• Korean Journal of Microbiology
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• v.51 no.1
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• pp.68-74
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• 2015

Based on the genomic analysis of Thermococcus pacificus P-4, we identified a putative GH1 ${\beta}$-glucosidase-encoding gene (Tpa-glu). The gene revealed a 1,464 bp encoding 487 amino acid residues, and the deduced amino acid residues exhibited 77% identity with Pyrococcus furiosus ${\beta}$-glucosidase (accession no. NP_577802). The gene was cloned and expressed in Escherichia coli system. The recombinant protein was purified by metal affinity chromatography and characterized. Tpa-Glu showed optimum activity at pH 7.5 and $75^{\circ}C$, and thermostability with a half life of 6 h at $90^{\circ}C$. Tpa-Glu exhibited hydrolyzing activity against various pNP-glycopyranosides, with kcat/Km values in the order of pNP-${\beta}$-glucopyranoside, pNP-${\beta}$-galactopyranoside, pNP-${\beta}$-mannopyranoside, and pNP-${\beta}$-xylopyranoside. In addition, the enzyme exhibited exo-hydrolyzing activity toward ${\beta}$-1,3-linked polysaccharide (laminarin) and ${\beta}$-1,3- and ${\beta}$-1,4-linked oligosaccharides. This is the first description of an enzyme from hyperthermophilic archaea that displays exo-hydrolyzing activity toward ${\beta}$-1,3-linked polysaccharides and could be applied in combination with ${\beta}$-1,3-endoglucanase for saccharification of laminarin.

• 한국생물공학회:학술대회논문집
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• 2000.04a
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• pp.347-350
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• 2000

Enzymatic deinking of office-waste paper was studied using crude cellulase and papain-hydrolyzed cellulase from Trichoderma reesei Rut C-30 in small-scale and mid-scale. The results were compared with deinkings using commercial enzyme(Novozym 342) and conventional chemical methods. Maximum brightness and freeness were obtained at 3 units/g Oven Dry Paper(ODP) of CMCase activity using crude cellulase in mid-scale deinking experiments. The deinked pulp had higher physical strength and brightness, and lower freeness and yield than the pulp deinked in small scale. In small scale deinking, maximum brightness and freeness were obtained at 2 unit/g ODP. Deinking by papain-hydrolyzed cellulase showed similar results with one by Novozym 342. It was better in brightness and freeness, but showed lower physical strength and yield, than the conventional deinking by sodium hydroxide. The ratio of endo-1,4-glucanase and exo-1,4-glucanase components in papain hydrolyzed cellulase from T. reesei Rut C-30 was similar to that of commercial enzyme, Novozym 342, implicating a successful application as a deinking enzyme.

• Microbiology and Biotechnology Letters
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• v.24 no.5
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• pp.567-573
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• 1996

Chitinase (EC 3.2.1.14) from culture fluid of Bacillus licheniformis KFB-C14 was purified 66-folds to homogenity in overall yield of 21% by ammonium sulfate fractionation, DEAE-Toyopearl, Butyl-Toyopearl and TSK-Gel HW-55F column chromatography. The enzyme protein had a molecular weight of about 86,000 and was composed of one subunit. The enzyme was significantly stable not only at high temperature but also on treatment with organic solvents and protein denaturants such as SDS, urea and guanidine-HC1. The optimum temperature and pH for reaction was 60$\circ $C and 6.0, respectively. The enzyme activity was inhibited by only Mn$^{2+}$ ion, but not inhibited by EDTA, N- ethylmaleimide and pCMB. The enzyme had high activity with colloidal chitin (V$_{max}$: 421) and commercial chitin (V$_{max}$: 480), but not with typical substrates of exo type chitinase. The thermostable chitinase had an useful reactivity for producing functional chitooligosaccharide, showing the production of (GlcNAc)$_{1}, (GlcNAc)$_{3}$, and (GlcNAc)$_{2}$ as major product.

