• 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.

• Journal of Microbiology and Biotechnology
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• 제22권12호
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• pp.1724-1730
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• 2012

An ${\alpha}$-L-arabinofuranosidase (TmAFase) from Thermotoga maritima MSB8 is a highly thermostable exo-acting hemicellulase that exhibits a relatively higher activity towards arabinan and arabinoxylan, compared with other glycoside hydrolase 51 family enzymes. In the present study, we carried out the enzymatic characterization and structural analysis of TmAFase. Tight domain associations found in TmAFase, such as an inter-domain disulfide bond (Cys306 and Cys476) in each monomer, a novel extended arm (amino acids 374-385) at the dimer interface, and total 12 salt bridges in the hexamer, may account for the thermostability of the enzyme. One of the xylan binding determinants (Trp96) was identified in the active site, and a region of amino acids (374-385) protrudes out forming an obvious wall at the substrate-binding groove to generate a cavity. The altered cavity shape with a strong negative electrostatic distribution is likely related to the unique substrate preference of TmAFase towards branched polymeric substrates.

• 한국미생물생명공학회:학술대회논문집
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• 한국미생물생명공학회 2004년도 Annual Meeting BioExibition International Symposium
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• pp.273-277
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• 2004

$\alpha$-L-Arabinofuranosidase ($\alpha$-L-AFase, EC 3.2.1.55) was isolated from hyperthermophilic microorganism, Thermotoga maritima. The open reading frame (ORF) of $\alpha$-L-AFase gene is 1,455 bp long and encodes 484 amino acid residues with a molecular weight of 55,265 Da. The ORF of $\alpha$-L-AFase gene was introduced into the E. coli expression vector, $_p/RSET-B, and overexpressed in E. coli BL21. The purified recombinant $\alpha$-L-AFase showed the highest activity at 10$0^{\circ}C$ and pH 5.5. The purified enzyme appeared to have no metal cofactor requirement. The Km and specific activity values of the recombinant enzyme were 0.99 mM and 1,200 U/mg on p-nitrophenyl-$\alpha$-L-arabinofuranoside. It released only L-arabinose from sugar beet arabinan, sugar beet debranched arabinan and oat spelts arabinoxylan but had no activity onarabinogalactan and gum arabic. This result suggests that L-arabinose could be produced from natural polysaccharides using this enzyme. Mutant enzymes which Glu26, Glu172 and Glu281 residues were replaced to alanine, aspartic acid or glutamine caused Kcat to decrease by a factor of between 10$^3$ and 10$^4$. Glu172 and Glu281 residues of $\alpha$-L-AFase are seemed to be the acid/base and nucleophile in catalytic reaction, respectively, and Glu26 is supposed to playa key role in substrate binding.ng.

• 한국생물공학회:학술대회논문집
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• 한국생물공학회 2003년도 생물공학의 동향(XII)
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• pp.587-587
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• 2003

• 한국균학회소식:학술대회논문집
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• 한국균학회 2015년도 추계학술대회 및 정기총회
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• pp.23-23
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• 2015

Rice blast disease caused by M. oryzae is the most devastating fungal disease in rice. During the infection process, M. oryzae secretes a large number of glycosyl hydrolase (GH) proteins into the apoplast to digest host cell wall and assist fungal ingress into host tissues. In this study, we identified a novel M. oryze arabinofuranosidase B (MoAbfB) which is secreted during fungal infection. Live-cell imaging exhibited that fluorescent labeled MoAbfB was highly accumulated in fungal invasive structures such as appressorium, tips of penetration peg, biotrophic interfacial complex (BIC), as well as invasive hyphal tip. Deletion of MoAbfB mutants extended biotrophic phase followed by enhanced disease severity, whereas, over-expression of OsMoAbfB mutant induced rapid defense responses and enhanced rice resistance to M. oryzae infection. Furthermore, exogenous treatment of MoAbfB protein showed inhibition of fungal infection via priming of defense gene expression. We later found that the extract of MoAbfB degraded rice cell wall fragments could also induce host defense activation, suggesting that not MoAbfB itself but oligosaccharides (OGs) derived from MoAbfB dissolved rice cell wall elicited rice innate immunity.

