• 한국미생물·생명공학회지
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• 제20권2호
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• pp.136-142
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• 1992

토양 분리균인 B.stearothermophilus chromosome의 유전자 은행으로부터 E.coli HB101 균주에 $\beta$-D-xylosidase 생산능력을 갖게하는 5.4Kb와 6.4Kb의 두 DNA 절편을 분리, pBR322에 크로닝하여 각각 pMG01과 pMG02의 재조합 플라스미드를 얻었다. 상기 두 B.stearothermophilus DNA 절편의 restriction map을 작성하고 이것을 기초로 하여 $\beta$-D-xylosidase 유전자인자의 위치를 확인함과 동시에 pUC18에 subcloning하여 각각 2.2kb와 1.0kb의 DNA 단편이 삽입된 $\beta$-D-xylosidase 양성의 pMG1와 pMG2를 분리하였다.

• 한국미생물·생명공학회지
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• 제17권6호
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• pp.574-579
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• 1989

Chromosomal DNA fragments of Bacillus sp. YA-14, isolated from soil as a potent $\beta$-xylosidase producing bacterium, were ligated to a vector plasmid pBR322 and used to transfer Escherichia coli HB101 cells. The recombinant plasmid pYXL22 was found to enable the transformants to produce $\beta$-xylosidase. pYXL22 was found to contain the 7.0 kb HindIII DNA fragment originated from the Bacillus sp. YA-14 chromosomal DNA by Southern hybridization. The optimum temperature for the reaction of $\beta$-xylosidase produced by E. coli HB101 (pYXL22) was appeared at 3$0^{\circ}C$. The enzyme was maintained stably up to 4$0^{\circ}C$ when stored 1hr at 4$0^{\circ}C$. The $\beta$-xylosidase was repressed completely by 0.4% (w/v) glucose concentration in E. coli HB101 (pYXL22). The optimum concentration of xylose for the $\beta$-xylosidase production in Bacillus sp. YA-14 was 0.2% (w/v).

• 한국미생물·생명공학회지
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• 제31권2호
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• pp.111-116
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• 2003

Xylan 분해 균주인 Bacillus stearothermophilus No. 236 분리균의 $\beta$-xylosidase 생산 유전자(xylA)의 염기 서열 및 transcription start site를 결정한 이전 연구 결과에 의하면 xylA 유전자는 매우 특이하게 UUG codon에서 translation이 시작되며 initiation codon 15dp 윗쪽에는 promoter로 추정되는 염기 서열을 가지고 있는 것으로 분석되었다. 이와 같은 xylA 유전자 promoter region의 구조는 E. coli에 클로닝된 xalA 유전자를 이용한 실험 결과로도 확인되었다. xalA promoter의 -10 element는 CATAAT로서 6개의 염기 중 5개가 그리고 -35 element의 경우는 TTGTTA로서 6개의 염기 중 4개가 consensus sequence와 일치되었으나 두 hexamer 사이의 거리가 최적 거리에서 크게 벗어난 12 bp인 것으로 분석되었다. 본 연구에서는 $\beta$-xylosidase의 대량 생산을 위한 연구의 일환으로 xalA promoter sequence의 체계적 구조 변화에 의한 promoter strength에 미치는 효과를 E. coli와 B. subtilis두 숙주 세포에서 조사 분석해 본 결과, 첫째로 두 promoter elements사이의 거리를 최적거리인 17 bp로 바꾸었을 때 xalA의 발현율은 E. coli에서는 1.6배, B. subtilis에서는 2.5배 정도 증가함을 보여주었다. 그리고 -35 element는 consensus sequence와 같이 5'쪽에서 네번째 위치에 있는 T$\longrightarrow$A로 변이 시켰을 때 E. coli경우 2.3배, 특히 B. subtilis에서는 35배나 되는 가장 높은 promoter 활성의 증가를 보였다. 그러나 -10 sequence의 경우 consensus sequence와 같이 5' 쪽에서 첫번째 위치에 있는 C$\longrightarrow$T로 transition시켰을 때 예상외로 오히려 발현율이 5~15배까지 낮아지는 특이한 결과를 얻었다. 따라서 본 연구 결과 xalA promoter의 경우 -10 sequence인 CATAAT의 C와 -35 element의 두 염기가 promoter활성에 있어 가장 중요한 염기임을 알 수 있었다.