• Food Science and Preservation
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• v.21 no.3
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• pp.442-450
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• 2014

In this study, we isolated a cellulase-producing bacterium isolated from traditional Korean fermented soybean paste and investigated the effect of culture conditions on the production of cellulase. This bacterium, which was identified as Bacillus subtilis 4-1 through 16S rRNA gene sequence analysis, showed the highest cellulase activity when the cells were grown at $45^{\circ}C$ for 24 hours in the CMC medium supplemented with 1.0% of soluble starch and 0.1% yeast extract. The initial optimum pH of the medium was observed in the range of 5.0~9.0. The optimal pH and temperature for the production of cellulase from B. subtilis 4-1 were pH 9.0 and $60^{\circ}C$ respectively. In addition, the enzyme showed significant activity in the temperature range of $20{\sim}90^{\circ}C$, which indicates that B. subtilis 4-1 cellulase is an alkaline-resistance and thermo-stable enzyme. This enzyme showed higher activity with CMC as the substrate for endo-type cellulase than avicel or pNPG as the exo-type substrates for exo-type cellulase and ${\beta}$-glucosidase. These results suggest that the cellulase produced from B. subtilis 4-1 is a complex enzyme rather than a mono-enzyme.

• Korean Journal Plant Pathology
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• v.13 no.4
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• pp.225-231
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• 1997

Activities of polygalacturonase, laccase, and intra- and extra-cellular $\beta$-glucosidase produced by 20 Botrytis cinerea isolates in liquid culture media containing cucumber cell was as a carbon source were measured and their relationships to the pathogenicity were analyzed. No significant correlations between these enzyme activities and the pathogenicity of B. cinerea were found. Mycelial growth rate on Bayendamm media, however, was higthly correlated with the pathogenicity (r=0.522) anong these isolates. Immuno-blot analysis of the culture filtrate using antibody against against exo-polygalacturonase revealed that only one band with molecular weight of 66 kDa was detected amone 34 tested isolates. It appears that these enzymes may not be primary factors in dermining the pathogenicity of B. cinerea.

• Microbiology and Biotechnology Letters
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• v.17 no.4
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• pp.289-294
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• 1989

A bacterial strain capable of producing high level of extracellular xylanase was isolated from soil. The characteristics of the isolated strain No.236 were identified to be Bacillus stearothermophilus. The maximal xylanase production was observed in the medium containing 0.75% xylan, 0.35% yeast extract, 1.06% $K_2$HPO$_4$and 0.05% CaCO$_3$with initial pH of 6.5 when the strain was cultured at 5$0^{\circ}C$ for 28 hrs with reciprocal shaking. Hydrolysis of xylan by the xylanase revealed that xylose was the only product of the reaction. This suggested that the enzyme produced by Bacillus stearothermophilus No. 236 was an exe-acting xylanase.

• Microbiology and Biotechnology Letters
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• v.22 no.3
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• pp.283-289
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• 1994

The production of maltose utilizing swollen extrusion starch seems to have many technical advantages, such as, high reaction rate and high yield, production of high purity concentrated maltose, and low energy consumption, over the conventional method utilizing liquefied starch. The characteristics of maltose formation in heterogeneous enzyme reaction system comtaining swollen extrusion starch was investigated using fungal $\alpha $-amylase. The influence of extrusion conditions on structure of extruded starch, such as, degree of gelatinization, water absorption index, and water solubility index was analyzed. The relationship between the structural features and maltose forming reaction was investigated, and the result was analyzed in terms of surface reaction of insoluble extruded swollen starch. The characteristics of maltose formation from swollen sxtrusion starch was compared using endo-type fungal $\alpha $-amylase and exo-type $\beta $anylase, and the structural trasformation of extruded starch was also observed to clarify the reaction mechanism.

