• Journal of Microbiology and Biotechnology
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• v.16 no.6
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• pp.921-926
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• 2006

The production and location of xylanolytic enzymes in alkaliphilic Bacillus sp. K-1, isolated from the wastewater treatment plant of the pulp and paper industry, was studied. When grown in alkaline xylan medium, the bacteria produced xylanolytic enzymes such as xylanase, $\beta$-xylosidase, arabinofuranosidase, and acetyl esterase. Two types of xylanases (23 and 45 kDa) were found to be extracellular, but another type of xylanase (35 and/or 40 kDa) was detected as pellet-bound that was eluted with 2% triethylamine from the residual xylan of the culture. The xylanases were different in their molecular weight and xylan-binding ability. Arabinofuranosidase and $\beta$-xylosidase were found to be intracellular and extracellular, respectively, and acetyl esterase was found to be extracellular. The extracellular xylanolytic enzymes effectively hydrolyzed insoluble xylan, lignocellulosic materials, and xylans in kraft pulps.

• Proceedings of the Korean Society for Applied Microbiology Conference
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• 2004.06a
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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.

• Journal of Microbiology and Biotechnology
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• v.31 no.2
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• pp.272-279
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• 2021

Two genes encoding probable α-ʟ-arabinofuranosidase (E.C. 3.2.1.55) isozymes (ABFs) with 92.3% amino acid sequence identity, ABF51A and ABF51B, were found from chromosomes 3 and 5 of Saccharomycopsis fibuligera KJJ81, an amylolytic yeast isolated from Korean wheat-based nuruk, respectively. Each open reading frame consists of 1,551 nucleotides and encodes a protein of 517 amino acids with the molecular mass of approximately 59 kDa. These isozymes share approximately 49% amino acid sequence identity with eukaryotic ABFs from filamentous fungi. The corresponding genes were cloned, functionally expressed, and purified from Escherichia coli. SfABF51A and SfABF51B showed the highest activities on p-nitrophenyl arabinofuranoside at 40~45℃ and pH 7.0 in sodium phosphate buffer and at 50℃ and pH 6.0 in sodium acetate buffer, respectively. These exoacting enzymes belonging to the glycoside hydrolase (GH) family 51 could hydrolyze arabinoxylo-oligosaccharides (AXOS) and arabino-oligosaccharides (AOS) to produce only ʟ-arabinose, whereas they could hardly degrade any polymeric substrates including arabinans and arabinoxylans. The detailed product analyses revealed that both SfABF51 isozymes can catalyze the versatile hydrolysis of α-(1,2)- and α-(1,3)-ʟ-arabinofuranosidic linkages of AXOS, and α-(1,2)-, α-(1,3)-, and α-(1,5)-linkages of linear and branched AOS. On the contrary, they have much lower activity against the α-(1,2)- and α-(1,3)-double-substituted substrates than the single-substituted ones. These hydrolases could potentially play important roles in the degradation and utilization of hemicellulosic biomass by S. fibuligera.

• Food Science and Biotechnology
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• v.18 no.6
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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.

• Journal of Microbiology and Biotechnology
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• v.25 no.2
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• pp.227-233
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• 2015

Two recombinant arabinosyl hydrolases, α-_L-arabinofuranosidase from Geobacillus sp. KCTC 3012 (GAFase) and endo-(1,5)-α-_L-arabinanase from Bacillus licheniformis DSM13 (BlABNase), were overexpressed in Escherichia coli, and their synergistic modes of action against sugar beet (branched) arabinan were investigated. Whereas GAFase hydrolyzed 35.9% of _L-arabinose residues from sugar beet (branched) arabinan, endo-action of BlABNase released only 0.5% of _L-arabinose owing to its extremely low accessibility towards branched arabinan. Interestingly, the simultaneous treatment of GAFase and BlABNase could liberate approximately 91.2% of _L-arabinose from arabinan, which was significantly higher than any single exo-enzyme treatment (35.9%) or even stepwise exo- after endo-enzyme treatment (75.5%). Based on their unique modes of action, both exo- and endo-arabinosyl hydrolases can work in concert to catalyze the hydrolysis of arabinan to _L-arabinose. At the early stage in arabinan degradation, exo-acting GAFase could remove the terminal arabinose branches to generate debranched arabinan, which could be successively hydrolyzed into arabinooligosaccharides via the endo-action of BlABNase. At the final stage, the simultaneous actions of exo- and endo-hydrolases could synergistically accelerate the _L-arabinose production with high conversion yield.

• Korean Journal of Food Science and Technology
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• v.51 no.4
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• pp.336-342
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• 2019

To elucidate structure-function relationship of polysaccharide from Angelica gigas, the AGE-2c-I was purified by two successive chromatography steps. AGE-2c-I showed a potent anti-complementary activity in a dose-dependent manner. AGE-2c-I with a molecular weight of 140 kDa comprised four monosaccharides and 13 glycosyl linkages, and strongly reacted with ${\beta}$-glucosyl Yariv reagent. For the fine structure analysis of AGE-2c-I, it was sequentially digested by exo-arabinofuranosidase and endo-galactanase. The results indicated that AGE-2c-I was a typical RG-I polysaccharide with side chains such as highly branched ${\alpha}$-arabinan, ${\beta}$-($1{\rightarrow}4$)-galactan and arabino-${\beta}$-3,6-galactan. To characterize the active moiety of AGE-2c-I, the anti-complementary activities of AGE-2c-I and its subfractions were assayed. It was observed that the anti-complementary activity of AGE-2c-I was due to the entire structure that resembled RG-I. In addition, arabino-${\beta}$-3,6-galactan side chain (GN-I) in AGE-2c-I probably plays a crucial role in the anti-complementary activity, whereas ${\alpha}$-arabinan side chain (AFN-I) consisting of 5-linked Araf and 3,5-branched Araf partially contributes to the activity.

