• Journal of Microbiology and Biotechnology
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• v.24 no.2
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• pp.236-244
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• 2014

The gene encoding ${\alpha}-\small{L}$-arabinofuranosidase (AFase) from Paenibacillus sp. DG-22 was cloned, sequenced, and expressed in Escherichia coli. The AFase gene (abfA) comprises a 1,509 bp open reading frame encoding 502 amino acids with a molecular mass of 56,520 daltons. The deduced amino acid sequence of the gene shows that AbfA is an enzyme consisting of only a catalytic domain, and that the enzyme has significant similarity to AFases classified into the family 51 of the glycosyl hydrolases. abfA was subcloned into the pQE60 expression vector to fuse it with a six-histidine tag and the recombinant AFase (rAbfA) was purified to homogeneity. The specific activity of the recombinant enzyme was 96.7 U/mg protein. Determination of the apparent molecular mass by gel-filtration chromatography indicated that AbfA has a tetrameric structure. The optimal pH and temperature of the enzyme were 6.0 and $60^{\circ}C$, respectively. The enzyme activity was completely inhibited by 1 mM $HgCl_2$. rAbfA was active only towards p-nitrophephenyl ${\alpha}-\small{L}$-arabinofuranoside and exhibited $K_m$ and $V_{max}$ values of 3.5 mM and 306.1 U/mg, respectively. rAbfA showed a synergistic effect in combination with endoxylanase on the degradation of oat spelt xylan and wheat arabinoxylan.

• BMB Reports
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• v.45 no.12
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• pp.748-753
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• 2012

A sequence of 10 amino acids at the C-terminus region of methylglyoxal synthase from Escherichia coli (EMGS) provides an arginine, which plays a crucial role in forming a salt bridge with a proximal aspartate residue in the neighboring subunit, consequently transferring the allosteric signal between subunits. In order to verify the role of arginine, the gene encoding MGS from a thermophile species, Thermus sp. GH5 (TMGS) lacking this arginine was cloned with an additional 30 bp sequence at the 3'-end and then expressed in form of a fusion TMGS with a 10 residual segment at the C-terminus ($TMGS^+$). The resulting recombinant enzyme showed a significant increase in cooperativity towards phosphate, reflected by a change in the Hill coefficient (nH) from 1.5 to 1.99. Experiments including site directed mutagenesis for Asp-10 in TMGS and $TMGS^+$, two dimentional structural survey, fluorescence and irreversible thermoinactivation were carried out to confirm this pathway.

• Journal of Microbiology and Biotechnology
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• v.27 no.4
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• pp.775-784
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• 2017

A neutral xylanase (CcXyn) was identified from Coprinus cinereus. It has a single GH10 catalytic domain with a basic amino acid-rich extension (PVRRK) at the C-terminus. In this study, the wild-type (CcXyn) and C-terminus-truncated xylanase ($CcXyn-{\Delta}5C$) were heterologously expressed in Pichia pastoris and their characteristics were comparatively analyzed with aims to examine the effect of this extension on the enzyme function. The circular dichorism analysis indicated that both enzymes in general had a similar structure, but $CcXyn-{\Delta}5C$ contained less ${\alpha}-helices$ (42.9%) and more random coil contents (35.5%) than CcXyn (47.0% and 32.8%, respectively). Both enzymes had the same pH (7.0) and temperature ($45^{\circ}C$) optima, and similar substrate specificity on different xylans. They all hydrolyzed beechwood xylan primarily to xylobiose and xylotriose. The amounts of xylobiose and xylotriose accounted for 91.5% and 92.2% (w/w) of total xylooligosaccharides (XOS) generated from beechwood by CcXyn and $CcXyn-{\Delta}5C$, respectively. However, truncation of the C-terminal 5-amino-acids extension significantly improved the thermostability, SDS resistance, and pH stability at pH 6.0-9.0. Furthermore, $CcXyn-{\Delta}5C$ exhibited a much lower $K_m$ value than CcXyn (0.27 mg/ml vs 0.83 mg/ml), and therefore, the catalytic efficiency of $CcXyn-{\Delta}5C$ was 2.4-times higher than that of CcXyn. These properties make $CcXyn-{\Delta}5C$ a good model for the structure-function study of $({\alpha}/{\beta})_8$-barrel-folded enzymes and a promising candidate for various applications, especially in the detergent industry and XOS production.