• Food Science and Biotechnology
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• 제17권5호
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• pp.990-995
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• 2008

A thermostable $\alpha$-L-arabinofuranosidases ($\alpha$-L-AFase) is an industrially important enzyme for recovery of L-arabinose from hemicellulose. The recombinant $\alpha$-L-AFase from Thermotoga maritima was expressed in Escherichia coli by using a constitutive pHCE or an inducible pRSET vectors. In batch fermentation, the constitutive expression system resulted in slightly faster growth rate (0.78 vs. 0.74/hr) but lower enzyme activity (2,553 vs. 3,723 units/L) than those of the induction system. When fed-batch fermentation was performed, biomass and enzyme activity reached the highest levels of 36 g/L and 9,152 units/L, respectively. The fed batch cultures performed superior results than batch culture in terms of biomass yield (4.62-5.42 folds) and enzyme synthesis (3.39-4.00 folds). In addition, the fed-batch induction strategy at high cell density resulted in the best productivity in cell growth as well as enzyme activity rather than the induction method at low cell density or the constitutive expression.

• 한국미생물·생명공학회지
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• 제26권4호
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• pp.297-302
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• 1998

Bacillus stearothermophilus No.236 xylB 유전자가 삽입된 재조합 플라스미드 pKMG12를 가지고 있는 E. coli HB101 균주를 이용하여 B. stearothermophilus $\beta$-xylosidase B을 생산, 정제하고 효소의 일반특성을 조사하였다. Ammonuim sulfate 분획, DEAE-Sepharose CL-6B 이온 교환 크로마토그래피, Sephacryl S-200 및 Superdex 200HR 젤 크로마토그래피의 과정을 거쳐 정제하였으며 정제된 효소는 SDS-PAGE 및 zymogram 실험을 통해 $\beta$-xylosidase B의 단백질임을 확인하였다. 정제 $\beta$-xylosidase B는 반응액의 수소이온 농도와 온도에 매우 민감하며 최적 활성 pH 및 온도는 각각 pH 6.5와 $50^{\circ}C$로 결정되었다. $\beta$-Xylosidase 활성은 1 mM $Mn^{2+}$ 첨가에 의해 약 35% 활성화됨을 보였으나 $Ag^{+}$, $Cu^{2+}$ 및 $Hg^{2+}$ 등의 중금속이온의 존재하에서는 거의 완전한 저해를 나타내었다. 또한 본 효소는 비록 높지는 않으나 $\alpha$-arabinofuranosidase 활성도 가지고 있어 B. stearothermophilus No 236의 $\beta$-xylosidase A 효소 보다 최소한 arabinoxylan의 분해에 있어서 더 우수한 효소로 판단되며 o-nitrophenyl-$\beta$-D-xylopyranoside 기질에 대한 $K_{m}$ 값과 $V_{max}$ 값은 각각 6.43 mM과 $1.45\mu$mole/min 로 계산되었다. 한편, $\beta$-xylosidase B 분자량은 gel 여과법으로는 약 160 kDa, 그리고 SDS-PAGE에 의해서는 약 54 kDa로 측정되어 본 효소는 trimer의 구조를 가지고 있음을 알 수 있었다.

• 한국생물공학회:학술대회논문집
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• 한국생물공학회 2005년도 생물공학의 동향(XVI)
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• pp.429-429
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• 2005

• 대한약학회:학술대회논문집
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• 대한약학회 2003년도 Proceedings of the Convention of the Pharmaceutical Society of Korea Vol.1
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• pp.224.2-225
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• 2003

Ginsenoside, major components of ginseng have been reported to show various biological activities including an increase of cholesterol metabolism. stimulation of serum protein synthesis, immunomodulatory effects. To explain these pharmacological actions, it is thought that ginseng saponins should be metabolized by human intestinal bacteria after they are orally administered. (omitted)

• Food Science and Biotechnology
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• 제18권6호
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• pp.1365-1370
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• 2009

Aspergillus kawachii extracellular pectinases were screened in liquid cultures with different carbon sources. The fungus grown on citrus pectin or lemon pomace produced at least one of these inducible pectinases: acidic polygalacturonase, pectin lyase, pectin methylesterase, $\alpha$-L-arabinofuranosidase, $\alpha$-1,5-endoarabinase, $\beta$-D-galactosidase/exogalactanase, and $\beta$-1,4-endogalactanase. The lemon-pomace filtrates also contained significant $\alpha$-L-rhamnosidase and $\beta$-D-fucosidase activities. Most of the screened pectinases were active at pH 2.0-2.5, indicating that the A. kawachii enzymes were acidophilic. Under the culture conditions employed we could not detect enzymatic degradation of soybean rhamnogalacturonan. The A. kawachii pectinase-production-related regulatory phenomena of induction-repression resemble those described for other Aspergillus sp.