotechnology Letters
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• v.30 no.4
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• pp.298-304
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• 2002

A family shuffling associating PCR-based and in vitro recombination and expression in Escherichia coli cdd mutant was carried out. Two cdd genes encoding cytidine deaminases (CDase) from thermophilic Bacillus caldolyticus and B. stearothermophilus were shuffled. Around 150 viable mutant colonies screened on AB minimal medium without uracil by E. coli cdd complementation were selected for cytidine deaminase assay and 4 candidates (SH1067, SH1077, SH1086, and SH1118) were chosen for the detailed study. The nucleotide sequence analyses of 4 selected mutants revealed that they have several point mutations and recombinations. Surprisingly, the SH 1067 showed 770 fold more specific CDase activity at $80^{\circ}C$ than that of T101 from parental B. stearothermophilus.