• Microbiology and Biotechnology Letters
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• 제13권4호
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• pp.349-354
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• 1985

A 4.7 kb Hind III fragment containing $\alpha$-amylase gene of Bacillus stearothermophilus IAM 11062 was cloned in Escherichia coil HB101, using plasmid pBR322 and runaway plasmid pSY343 as a vector. The cloned gene was stably maintained and expressed In E.coli. The constructed strain of E. coli have at least 3 times higher amylase activity than the donor strain, of B. stearothermophilus. About 75％ of the $\alpha$-amylase produced by the constructed strain of E. coli was localized in the periplasm and it was found that the enzymes can be released by an osmotic shock using EDTA. The enzymatic properties of L-amylase produced in E. coli were very similar to those produced by B. stearothermophilus in terms of optimum temperature, heat stability and molecular weight.

• Microbiology and Biotechnology Letters
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• 제21권6호
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• pp.542-548
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• 1993

Bacillus stearothermophilus was shown to express multiple xylanolytic enzymes including acetyl xylan esterase. Genomic DNA of the strain partially digested with HindIII was ligated into the HindIII site of pBR322, and expressed in E. coli HB101 cells in order to clone the gene for acetyl xylan esterase. One transformant among 4000 screened formed a clear zone around its colony on the LB agar supplemented with 1.0% tributyrin. The functional clone harbored the recombinant plasmid pKMG5 with an insert of 5.1kb.

• Journal of Microbiology and Biotechnology
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• 제6권2호
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• pp.79-85
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• 1996

By using a T7 expression system, a large amount of Bacillus stearothermophilus endo-xylanase A (XynA) could be produced in Escherichia coli cells. The overproduced enzyme formed inclusion bodies, and so the protein could be more easily purified to homogeneity. The molecular weight of the purified enzyme was estimated to be 22 kDa by SDS-polyacrylamide gel electrophoresis and 43 kDa by Sephacryl S-200 gel filtration, suggesting that the native enzyme was a homodimer. The pI value was determined to be 8.4. The Michaelis constants for birchwood xylan and oat spelts xylan were calculated to be 3.83 mg/ml and 5.03 mg/ml, respectively, and the $V_{max}$ max/ values for both xylans were 2.86 $\mu mole$/min. The purified enzyme was most active at $55^{\circ}C$ and pH 8.0, and stable up to $60^{\circ}C$ and in the near neutral pH range. From the zymogram, Bacillus stearothermophilus was found to have at least three xylanases and the purified one was the smallest among them.

• Microbiology and Biotechnology Letters
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• 제22권6호
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• pp.607-613
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• 1994

The Bacillus stearothermophilus arfI gene encoding a-arabinofuranosidase was isolated from the genomic library, cloned into pBR322, and subsequently transferred into the Escherichia coli HB101. The recombinant E. coli was selected from approximately 10,000 transformants screened by making use of its ability to produce a yellow pigment around the colony on the selective medium supplemented with p-nitrophenyl-$\alpha$-L-arabinofuranoside (pNPAf), a chromogenic substrate. The functional clone was found to harbor a recombinant plasmid, pKMG11 with an insertion of about 5 kb derived from the B. stearothermophilus chromosomal DNA. Identity of the arfI gene on the insert DNA was confirmed by a zymogram with 4-methylumbelliferyl-$\alpha$-L-arabinofuranoside as the enzyme substrate. The $\alpha$-arabinofuranosidase from the recombinant E. coli strain showed very high substrate specificity; the enzyme displayed high activity only with pNPAf among many other p- or $o$-nitrophenyl derivatives of several sugars, and acted only on arabinoxylan among various natural arabinose containing polysaccharides tested.

• Microbiology and Biotechnology Letters
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• 제22권6호
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• pp.636-642
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• 1994

Essential amino acids involving in the catalytic mechanism of the $\beta$-D-xylosidase of Bacillus stearothermophilus were determined by chemical modification studies. Among various che- mical modifiers tested N-bromosuccinimide (NBS), $\rho$-hydroxymercurybenzoate (PHMB), N-ethylma- leimide, 1-[3-(di-ethylamino)-propyl]$-3-ethylcarbodi-imide (EDC), and Woodward's Reagent K(WRK)inactivated the enzyme, resulting in the residual activity of less than 20%. WRK reduced the enzyme activity by modifying carboxylic amino acids, and the inactivation reacion proceeded in the form of pseudo-first-order kinetics. The double-lagarithmic plot of the observed pseudo-first- order rate constant against the modifier concentration yielded a reaction order of 2, indicating that two carboxylic amino acids were essential for the enzyme activity. The $\beta$-D-xylosidase was also inactivated by N-ethylmaleimide which specifically modified a cysteine residue with a reaction order of 1, implying that one cysteine residue was important for the enzyme activity. Xylobiose protected the enzyme against inactivation by WRK and N-ethylmaleimide, revealing that carboxylic amino acids and a cysteine residue were present at the substrate-binding site of the enzyme molecule.