• Journal of Microbiology and Biotechnology
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• v.24 no.11
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• pp.1525-1535
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• 2014

The xynC gene, which encodes high molecular weight xylanase from Paenibacillus sp. DG-22, was cloned and expressed in Escherichia coli, and its nucleotide sequence was determined. The xynC gene comprised a 4,419bp open reading frame encoding 1,472 amino acid residues, including a 27 amino acid signal sequence. Sequence analysis indicated that XynC is a multidomain enzyme composed of two family 4_9 carbohydrate-binding modules (CBMs), a catalytic domain of family 10 glycosyl hydrolases, a family 9 CBM, and three S-layer homologous domains. Recombinant XynC was purified to homogeneity by heat treatment, followed by Avicel affinity chromatography. SDS-PAGE and zymogram analysis of the purified enzyme identified three active truncated xylanase species. Protein sequencing of these truncated proteins showed that all had identical N-terminal sequences. In the protein characterization, recombinant XynC exhibited optimal activity at pH 6.5 and $65^{\circ}C$ and remained stable at neutral to alkaline pH (pH 6.0-10.0). The xylanase activity of recombinant XynC was strongly inhibited by 1 mM $Cu^{2+}$ and $Hg^{2+}$, whereas it was noticeably enhanced by 10 mM dithiothreitol. The enzyme exhibited strong activity towards xylans, including beechwood xylan and arabinoxylan, whereas it showed no cellulase activity. The hydrolyzed product patterns of birchwood xylan and xylooligosaccharides by thin-layer chromatography confirmed XynC as an endoxylanase.

• KSBB Journal
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• v.15 no.1
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• pp.42-48
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• 2000

Urokinase (UK), a thrombolytic enzyme used to clear catheters obstructed by blood clots, can be also used industrially in the recombinant protein purification system to cleave a fusion protein linked with a certain fragment of GST. We have immobilized UK by covalent attachment to activated Sepharose 6B-Cl gels and evaluated its performance to cleave a fusion protein of hGH and GST. The Sepharose gels were activated by etherification with glycidol (2,3-epoxypropanol) and further oxidized with periodate resulting in glyceryl-Sepharose gels. After the activation treatment, surface density of the aldehyde groups was 7-30 $\mu$mol-aldehde/mL-gel. Immobilization yield was higher than 99% at high pH (10.5), and the immobilized UK maintained ca. 80% specific activity of the soluble UK. In a column reaction the cleavage yield heavily depended on the feed rate, and it was nearly 86% of that from soluble UK. And the immobilized UK was successfully regenerated by unfolding and refolding with 6M GuHCl. After cleavaging reaction, the monomeric hGH was purified by using expanded bed adsorption chromatography.

• Journal of Microbiology and Biotechnology
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• v.31 no.10
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• pp.1446-1454
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• 2021

Herein, we cloned and expressed an endo-β-1,4-glucanase gene (celA1805) from Bacillus subtilis B111 in Escherichia coli. The recombinant celA1805 contains a glycosyl hydrolase (GH) family 8 domain and shared 76.8% identity with endo-1,4-β-glucanase from Bacillus sp. KSM-330. Results showed that the optimal pH and temperature of celA1805 were 6.0 and 50℃, respectively, and it was stable at pH 3-9 and temperature ≤50℃. Metal ions slightly affected enzyme activity, but chemical agents generally inhibited enzyme activity. Moreover, celA1805 showed a wide substrate specificity to CMC, barley β-glucan, lichenin, chitosan, PASC and avicel. The K_m and V_max values of celA1805 were 1.78 mg/ml and 50.09 µmol/min/mg. When incubated with cellooligosaccharides ranging from cellotriose to cellopentose, celA1805 mainly hydrolyzed cellotetrose (G4) and cellopentose (G5) to cellose (G2) and cellotriose (G3), but hardly hydrolyzed cellotriose. The concentrations of reducing sugars saccharified by celA1805 from wheat straw, rape straw, rice straw, peanut straw, and corn straw were increased by 0.21, 0.51, 0.26, 0.36, and 0.66 mg/ml, respectively. The results obtained in this study suggest potential applications of celA1805 in biomass saccharification.

• Journal of Microbiology and Biotechnology
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• v.28 no.5
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• pp.776-783
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• 2018