• Journal of Microbiology and Biotechnology
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• v.32 no.2
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• pp.187-194
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• 2022

Two α-ʟ-arabinofuranosidases (BfdABF1 and BfdABF3) and a β-ᴅ-xylosidase (BfdXYL2) genes were cloned from Bifidobacterium dentium ATCC 27679, and functionally expressed in E. coli BL21(DE3). BfdABF1 showed the highest activity in 50 mM sodium acetate buffer at pH 5.0 and 25℃. This exo-enzyme could hydrolyze p-nitrophenyl arabinofuranoside, arabino-oligosaccharides (AOS), arabinoxylo-oligosaccharides (AXOS) such as 3²-α-ʟ-arabinofuranosyl-xylobiose (A³X), and 2³-α-ʟ-arabinofuranosyl-xylotriose (A²XX), whereas hardly hydrolyzed polymeric substrates such as debranched arabinan and arabinoxylans. BfdABF1 is a typical exo-ABF with the higher specific activity on the oligomeric substrates than the polymers. It prefers to α-(1,2)-ʟ-arabinofuranosidic linkages compared to α-(1,3)-linkages. Especially, BfdABF1 could slowly hydrolyze 2³,3³-di-α-ʟ-arabinofuranosyl-xylotriose (A²⁺³XX). Meanwhile, BfdABF3 showed the highest activity in sodium acetate at pH 6.0 and 50℃, and it has the exclusively high activities on AXOS such as A³X and A²XX. BfdABF3 mainly catalyzes the removal of ʟ-arabinose side chains from various AXOS. BfdXYL2 exhibited the highest activity in sodium citrate at pH 5.0 and 55℃, and it specifically hydrolyzed p-nitrophenyl xylopyranoside and xylo-oligosaccharides (XOS). Also, BfdXYL2 could slowly hydrolyze AOS and AXOS such as A³X. Based on the detailed hydrolytic modes of action of three exo-hydrolases (BfdABF1, BfdABF3, and BfdXYL2) from Bf. dentium, their probable roles in the hemiceullose-utilization system of Bf. dentium are proposed in the present study. These intracellular exo-hydrolases can synergistically produce ʟ-arabinose and ᴅ-xylose from various AOS, XOS, and AXOS.

• Microbiology and Biotechnology Letters
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• v.18 no.3
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• pp.260-267
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• 1990

The mechanism of enzymatic hydrolysis of raw corn starch by the purified glucoamylase and a - amylase in an agitated bead reaction system was studied by investigating the changes of sugar profiles produced by each enzyme, the granular structure of raw corn starch, the amount of enzyme adsorption on residual starch, and the amylose content in residual raw starch. The sugar profiles produced by the action of exo-type glucoamylase or endo-type $\alpha$ -amylase in an agitated bead system were not recognizably differed with those produced in reaction system without bead. Without enzyme the intergenic microcrystalline structure of starch granule was not changed by the simple mechanical impact of solid media, but it was cleaved. However, starch granule was fragment into large number of small particles by the synergistic action of enzyme and attrition-milling media, identified to be the major saccharification enhancing mechanism along with the increased amount of enzyme adsorption. The amylose content decreased more readily in an agitated bead reaction system, especially by $\alpha$ -amylase.

• Food Science and Biotechnology
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• v.16 no.3
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• pp.482-484
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• 2007

To investigate production and hydrolysis of levan, the levansucrase enzymes from Zymomonas mobilis ATCC 10988 and Rahnella aquatilis ATCC 33071 were used. The optimum temperature of R. aquatilis levansucrase for levan formation was $37^{\circ}C$, whereas that of Z. mobilis was $4^{\circ}C$, under the experimental conditions. Both levansucrases also catalyzed the reverse levan hydrolysis reaction. Levan hydrolysis reactions from both levansucrases were temperature dependent; high temperature ($20^{\circ}C$) was more favorable than low temperature ($4^{\circ}C$) by 4 times. Fructose was the only product from hydrolysis reaction by both levansucrases, showing that both levansucrases mediated the hydrolysis reaction of exo-enzyme acting. In both enzymes, initial levan hydrolysis activity was almost accounted to 1% of initial levan formation activity. The results allow the estimation of the fructose release rate in enzyme processing conditions.