• Korean Journal of Food Science and Technology
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• 제26권4호
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• pp.375-381
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• 1994

A bacterial strain No. KY-126, which produced extracellular cyclodextrin glycosyltransferase(CGTase), was isolated from soil and identified as Bacillus stearothermophilus KY-126. The enzyme was purified by the treatments of ammonium sulfate precipitation, DEAF-Sephadex, Sephadex G-100 column chromatography. The optimal pH and temperature for the enzyme activity were pH 5.5 and $65^{\circ}C$, respectively. And the enzyme was stable at pH values from 6.0 to 11.0 at $55^{\circ}C$ for 30 min and stable up to $60^{\circ}C$ for 30 min.. The enzyme was inhibited by $HgCl_{2}$. The molecular weight of the enzyme was estimated to be 67,000 by using SDS-PAGE. The maximum conversion from starch to cyclodextrin (CD) by CGTase was 43% and obtained at 6 hr reaction and the ratio of ${\alpha}-,\;{\beta}-,\;{\gamma}-$, CD production at this time was 2.9 : 2.1 : 1.0.

• Applied Chemistry for Engineering
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• 제16권1호
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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.

• Microbiology and Biotechnology Letters
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• 제26권4호
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• pp.297-302
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• 1998

$\beta$-Xylosidase B was produced by Escherichia coli HB101/pKMG12 carrying the xylB gene of Bacillus stearothermophilus No.236 on its recombinant plasmid. The $\beta$-xylosidase B produced was purified by ammonium sulfate fractionation, DEAE-Sepharose CL-6B, Sephacryl S-200 and Superdex 200 HR gel filtration. The purified enzyme showed the highest activity at pH 6.5 and 5$0^{\circ}C$. But, the enzyme was observed to be very sensitive to the pH and temperature of the reaction mixture. The enzyme was activated about 35% of its original activity in the presence of 1 mM of $Mn^{2+}$ but it was completely inhibited by $Ag^{+}$, $Cu^{2+}$and $Hg^{2+}$ions. In contrast with the $\beta$-xylosidase A, the B enzyme was found to have $\alpha$-arabinofuranosidase activity though the activity was fairly low compared with the $\alpha$-arabinofuranosidase produced from the arfI gene of the same Bacillus stearothermophilus. Therefore, $\beta$-xylosidase B is considered to be more suitable than $\beta$-xylosidase A at least for the biodegradation of arabinoxylan. The $K_{m}$ and V$_{max}$ values of the $\beta$-xylosidase B for o-nitrophenyl-$\alpha$-D-xylopyranoside were 6.43 mM and 1.45 $\mu$mole/min, respectively. Molecular mass of the enzyme was determind to be about 54 kDa by SDS-PAGE and 160 kDa by Superdex 200HR gel filtration, indicating that the functional $\beta$-xylosidase B was composed of three identical subunits.s.

• Journal of Microbiology and Biotechnology
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• 제7권5호
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• pp.305-309
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• 1997

Bacillus stearothermophilus No. 236, an effective xylanolytic bacterium, produced an extracellular carboxymethyl cellulase when the strain was grown on xylan. The carboxymethyl cellulase was purified to homogeneity as judged by SDS-PAGE and zymogram, The carboxymethyl cellulase had a pI of 4.0, and a molecular mass of 95 kDa. The highest level of enzyme activity was observed at pH 6.5 and $60^{\circ}C$. The $K_m$, and $V_{max}$ values of the enzyme to carboxymethyl cellulose were 20.8 mg/ml and $0.63 {\mu}mole$/min/mg protein, respectively, The enzyme was found to act also on filter paper and xylan as well as carboxymethyl cellulose. Therefore, it is expected that this xylanolytic strain isolated from soil could be efficiently used for xylan biodegradation.

• Journal of Microbiology and Biotechnology
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• 제16권3호
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• pp.484-486
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• 2006

Maltosyl (G2)-mannitol, produced by the transglycosylation of mannitol with maltotriose by Bacillus stearothermophilus maltogenic amylase, was not found to support lactic acid production by Streptococcus sobrinus NRRL 14555. Furthermore, the synthesis of water-insoluble glucans from maltosyl-mannitol by S. sobrinus NRRL 14555 was much lower than that from xylitol or mannitol. Consequently, these results suggest that maltosyl-mannitol could be used as a noncariogenic sugar substitute in food products.