• Korean Journal of Fisheries and Aquatic Sciences
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• v.28 no.6
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• pp.728-735
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• 1995

The peptidyl prolyl cis-trans isomerase(PPlase, EC 5.2.2.8) from Bacillus stearothermophilus SIC1 was extracted from the cells treated with by lysozyme. PPlase was purified from the cell extracts by heat treatment, ammonium sulfate precipitation, ion exchange chromatography and finally gel filtration (FPLC). The purity of purified the enzyme after Superose 12 column chromatography was examined by sodium dodecyl sulfate polyacrylamide gel electrophoresis(SDS-PAGE). The molecular weight of the purified PPlase was estimated as 18,000 by SDS-PAGE. The 39 amino acid residues from the N-terminus were determined by the protein sequencer. The enzyme showed the optimum pH at 8.0 and was stable at the range of pH 7.0 to 8.0. The enzyme was considerably stable after heat treatment at $60^{\circ}C$ for 30 minutes, and the enzyme was quite stable up to $65^{\circ}C$. The presence of the PPlase in the refolding solution accelerated the isomerization rate of the assay peptide.

• 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.18 no.6
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• pp.585-590
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• 1990

The cyclodextrin glucanotransferase (CGTase) was purified from the culture broth of Bacillus stearothermophilus by ammonium sulfate precipitation and affinity chromatography. The specific activity of the CGTase increased by about 31-fold from 111.5 U/mg protein to 3445.0 Ulmg protein. The SDSPAGE indicated that the purified CGTase was homogeneous and the molecular weight of the purified CGTase was about 78,000. The optimum pH and temperature was 6.0 and $60^{\circ}C$, respectively. This enzyme was stable from pH 5.5-10.0. The enzyme retained its full activity at the incubation temperature up to $60^{\circ}C$ and calcium ion increased the thermal stability. The isoelectric point was about 4.8.

• Microbiology and Biotechnology Letters
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• v.22 no.2
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• pp.134-142
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• 1994

The neucleotide sequences of the xylA gene encoding $\beta $-xylosidase of Bacillus stearothermophilus and is its flanking regions were datermined. Three open reading frame(ORFs) were found, one of which(ORF1) appeared to code for the $\beta $-xylosidase. The 1830 base pair ORF1 encoded 609 amino acids starting from a TTG initiation codon. The molecular weight deduced from the nucleotide sequence(68 KD) was in agreement with that estimated by SDS-polyacrylamide gel electrophoresis of the purified enzyme(66 KD). The Shine-Dalgarno sequence(5'-AGGAGG-3') was found 11 bp upstream of the initiation codon. Further 15 bp upstream, there observed a potential transcription initiation signals. The putative -10 sequence(CATAAT) and -35 sequence(TTGTTA) coresponded closely to the consensus sequences for Bacillus subtilis RNA polymerase with major sigma factor. The guanine-plus-cytosine content of the coding region of the xylA gene was 56mol% while that of the third position of the codons was 63 mol%. Based on the comparison with the amino acid sequences of several other carbohydrate degrading enzymes, two conserved regions, possibly participating in the catalytic mechamism of $\beta $-xylosidase xylA, were identified in 278-298 and 329-350 regions of the translated xylA gene. The nucleotide sequence of the xylA was found to exhibit no homology to any other genes so far reproted.

• 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).

• 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.

• 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.25 no.1
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• pp.23-29
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• 1997

The nucleotide sequence of the estI gene encoding acetylxylan esterase I of Bacillus stearothermophilus was determined and analyzed. The estI gene was found to consist of a 810 base pair open reading frame coding for a polypeptide of 270 amino acids with a deduced molecular weight of 30 kDa. This was in well agreement with the molecular weight (29 kDa) estimated by SDS-PAGE of the purified esterase. The coding sequence was preceded by a putative ribo some binding site 10 bp upsteam of the ATG codon. Further 53 bp upstream, the transcription initiation signals were identified. The putative $_{-}$10 sequence (TCCAAT) and $_{-}$35 seqence (TTGAAT) corresponded closely to the respective consensus sequences for the Bacillus subtiis major RNA polymerase. The G+C content of the coding region of the estI was 51% whereas that of the third position of codone was 60.2%. The N-terminal amino acid sequence of the EstI deduced from the nucleotide sequence perfectly matched the corresponding region of the purified esterase described previously. Comparison with the amino acid sequence of other esterases and lipases reported so far allowed us to identify a sequence, GLSMG at positions 123 to 127 of the EstI which was reported to be the highly conserved active site sequence for those enzymes. The nucleotide sequence of the estI revealed 55.7% homology to that of the xylC coding for the acetylxylan esterase of Caldocellum saccharolyticum.

• Journal of Microbiology and Biotechnology
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• v.5 no.3
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• pp.117-124
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• 1995

A gene (xynA) encoding the endo-xylanase (E.C.3.2.1.8) from Bacillus stearothermophilus was cloned in E. coli, and its complete nucleotide sequence was determined. The xynA gene consists of a 636 base pairs open reading frame coding for a protein of 212 amino acids with a deduced molecular weight of 23, 283 Da. A putative signal sequence of 27 amino acid residues shows the features comparable with the Bacillus signal sequences; namely, the signal contains a positively charged region close to the N-terminus followed by a long hydrophobic string. The coding sequence is preceded by a possible ribosome binding site with a free energy value of -16.6 kcal/mol and the transcription initiation signals are located further upstream. The translation termination codon (TAA) at the 3 end of the coding sequence is followed by two palindrome sequences, one of which is thought to act as a terminator. The xynA gene has a high GC content, especially in the wobble position of codons (64%). Comparison of the primary protein sequence with those of other xylanases shows a high homology to the xylanases belonging to family G.

• Microbiology and Biotechnology Letters
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• v.19 no.6
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• pp.592-597
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• 1991

An extracellular xylanase of Bacillus stearothemophilus was purified to a single protein through a sequency of operations including ammonium sulfate fractionation, DEAE Sepharose CL-6B ion exchange chromatography, Sephadex G-100 gel filtration and heat treatment. The purified enzyme had a moleular weight of 170, 000. the pH and temperature optima for the enzyme activity were pH 9.0 and $55^{\circ}C$, respectively. The activity was enhanced by $co^{2+} \; and\; Mn^{2+}$, and inhibited by $Hg^{2+}$. Pattern of hydrolysis demonstrated that the xylanase was an endo-splitting enzyme able to break down larchwood xylan at random giving xylobiose and xylotriose as the main end products.