• Microbiology and Biotechnology Letters
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• v.17 no.4
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• pp.289-294
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• 1989

A bacterial strain capable of producing high level of extracellular xylanase was isolated from soil. The characteristics of the isolated strain No.236 were identified to be Bacillus stearothermophilus. The maximal xylanase production was observed in the medium containing 0.75% xylan, 0.35% yeast extract, 1.06% $K_2$HPO$_4$and 0.05% CaCO$_3$with initial pH of 6.5 when the strain was cultured at 5$0^{\circ}C$ for 28 hrs with reciprocal shaking. Hydrolysis of xylan by the xylanase revealed that xylose was the only product of the reaction. This suggested that the enzyme produced by Bacillus stearothermophilus No. 236 was an exe-acting xylanase.

• Journal of Microbiology and Biotechnology
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• v.7 no.5
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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.

• 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.14 no.3
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• pp.474-482
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• 2004

The $\alpha$-L-arabinofuranosidase (Arfase) gene of Bacillus stearothermophilus No. 236 was cloned and sequenced. The ORF of the gene, designated arfA, encoded a 507 -residue polypeptide with calculated molecular mass of 57 kDa. The Arfase produced by a recombinant Escherichia coli strain containing the arfA gene was purified to apparent homogeneity and characterized. The molecular mass of the Arfase determined by SDS-PAGE was 60 kDa. However, according to gel filtration, it was estimated to be approximately 190 kDa. These results indicated that the functional form of the Arfase is trimeric. The optimal pH and temperature for the enzyme activity were pH 6.5 and $55^{\circ}C$, respectively. The half-life of the enzyme at $60^{\circ}C$ was about 6 h. Kinetic experiments at $45^{\circ}C$ with pNPM (p-nitrophenyl $\alpha$-L-arabinofuranoside) as a substrate gave the $K_m and V_{max}$ values of 1.19 mM and 26.1 U/ mg, respectively. When the enzyme was combined with Bacillus stearothermophilus No. 236 endoxylanase and $\beta$-xylosidase, it hydrolyzed arabinoxylan into L-arabinose and xylose more efficiently than Arfase alone. This synergistic effect suggested that the complete hydrolysis of xylan with large amounts of arabinose side chains required Arfase as well as endoxylanase and $\beta$-xylosidase.

• 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.

• Microbiology and Biotechnology Letters
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• v.31 no.2
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• pp.111-116
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• 2003

The xylA gene of Bacillus stearothermophilus No. 236 encoding $\beta$-xylosidase was cloned and sequenced previously. The transcriptional start site of the xylA gene cloned in E. coli was identified to be the guanine (G) by primer extension analysis. This supports that the expression of xylA gene is also directed in the E. coli cells by the previously determined transcription initiation signals, -10 sequence (CATAAT) and -35 sequence (TTGTTA) separated by 12 bp. To increase the expression of $\beta$-xylosidase, firstly the spacer region of xylA promoter was extended from 12 to 17 bp, and then the -10 and -35 elements were converted into their respective consensus sequences. The mutant promoters thus obtained were tested for their activities in both the E. coli and B. subtilis host cells. The change of the length of the spacer region from 12 to 17 bp resulted in a 1.6- and 2.5-fold increase in promoter strength in comparison with the wild type promoter in E. coli and B. subtilis cells, respectively. Also, strength of the promoter with the fourth T to A transversion on its -35 element increased in the transcription level by about 35 times compared with that of wild-type promoter. However, surprisingly the 5' end C-to-T transition of the -10 hexamer showed a 5- to 15-fold reduction in $\beta$-xylosidase activity in both E. coli and B. subtilis. Together, the present data demonstrated that the 5' end nucleotide C of the -10 sequence CATAAT and the fourth nucleotide A of the -35 hexamer are two most critical nucleotides for the promoter activity in the context of the xylA promoter.

• 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.

• Journal of Microbiology and Biotechnology
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• v.13 no.4
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• pp.584-590
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• 2003

The xylA gene of Bacillus stearothermophilus No. 236 encoding ${\beta}-xylosidase$, a major xylanolytic enzyme, was previously cloned and sequenced by the present authors. Sequence analysis indicated that translation of the xylA gene was initiated from the noncanonical initiation codon UUG, confirmed by analyzing three different amber (UAG) mutants of the xylA gene. In the present study, the UUG initiation codon was mutated into AUG or GUG, and the effects of the mutations on the XylA synthesis were examined. The AUG initiation codon was found to direct the highest level of ${\beta}-xylosidase$ synthesis; three-fold and fourteen-fold more enzyme activity than the UUG codon in E. coli and B. subtilis cells, respectively. Surprisingly, contrary to other systems reported to date, the UUG start codon was found next to AUG in the relative order of translational efficiency in both organisms. In addition, a greater abundance of the xylA mRNA was detected with the AUG start codon in both of these host cells than with GUG or UUG. Northern blot and Toeprint assays revealed that this was due to enhanced stability of mRNA with the AUG initiation codon. As expected, the ${\beta}-xylosidase$ protein level in the bacterial cells containing mRNA with the AUC start codon was also much higher than the levels with the other two different mRNAs.

• Journal of Microbiology and Biotechnology
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• v.8 no.4
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• pp.378-387
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• 1998

The DNA sequence immediately following the xynA gene of Bacillus stearothermophilus 236 ［about l-kb region downstream from the translational termination codon (TAA) of the xynA gene］was found to have an ability to enhance the xylanase activity of the upstream xynA gene. An 849-bp ORF was identified in the downstream region, and the ORF was confirmed to encode a novel protein of 283 amino acids designated as XaiF (xylanase-activity-increasing factor). From the nucleotide sequence of the xaiF gene, the molecular mass and pI of XaiF were deduced to be 32,006 Da and 4.46, respectively. XaiF was overproduced in the E. coli cells from the cloned xaiF gene by using the T7 expression system. The transcriptional initiation site was determined by primer extension analysis and the putative promoter and ribosome binding regions were also identified. Blast search showed that the xaiF and its protein product had no homology with any gene nor any protein reported so far. Also, in B. subtilis, the xaiF trans-activated the xylanase activity at the same rate as in E. coli. In contrast, xaiF had no activating effect on the co-expressed ${\beta}-xylosidase$ of the xylA gene derived from the same strain of B. stearothermophilus. In addition, the intracellular and extracellular fractions from the E. coli cells carrying the plasmid-borne xaiF gene did not increase the isolated xylanase activity, indicating that the protein-protein interaction between XynA and XaiF was not a causative event for the xylanase activating effect of the xaiF gene.