• Journal of Microbiology and Biotechnology
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• v.10 no.6
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• pp.737-752
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• 2000

Wood-rotting basidiomycetous fungi are the most efficient degraders of lignin on earth. The white-rot fungus Phanerochaete chrysosporium has been used as a model microorganism in the study of enzymology and its application. Because of the ability of the white-rot fungus to degrade lignin, which has an irregular structure and large molecular mass, this fungus has also been studied in relation to degrading and mineralizing many environmental pollutants. The fungus includes an array of enzymes, such as lignin peroxidase (LiP), manganese-dependent peroxidase (MnP), cellobiose:quinone oxidoreductase, and $H_2O_2$-producing enzymes and also produces many other components of the ligninolytic system, such as veratryl alcohol (VA) and oxalate. In addition, the fungus has mechanisms for the reduction of degradation intermediates. The ligninolytic systems have been proved to provide reductive reactions as well as oxidative reactions, both of which are essential for the degradation of lignin and organopollutants. Further study on the white-rot fungus may provide many tools to both utilize lignin, the most abundant aromatic polymer, and bioremediate many recalcitrant organopollutants.

• Journal of Microbiology and Biotechnology
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• v.17 no.5
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• pp.745-752
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• 2007

A $\beta$-glucosidase from the algal lytic bacterium Sinorhizobium kostiense AFK-13, grown in complex media containing cellobiose, was purified to homogeneity by successive ammonium sulfate precipitation, and anion-exchange and gel-filtration chromatographies. The enzyme was shown to be a monomeric protein with an apparent molecular mass of 52 kDa and isoelectric point of approximately 5.4. It was optimally active at pH 6.0 and $40^{\circ}C$ and possessed a specific activity of 260.4 U/mg of protein against $4-nitrophenyl-\beta-D-glucopyranoside$(pNPG). A temperature-stability analysis demonstrated that the enzyme was unstable at $50^{\circ}C$ and above. The enzyme did not require divalent cations for activity, and its activity was significantly suppressed by $Hg^{+2}\;and\;Ag^+$, whereas sodium dodecyl sulfate(SDS) and Triton X-100 moderately inhibited the enzyme to under 70% of its initial activity. In an algal lytic activity analysis, the growth of cyanobacteria, such as Anabaena flos-aquae, A. cylindrica, A. macrospora, Oscillatoria sancta, and Microcystis aeruginosa, was strongly inhibited by a treatment of 20 ppm/disc or 30 ppm/disc concentration of the enzyme.

• Journal of Microbiology and Biotechnology
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• v.17 no.5
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• pp.800-805
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• 2007

Two endoglucanases with processive cellulase activities, produced from Fomitopsis palustris grown on 2% microcrystalline cellulose(Avicel), were purified to homogeneity by anion-exchange and gel filtration column chromatography systems. SDS-PAGE analysis indicated that the molecular masses of the purified enzymes were 47 kDa and 35 kDa, respectively. The amino acid sequence analysis of the 47-kDa protein(EG47) showed a sequence similarity with fungal glycoside hydrolase family 5 endoglucanase from the white-rot fungus Phanerochaete chrysosporium. N-terminal and internal amino acid sequences of the 35-kDa protein(EG35), however, had no homology with any other glycosylhydrolases, although the enzyme had high specific activity against carboxymethyl cellulose, which is a typical substrate for endoglucanases. The initial rate of Avicel hydrolysis by EG35 was relatively fast for 48 h, and the amount of soluble reducing sugar released after 96 h was $100{\mu}g/ml$. Although EG47 also hydrolyzed Avicel, the hydrolysis rate was lower than that of EG35. Thin layer chromatography analysis of the hydrolysis products released from Avicel indicated that the main product was cellobiose, suggesting that the brown-rot fungus possesses processive EGs capable of degrading crystalline cellulose.

• Bulletin of the Korean Chemical Society
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• v.15 no.9
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• pp.719-724
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• 1994

Major cellulase components, such as three endoglucanases (endoglucanases I, II, and III) and one exoglucanase (exoglucanase II), were isolated from a commercial cellulase (Meicelase TP 60) derived from the fungus Trichoderma viride by a series of chromatography procedures. These procedures were the gel filtration on Bio-Gel, the anion exchange on DEAE-Bio-Gel A, the cation exchange on SP-Sephadex C50, and the affinity chromatography on Avicel cellulose. The average molecular weights determined by SDS-polyacrylamide gel electrophoretic analysis were 51,000, 59,000, 41,000 and 62,000 Da for endoglucanases I, II and III and exoglucanase II, respectively. The extinction coefficients, ${\varepsilon}^{1%}$ 280 nm, of these enzymes were 11.7, 3.3, 7.2 and 11.3, respectively. Among them, the endoglucanase II showed the very low value of the coefficient compared with the others. On the other hand, it was found that endoglucanase II and III were of more random hydrolytic mode on carboxymethylcellulose as compared with those of endoglucanase I and exoglucanase II. Especially, endoglucanase I showed less random action than that of exoglucanase II. In the hydrolysis of insoluble cellulose by the enzyme components, cellobiose was the major product, but glucose was the major product by endoglucanase III.