• Journal of Microbiology and Biotechnology
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• 제8권1호
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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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• 제8권3호
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• pp.258-263
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• 1998

${\beta}$-Xylosidase was purified from the recombinant Escherichia coli carrying the Bacillus sp. KK-1 ${\beta}$-xylosidase gene (xylB). The molecular mass of the purified enzyme was estimated to be 62 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. However, the apparent molecular mass of the ${\beta}$-xylosidase was 140 kDa, indicating that the native ${\beta}$-xylosidase has an oligomeric structure composed of two identical subunits. The isoelectric point was determined to be pH 5.5. The enzyme was highly active on p-nitrophenyl-$\beta$-D-xylopyranoside but it barely hydrolyzed xylan substrates, and did not exhibit activity towards carboxymethylcellulose and p-nitrophenyl-${\beta}$-D- glucopyranoside. The enzyme had a pH optimum for its activity at pH 6.5 and a temperature optimum at $40^{\circ}C$. The enzyme activity was completely inhibited by the presence of $Hg^{++}$, and also markedly inhibited by D-xylose and D-glucose.

• Journal of Microbiology and Biotechnology
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• 제13권4호
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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.

• 한국미생물·생명공학회지
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• 제17권5호
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• pp.436-439
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• 1989

고온, 호알칼리성 Bacillus속 K-17 균주에서 $\beta$-xylosidase 유전자를 pBR322를 벡터로 이용하여 클로닝시켰다. p-Nitrophenyl-$\beta$-xylopyranoside를 함유하는 LB 한천배지에서 노란색을 형성하는 대장균 형질전환주에서 재조합 플라스미드 pAX278을 분리하였으며, 본 pAX278은 pBR322와 고온, 호알칼리성 Bacillus K-17 균주 염색체 DNA의 5.0 kb HindIII절편으로 구성되어 있었다. Biotin으로 로식된 pAX278을 probe로 하여 상동성 시험을 하여 본 결과, pAX278에 존재하는 5.0 kb HindIII 절편은 Bacillus K-17 균주의 염색체 DNA HindIII 절편 중에서 5.0 kb 분만 아니라 0.9 kb 절편과도 상동성이 있었다. pAX278의 5.0 kb 절편을 pGR71에 연결시켜 B. subtilis에서도 발현시켰다. pAX278을 가지는 E. coli 균주가 생성하는 $\beta$-xylosidase는 균체외에 존재하였으며 그 효소학적 성질은 Bacillus속 K-17의 $\beta$-xylosidase와 동일하였다.

• Journal of Microbiology and Biotechnology
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• 제6권5호
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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.

• Journal of Animal Science and Technology
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• 제65권3호
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• pp.679-682
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• 2023

The Lactococcus taiwanensis strain K_LL004 was isolated from the gut of a grasshopper (Oxya chinensis sinuosa) collected from local farm in Korea. L. taiwanensis strain K_LL004 is the functional probiotic candidate with an ability to hydrolyse plant polysaccharides. The complete genome of the L. taiwanensis strain K_LL004 contains one circular chromosome (1,995,099 bp) with a guanine + cytosine (GC) content of 38.8%. Moreover, 1,929 Protein-coding sequence, 19 rRNA genes, and 62 tRNA genes were identified based on results of annotation. L. taiwanensis strain K_LL004 has a gene, which encodes hydrolytic enzymes such as beta-glucosidase and beta-xylosidase, that hydrolyzes plant polysaccharides.

• Journal of Microbiology and Biotechnology
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• 제14권3호
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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.