• Journal of Food Hygiene and Safety
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• v.38 no.5
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• pp.356-360
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• 2023

We analyzed the heat resistance of non-pathogenic Bacillus atrophaeus, Bacillus subtilis, and Geobacillus stearothermophilus spores which exhibit strong heat resistance and evaluated the possibility of using them to determine direct sterilization when manufacturing retort foods. The D₁₂₁-values of B. subtilis, G. stearothermophilus, and B. atrophaeus spores were 2.9±0.1 min, 4.3±0.1 min, and 3.7±0.1 min, respectively. The Z-values of B. subtilis, G. stearothermophilus, and B. atrophaeus spores were 43.0±1.4℃, 25.0±1.6℃, and 35.8±1.4℃, respectively. The D₁₂₁-values of B. subtilis, G. stearothermophilus, and B. atrophaeus spores were all higher than that of Clostridium botulinum spores used to confirm retort food sterilization. Considering these results, B. subtilis, G. stearothermophilus, and B. atrophaeus spores can be used instead of the pathogenic spore-forming bacteria C. botulinum when sterilizing retort food. In addition, sterilization can be confirmed in 2 to 3 days, a shorter time than the 13 days required for existing bacterial growth experiments based on the Korean food code.

• Research in Plant Disease
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• v.8 no.4
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• pp.234-238
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• 2002

In vitro and in vivo activities of a biocontrol agent, Bacillus stearothermophilus strain YC4194 was evaluated for the control of Pythium damping-off of cucumber. B. stearothermophilus YC4194 inhibited germination of cystospores and formation of zoosporangia of Pythium aphanidermatum in vitro. Incorporation of a bentonite and talc based formulation(10$^{9}$ cfu/g) of B. stearothermophilus YC4194 to the nursery soils (10 g/ι soil) resulted In a significant (p=0.01) reduction in the disease severity of cucumber damping-off after inoculation with P. aphanidermatum. The control efficacy of B. stearothermophilus YC4194 formulation was not different from that of the fungicides, dimethomorph, metalaxyl, ethaboxam. When the cucumber plants were transplanted to the soil inoculated with P. aphanidermatum zoospores, the B. stearothermophilus YC4194 maintained the high population density in rhizosphere soil upto 10$^{7}$ cfu/g until 15 days after treatment.

• Microbiology and Biotechnology Letters
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• v.13 no.4
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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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• v.22 no.6
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• pp.599-606
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• 1994

Bacillus stearothermomophilus, a strong xylan degrader, was confirmed to express multiple esterase activities in addition to the major xylanolytic enzymes. One of the genes encoding the esterases was isolated from the genomic library of B. stearothermophilus constructed with EcoRl restriction endonuclease and pBR322 plasmid. Three recombinant plasmids showing the tributyrin degrading activity were selected from approximately 7, 000 E. coli HB101 transformants, and were found to have the same insert of a 3.2 kb DNA fragment. Restriction mapping and hybridization studies revealed that the gene(estII) on the hybrid plasmid (pKMG7) had originated from the B. stearothermophilus chromosome, and was distinct from the estl, another esterase gene of B. stearothermophilus isolated in the previous work. The E. coli cells harboring pKMG7 produced an acetylxylan esterase that exibited similar substrate specificity to the esterase encoded by the estI gene.

• Microbiology and Biotechnology Letters
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• v.30 no.4
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• pp.293-297
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• 2002

The cyclodextrin glucanotransferase (CGTase) gene from Bacillus stearothermophilus NO2 was expressed in Saccharomyces cerevisiae 2805 under the adhl promoter. The CGTase was purified from S. cerevisiae 2805/pVT-CGTS. The purified enzyme exhibited a optima of activity around pH 7.0 and $65^{\circ}C$. Thermal stability of the enzyme was increased fairly as compared with the CGTase of B. stearothermophilus NO2. The conversion yield of cyclodextrin (CD) and the production ratio of $\alpha$-, $\beta$,-, ${\gamma}$-CD from starch were showed similarly aspect to the CGTase of B. stearothermophilus NO2.