• Microbiology and Biotechnology Letters
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• v.50 no.3
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• pp.395-403
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• 2022

In this study, we identified that the fermentation of Korean indigenous probiotics and red ginseng produced ginsenoside compound K (CK) from major ginsenosides. Based on whole genome sequencing of 19 probiotics species, β-glucosidase, α-arabinofuranosidase, β-xylosidase, and α-rhamnosidase related to bioconversion of ginsenosides are identified in the genome of 19 species, 3 species, 6 species, and 8 species, respectively. Among the 19 probiotics species, Bifidobacterium longum CBT BG7 converted from ginsenoside Rb1 to CK, and both B. breve CBT BR3 and B. lactis CBT BL3 converted ginsenoside Rb1 to Rd. The final concentration and yield of ginsenoside F2 and CK were higher in the fermentation with the nondisrupted cells than with disrupted cells. The combination of both CBT BG7 and BL3, and CBT BG7 and BR3 showed higher amounts of F2 than CBT BG7 only. CBT BG7 with adding α-amylase increased the amounts of F2. In this study, we identified that the fermentation of both Korean indigenous probiotic bacteria CBT BG7, BR3 and BL3, and red gingseng is able to produce CK, a bioactive compound that promotes health benefits.

• Journal of Microbiology and Biotechnology
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• v.17 no.3
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• pp.481-489
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• 2007

[ $\alpha$ ]-L-Arabinofuranosidases (AFases; EC 3.2.1.55) are exo-type enzymes, which hydrolyze terminal nonreducing arabinose residues from various polysaccharides such as arabinan and arabinoxylan. Genome-wide BLAST search showed that various bacterial strains possess the putative AFase genes with well-conserved motif sequences at the nucleotide and amino acid sequence levels. In this study, two sets of degenerate PCR primers were designed and tested to detect putative AFase genes, based on their three highly conserved amino acid blocks (PGGNFV, GNEMDG; and DEWNVW). Among 20 Bacillus-associated species, 13 species were revealed to have putative AFase genes in their genome and they share over 67% of amino acid identities with each other. Based on the partial sequence obtained from an isolate, an AFase from Geobacillus sp. was cloned and expressed in E. coli. Enzymatic characterization has verified that the resulting enzyme corresponds to a typical AFase. Accordingly, degenerate PCR primers developed in this work can be used for fast, easy, and specific detection and isolation of putative AFase genes from bacterial cells.

• Asian-Australasian Journal of Animal Sciences
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• v.20 no.5
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• pp.802-810
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• 2007

Fibrobacter succinogenes, a Gram-negative, anaerobic ruminal bacterium is a major fibre digesting species in the rumen. It intensively degrades plant cell walls by an erosion type of mechanism, burrowing its way through the complex matrix of cellulose and hemicellulose with the release of digestible and undigested cell wall fragments. The enzymes involved in this process include a combination of glucanases, xylanases, arabinofuranosidase(s) and esterases. The genome of the bacterium has been sequenced and this has revealed in excess of 100 putative glycosyl hydrolase, pectate lyase and carbohydrate esterase genes, which is greater than the numbers reported present in other major cellulolytic organisms for which genomes have been sequenced. Modelling of the amino acid sequences of two glycanases, CedA and EGB, by reference to crystallized homologs has enabled prediction of the major features of their tertiary structures. Two dimensional gel electrophoresis in conjunction with mass spectroscopy has permitted the documentation of proteins over expressed in F. succinogenes grown on cellulose, and analysis of the cell surfaces of mutant strains unable to bind to cellulose has enabled the identification of candidate proteins with roles in adhesion to the plant cell wall substrate, the precursor to cellulose biodegradation.

• Journal of Applied Biological Chemistry
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• v.48 no.1
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• pp.7-10
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• 2005

Trichoderma sp. SY most effectively produces an extracellular ${\gamma}$-L-arabinofuranosidase (AF) using arabinose as a carbon source. AF grown on cellulose as a carbon source was purified 28-fold with 4.4% yield by DEAE exchange and HQ/20 cation exchange chromatographies The purified enzyme was found to be homogeneous on SDS-PAGE with molecular weight of 89 kDa. It exhibited a high level of activity with p-nitrophenyl ${\alpha}$-L-arabinofuranoside, showing $K_m$ and $V_{max}$ values of $0.15\;{\mu}M$ and $239.85U{\cdot}mg^{-1}$, respectively and did not require any metal ion for activity. It also released p-nitrophenol from p-nitrophenol conjugated ${\beta}$-D-xylopyranoside, and ${\beta}$-D-galactopyranoside not from ${\beta}$-D-glucopyranoside.