• Journal of Microbiology and Biotechnology
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• v.17 no.8
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• pp.1291-1299
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• 2007

Two ${\beta}$-1,4-glucanases (DI and DIII fractions) were purified to homogeneity from the culture filtrate of a cellulolytic bacteria, Cellulomonas sp. CS 1-1, which was classified as a novel species belonging to Cellulomonas uda based on chemotaxanomic and phylogenetic analyses. The molecular mass was estimated as 50,000 Da and 52,000 Da for DI and DIII, respectively. Moreover, DIII was identified as a glycoprotein with a pI of 3.8, and DI was identified as a non-glycoprotein with a pI of 5.3. When comparing the ratio of the CMC-saccharifying activity and CMC-liquefying activity, DI exhibited a steep slope, characteristic of an endoglucanase, whereas DIII exhibited a low slope, characteristic of an exoglucanase. The substrate specificity of the purified enzymes revealed that DI efficiently hydrolyzed CMC as well as xylan, whereas DIII exhibited a high activity on microcrystalline celluloses, such as Sigmacells. A comparison of the hydrolysis patterns for pNP-glucosides (DP 2-5) using an HPLC analysis demonstrated that the halosidic bond 3 from the nonreducing end was the preferential cleavage site for DI, whereas bond 2, from which the cellobiose unit is split off, was the preferential cleavage site for DIII. The partial N-terminal amino acid sequences for the purified enzymes were $^1Ala-Gly-Ser-Thr-Leu-Gln-Ala-Ala-Ala-Ser-Glu-Ser-Gly-Arg-Tyr^{15}$-for DI and $^1Ala-Asp-Ser-Asp-Phe-Asn-Leu-Tyr-Val-Ala-Glu-Asn-Ala-Met-Lys^{15}$-for DIII. The apparent sequences exhibited high sequence similarities with other bacterial ${\beta}$-1,4-glucanases as well as ${\beta}$-1,4-xylanases.

• Journal of Microbiology and Biotechnology
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• v.4 no.2
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• pp.102-107
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• 1994

The $\beta$-1,4-endoglucanase gene from Actinomyces sp. 40 was cloned into Escherichia coli DH5$\alpha$ with pUC19. Chromosomal DNA from Actinomyces sp. 40 was cleaved with the restriction enzyme Sau3AI and ligated into pUC19 for the transformation of Escherichia coli DH5$\alpha$. Positive clones of $\beta$-1,4-endoglucanase gene were detected as the clear zones on a medium supplemented with carboxymethylcellulose (CMC). This transformant possessed a single plasmid, designated pDS1, which contained the vector DNA and a 3.5 kilobase (kb) Sau3AI insertion fragment encoding endoglucanase. The size of the cloned fragment was reduced to 2.0 kb. The endoglucanase activity produced by the E. coli DH5$\alpha$ (pDS6) was higher than that of Actinomyces sp. 40 strain. The optimum pH and temperature of the cloned enzyme were pH 4.0$\sim$5.0 and 55$^{\circ}C$, respectively. The cloned enzyme was stable at 55$^{\circ}C$ or below and in buffer ranging from pH 4.0 to 7.0. The enzyme degraded CMC but did not degrade xylan, cellobiose, and methyl-umbelliferylcellobiopyranoside (MUC).

• Korean Journal of Microbiology
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• v.11 no.4
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• pp.157-166
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• 1973

Fermented feed using rice, barley, wheat, and defatted rice brans as the raw materials were prepared by 3 species of wild yeasts which were selected among 35 strains of yeasts isolated, and their analytical values were examined. The results were as follows : 1. The three yeasts were identified as H.amomala var. anomala (No.225), Candida utilis (No.400), and Irpex-cellulase(consors) (no.403-A). 2. The optimum pH, and sugar concentration of these yeasts in liquid culture were pH 5.0 and Bllg. 10.deg. each. The optimum temperature was 30.deg.C for No.225 and No.403-A, 25.deg.C for No.400. The No.225 and No.403-A grow at higher temperature than 37.deg.C and 40.deg.C each. 3. The No.225 yeast had a large vegetative cell and strong sugar fermentability. The No.225 and 403-A could assimilate cellobiose, xylose, $KNO_2$ and $KNO_3$. These properties were fit for bran fermentation. 4. The No.403-A microorganism was a yeast-like microbe and showed cellulase activity which might help the propagation of other yeasts on the brans. 5. The analytical data of fermented feed indicated the following order of usable value ; rice-wheat-barley bran 4:4:2, rice-wheat bran 5:5, rice-barley bran 5:5, rice-defatted rice bran 5:5. 6. the fermented feed were prepared by mixing brans, 0.3% ammonium sulfate and 5%(w/w) inoculum of yeast suspension in 4% glucose solution. Water content 70-80%, fermentation temperature 25-30.deg.C, and fermentation time 2-3 days were given. 7. The rice-wheat bran 5:5 and rice-barley bran 5:5 fermented feed showed 11, 17-11.45% protein increase, and the rice-barley-wheat bran 4:4:2 and rice-defatted bran 5:5 showed 3.75-6.03% protein increase. 8. The fermented feed prepared in this experiment by the author might work as a nutritive feed using microbial cell body, enzymes produced by microbes and other microbial cell constituents.