• Microbiology and Biotechnology Letters
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• v.26 no.4
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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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• v.9 no.5
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• pp.687-690
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• 1999

Bacillus stearothermophilus No. 236 isolated from the soil is a strong xylan degrader producing all the xylanolytic enzymes. However, the strain was discovered to be highly intractable to its transformation. In the present study, we have developed a reliable method for transformation of B. stearothermophilus No. 236 by a systematic examination of several factors which might have an influence on the efficiency of electrotransformation. Notably, we found that the most critical factor influencing the transformation efficiency (TE) was the incubation temperature after pulsing, with its optimum incubation of $37^{\circ}C.\; At\; 50^{\circ}C$, the optimum growth temperature of the B. stearothermophilus strain, the transformants could not be obtained at a recognizable level. The combination of field strength of 7.5 kV/cm along with pulse duration of 10 msec (resistance of $400{\Omega}\; and\; capacitance\; of\; 25{\mu}F$) was shown to be the best electrical parameters at the incubation temperature of $37^{\circ}$. A higher TE was obtained when the cells were harvested at an early-exponential phase. Twenty percent of PEG-8000 in a suspension buffer and an addition of 0.1% glycine in the growth medium resulted in about 4-fold and 3-fold increases in TE, respectively. We also found that the plasmid DNA which had been cycled through the host B. stearothermophilus cells enhanced TE by one order of magnitude higher. Under the presently described conditions, $2.5{\times}10^{5} transformants per ${\mu}g$ DNA was attained.

• Journal of Microbiology and Biotechnology
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• v.6 no.5
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• pp.331-335
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• 1996

The second $\beta$-Xylosidase gene (xylB) from Bacillus stearothermophilus was isolated from the genomic library, cloned into pBR322, and subsequently transferred into Escherichia coli HB101. Six out of 10, 000 transformants were selected from the selective LB medium supplemented with p-nitrophenyl-$\alpha$-L-arabinofuranoside (pNPAf) and ampicillin ($50\mu g$/ml) based on their ability to form a yellow ring around the colony. One of the clones was found to harbor the recombinant plasmid with 5.0 kb foreign DNA, which was identical to the $\alpha$-L-arabinofuranosidase gene (arfI) previously cloned in this lab, while the other five had 3.5 kb of the foreign DNA. Southern blotting experiments confirmed that the 3.5 kb insert DNA was from B. stearothermophilus chromosomal DNA. A zymogram with 4-methylumbelliferyl-$\alpha$-L-arabinofuranoside as the enzyme substrate revealed that the cloned gene product was one of the mutiple $\alpha$-L-arabinofuranosidases produced by B. stearothermophilus. Unlike the arfI gene product, the product of the gene on the insert DNA (xylB) showed an activity not only on pNPAf but also on oNPX suggesting that the cloned gene product could be a bifunctional enzyme having both $\alpha$-L-arabinofuranosidase and $\beta$-xylosidase activities.

• Microbiology and Biotechnology Letters
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• v.24 no.4
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• pp.385-392
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• 1996

Bacillus stearotheymophilus, a potent xylanolytic bacterium isolated from soil, was tested for the strain's strategies of pentose utilization and the evidence of substrate preferences. The strain metabolized glucose, xylose, ribose, maltose, cellobiose, sucrose, arabinose and xylitol. The efficacy of the sugars as a carbon and energy source in this strain was of the order named above. The organism, however, could not grow on glycerol as a sole growth substrate. During cultivation on a mixture of glucose and xylose or arabinose, the major hydrolytic products of xylan, B. stearothermophilus displayed classical diauxic growth in which glucose was utilized during the first phase. On the other hand, the pentose utilization was prevented immediately upon addition of glucose. Cellobiose was preferred over xylose or arabinose. In contrast, maltose and pentose were co-utilized, and also no preference on between xylose and arabinose. Enzymatic studies indicated that B. stearothermophilus possessed constitutive hexokinase, a key enzyme of the glucose metabolic system. While, the production of $^{D}$-xylose isomerase, $^{D}$-xylulokinase and $^{D}$-arabinose isomerase essential for pentose phosphate pathway were induced by xylose, xylan, and xylitol but repressed by glucose. Taken together, the results suggested that the sequential utilization of B. stearothermophilus would be mediated by catabolite regulatory mechanisms such as catabolite inhibition or inducer exclusion.

• 한국생물공학회:학술대회논문집
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• 2000.11a
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• pp.56-59
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• 2000

Thermophilic bacterium, Bacillus stearothermophilus NUB3621, was engineered to produce ethanol from glucose by introducing cloned thermostable pyruvate decarboxylase and alcohol dehydrogenase genes. A novel promoter sequence was screened and used for the enhancement of these two enzymes. Successful redirection of metabolic flux into ethanol was obtained. In addition, gene expression profiling using Bacillus subtilis DNA microarray was analyzed to overcome the intrinsic low glucose utilization of B.stearothermophilus. Many known and unknown genes were identified to be up or down regulated under glucose-containing media.