The agarase gene gaa16a was identified from a draft genome sequence of Gilvimarinus agarilyticus JEA5, an agar-utilizing marine bacterium. Recently, three agarase-producing bacteria, G. chinensis, G. polysaccharolyticus, and G. agarilyticus, in the genus Gilvimarinus were reported. However, there have been no reports of the molecular characteristics and biochemical properties of these agarases. In this study, we analyzed the molecular characteristics and biochemical properties of agarases in Gilvimarinus. Gaa16A comprised a 1,323-bp open reading frame encoding 441 amino acids. The predicted molecular mass and isoelectric point were 49 kDa and 4.9, respectively. The amino acid sequence of Gaa16A showed features typical of glycosyl hydrolase family 16 (GH16) ${\beta}$-agarases, including a GH16 domain, carbohydrate-binding region (RICIN domain), and signal peptide. Recombinant Gaa16A (excluding the signal peptide and carbohydrate-binding region, rGaa16A) was expressed as a fused protein with maltose-binding protein at its N-terminus in Escherichia coli. rGaa16A had maximum activity at $55^{\circ}C$ and pH 7.0 and 103 U/mg of specific activity in the presence of 2.5 mM $CaCl_2$. The enzyme hydrolyzed agarose to yield neoagarotetraose as the main product. This enzyme may be useful for industrial production of functional neoagaro-oligosaccharides.

• Journal of Microbiology and Biotechnology
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• v.32 no.4
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• pp.447-457
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• 2022

Notoginsenoside R1 and ginsenoside Rg1 are the main active ingredients of Panax notoginseng, exhibiting anti-fatigue, anti-tumor, anti-inflammatory, and other activities. In a previous study, a GH39 β-xylosidase Xln-DT was responsible for the bioconversion of saponin, a natural active substance with a xylose group, with high selectivity for cleaving the outer xylose moiety of notoginsenoside R1 at the C-6 position, producing ginsenoside Rg1 with potent anti-fatigue activity. The optimal bioconversion temperature, pH, and enzyme dosage were obtained by optimizing the transformation conditions. Under optimal conditions (pH 6.0, 75℃, enzyme dosage 1.0 U/ml), 1.0 g/l of notoginsenoside R1 was converted into 0.86 g/l of ginsenoside Rg1 within 30 min, with a molar conversion rate of approximately 100%. Furthermore, the in vivo anti-fatigue activity of notoginsenoside R1 and ginsenoside Rg1 were compared using a suitable rat model. Compared with the control group, the forced swimming time to exhaustion was prolonged in mice by 17.3% in the Rg1 high group (20 mg/kg·d). Additionally, the levels of hepatic glycogen (69.9-83.3% increase) and muscle glycogen (36.9-93.6% increase) were increased. In the Rg1 group, hemoglobin levels were also distinctly increased by treatment concentrations. Our findings indicate that treatment with ginsenoside Rg1 enhances the anti-fatigue effects. In this study, we reveal a GH39 β-xylosidase displaying excellent hydrolytic activity to produce ginsenoside Rg1 in the pharmaceutical and food industries.

• Journal of Life Science
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• v.22 no.2
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• pp.266-280
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• 2012

Agar is a cell wall component of macro red algae that can be hydrolyzed by agarase. Agarases are classified into ${\alpha}$-agarase (E.C. 3.2.1.158) and ${\beta}$-agarase (E.C. 3.2.1.81), in accordance with their cleavage pattern, and can be grouped in the glycoside hydrolase (GH)-16, -58, -86, -96, and -118 family according to the amino acid sequences of the proteins. Many agarases and/or their genes have been detected, isolated, and recombinantly expressed from bacteria, and metagenomes have their origins in sea and terrestrial environments. Products of agarases, agarooligosaccharides and neoagarooligosaccharides, represent wide functions such as antitumor, immune stimulation, antioxidation, prebiotic, hepa-protective, antibacterial, whitening, and moisturizing effects; hence, broad applications would be possible in the food industry, cosmetics, and medical fields. In addition, agarases are also used as a tool enzyme for research. This paper reviews the sources, purifications and detection methods, and application fields of agarases. The role of agarases in agar metabolism and the function of their enzymatic products are also surveyed.

• Journal of the Korean Wood Science and Technology
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• v.46 no.6
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• pp.670-680
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• 2018

Acanthophysium sp. KMF001, a wood rotting fungus, produces a strong crude enzyme complex that efficiently produces simple sugars from wood. The transcriptomic analysis of Acanthophysium sp. KMF001 identified 14 genes for putative glycoside hydrolases. Among them, isotig01043 was expressed heterogeneously in Escherichia coli BL21(DE3), and the expressed protein exhibited an endo-${\beta}$-1,4-xylanase activity which showed the optimum reaction at pH 5.0 and $30^{\circ}C$. The enzyme kinetic values of $K_m$ and $V_{max}$ were 25.92 mg/ml and $0.628{\mu}mole/mg/ml$, respectively. The enzymatic characteristics of the expressed xylanase showed a typical fungal xylanase. However, the bioinformatics analysis suggested that the protein encoded by isotig01043 was a novel xylanase based on a low identity when it was compared with the closest protein in the NCBI database and a similar protein domain with GH16_fungal_Lam16A_glucanase, which had not been earlier suggested as a xylanase.