• Korean Journal of Microbiology
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• v.29 no.2
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• pp.85-91
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• 1991

.betha.-1,4-D-Glucan glucanohydrolase(EC 3.2.1.4;F-II-IV) purified from Trichoderma koningii was identified as a glycoprotein containing 9% carbohydrate. Isoelectric point of the enzyme was estimated to be 4.9 and molecular weight was determined to be approximately 58,000. The porducts of p-nitrophenyl-cellobioside ($PNPG_{2}$) catalyzed by the enzyme were p-nitrophenol(PNP) and p-nitrophenyl-glucoside($PNPG_{1}$). The Km value for $PNPG_{2}$ was estimated to be 0.97 mM in case of the holoside lindage and 10.4 mM in case of the aglycon linkage and their kcat values were $1.8*10^{5}$$ min^{-1}$ and $7.5*10^{5}$ $min^{-1}$ respectively. The product of p-nitrophenyl cellotriose($PNPG_{3}$) was only $PNPG_{1}$. The Km value for $PNPG_{3}$ was 69.5 .$\mu$M and kcat was $1*10^{8}$ $min^{-1}$ which implicates that the enzyme have higher affinity and higher hydrolysis rate toward $PNPG_{3}$ than toward $PNPG_{2}$. The enzyme showed its optimal activity at pH 4.0-4.5 and at 60.deg.C. The effect of gluconolactone on the activity toward $PNPG_{2}$ showed competitive inhibition pattern but glucose and cellobiose did not. The enzyme contained a high content of acidic and hydroxylated amino acids in contrast to basic amino acids.

• Journal of Microbiology and Biotechnology
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• v.17 no.3
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• pp.454-460
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• 2007

Enzymatic properties and substrate specificity of recombinant $\beta-glycosidases$ from a hyperthermophilic archaeon, Sulfolobus shibatae (rSSG), were analyzed. rSSG showed its optimum temperature and pH at $95^{\circ}C$ and pH 5.0, respectively. Thermal inactivation of rSSG showed that its half-life of enzymatic activity at $75^{\circ}C$ was 15 h whereas it drastically decreased to 3.9 min at $95^{\circ}C$. The addition of 10 mM of $MnCl_2$ enhanced the hydrolysis activity of rSSG up to 23% whereas most metal ions did not show any considerable effect. Dithiothreitol (DTT) and 2-mercaptoethanol exhibited significant influence on the increase of the hydrolysis activity of rSSG rSSG apparently preferred laminaribiose $(\beta1\rightarrow3Glc)$, followed by sophorose $(\beta1\rightarrow2Glc)$, gentiobiose $(\beta1\rightarrow6Glc)$, and cellobiose $(\beta1\rightarrow4Glc)$. Various. intermolecular transfer products were formed by rSSG in the lactose reaction, indicating that rSSG prefers lactose as a good acceptor as well as a donor. The strong intermolecular transglycosylation activity of rSSG can be applied in making functional oligosaccharides.

• Journal of Microbiology and Biotechnology
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• v.17 no.6
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• pp.905-912
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• 2007

A novel ${\beta}-glucosidase$ gene, bglA, was isolated from uncultured soil bacteria and characterized. Using genomic libraries constructed from soil DNA, a gene encoding a protein that hydrolyzes a fluorogenic analog of cellulose, 4-methylumbelliferyl ${\beta}-D-cellobioside$ (MUC), was isolated using a microtiter plate assay. The gene, bglA, was sequenced using a shotgun approach, and expressed in E. coli. The deduced 55-kDa amino acid sequence for bglA showed a 56% identity with the family 1 glycosyl hydrolase Chloroflexus aurantiacus. BglA included two conserved family 1 glycosyl hydrolase regions. When using $p-nitrophenyl-{\beta}-D-glucoside$ (pNPG) as the substrate, the maximum activity of the purified ${\beta}-glucosidase$ exhibited at pH 6.5 and $55^{\circ}C$, and was enhanced in the presence of $Mn^{2+}$. The $K_m\;and\;V_{max}$ values for the purified enzyme with pNPG were 0.16 mM and $19.10{\mu}mol/min$, respectively. The purified BglA enzyme hydrolyzed both pNPG and $p-nitrophenyl-{\beta}-D-fucoside$. The enzyme also exhibited substantial glycosyl hydrolase activities with natural glycosyl substrates, such as sophorose, cellobiose, cellotriose, cellotetraose, and cellopentaose, yet low hydrolytic activities with gentiobiose, salicin, and arbutin. Moreover, BglA was able to convert the major ginsenoside $Rb_1$ into the pharmaceutically active minor ginsenoside Rd within 24 h.