• Journal of the Korea Academia-Industrial cooperation Society
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• v.15 no.8
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• pp.5412-5418
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• 2014

The red seabream iridovirus (RSIV), which belongs to the iridoviridae, causes infectious fish diseases in many Asian countries, leading to considerable economic losses to the aquaculture industry. Using the yeast surface display (YSD) technique, a new experimental system was recently developed for the detection and identification of a variety of marine viruses. In this study, a coat protein gene of RSIV was synthesized based on the nucleotide sequence database and subcloned into the yeast expression vector, pCTCON2. The expression of viral coat proteins in the yeast strain, EBY100, was detected by flow cytometry and Western blot analysis. Finally, they were isolated from the yeast surface through a treatment with ${\beta}$-mercaptoethanol. The data suggests that the YSD system can be a useful method for acquiring coating proteins of marine viruses.

• Korean Journal of Food Science and Technology
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• v.29 no.5
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• pp.1021-1027
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• 1997

We examined some properties of yeast cell wall lytic enzyme produced by Dicyma sp. YCH-37. Several metal ions, reducing reagents, and chemical modifiers have little effects on the lytic activity, except guanidine-HCl. Yeast cells of early log phase were more susceptible to the enzyme than those of stationary phase, and heat-treated cells were more easily lysed than intact living ones. Yeast cells pretreated with organic solvents such as butanol and acetone were more susceptible to the enzyme than intact living ones. Yeast cells cultured in Yeast extract-Malt extract medium containing 0.5 M ammonium sulfate were easily lysed by the lytic enzyme, and yeast cells cultured without shaking were more easily lysed by the enzyme than those with shaking. When SDS, ${\beta}-mercaptoethanol$, Triton X-100, sodium sulfite, and KCl were added to enzyme reaction mixture each, lysis of yeast cells was more effective.

• Korean Journal of Food Science and Technology
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• v.22 no.4
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• pp.426-433
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• 1990

The conditions for immobilization of the partially purified ${\beta}-galactosidase$ form Bacillus subtilis HP4 and the properties of the immobilized enzyme have been investigated. The crude enzyme precipitated with cold acetone was purified about 68-fold through DEAE-cellulose and sephadex G-100 chromatography and its recovery was 19.9% The optimal conditions for Immobilization of enzyme were obtained in 2%(w/v) sodium alginate, 15%(v/v) enzyme solution and 2%(w/v) calcium chloride, and also the optimal stirring thme was 2 hours on the above conditions. The optimum temperature and pH values for immobilized enzyme were $55^{\circ}C$ and 6.5, respectively. Its residual activity was show 25% after heat treatment for an hour at $65^{\circ}C$, and found its high stability in pH 6.0 to 8.0. The enzyme activity was not affected b)· EDTA, 2-mercaptoethanol, KCN, protective agents, and other methal ions except Hg ion and Cu ion. The $K_m\;and\;V_{max}$ values of the immobilized enzyme on ONPG were $1.82{\times}10^{-2}M\;and\;3.57{\times}10^{-8}mole/min$, whereas those on lactose were $2.94{\times}10^{-2}M\;and\;1.68{\times}10^{-7} mole/min$, respectively. The remained enzyme activity for the immobilized enzyme was 95%t of original activity after storage of 40 days at $4^{\circ}C$, and when reused for 5 times was 81%. When skim milk(4.8% lactose) and 5% lactose solution were reacted with the immobilized enzyme(250 units/g) of lactose were 51% and 43%, respectively.

• Asian-Australasian Journal of Animal Sciences
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• v.9 no.5
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• pp.583-590
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• 1996

The purpose of this study was to evaluate some factors in the bovine embryonic development from one-cell to blastocyst using modified synthetic oviduct fluid medium (mSOFM), after maturation and in vitro fertilization of the oocytes. The embryonic development to the blastocyst stage was assessed at 7-10 days after in vitro fertilization, and the total cells in the blastocysts were counted by staining nuclei with fluorochrome. Some commercial calf sera (CS) and a superovulated cow serum had different effects on the embryonic development to the blastocyst stage (8.6-21.4%), dependent upon their product lots, although the development might not be affected at least by serum progesterone levels. ${\beta}$-Mercaptoethanol (${\beta}$-ME) supplemented into mSOFM was effective to the embryonic development (27.8%), as well as the co-culture system with cumulus cells (19.5%). In a serum- and feeder cell-free culture using mSOFM containing several growth factors and ${\beta}$-ME instead of CS plus co-cultured cumulus cells, bovine serum albumin (BSA, fraction V), but not polyvinyl alcohol (PVA), was highly effective in embryonic development to the blastocyst stage, almost comparable to CS in the serum-contained culture (CS, BSA and PVA; 27.8, 19.5 and 5.7%, respectively). However, fatty acid free BSA rather reduced the number of developed blastocysts, compared with fraction V BSA (7.3 vs 29.4%). In the serum- and feeder cell-free culture, supplement of glucose to the medium (final 2.0 mM) stimulated the cell proliferation of developing embryos 120 hr after in vitro fertilization. These results indicated that a serum-free medium supplemented with ${\beta}$-ME could successfully support the development of bovine one-cell embryos to the blastocyst stage. Moreover, supplement of glucose and fatty acids to the medium might support preferably the development and cell proliferation of embryos.

• Food Science and Preservation
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• v.4 no.1
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• pp.45-51
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• 1997

In our studies on the role of $\beta$-galactosidase in fruit softening, significant difficulty, was encountered in our attempts to extract RNA from persimmon(Diospyros kaki L. cv. Fuyu) fruit due to astringency and tannin content. Initial, unsuccessful RNA extractions involved methods using guanidinium isothiocyanate/CsCl with and without polyvinylpyrrolidone(PVP), phenol/sodium lauryl sulfate(SDS), guanidinium hydrochloride, as well as polysomal RNA purification method that used 0.2 M Tris-HCI (pH 9.0) containing KCI, Mg-acetate, EDTA, $\beta$-mercaptoethanol, and sucrose. A method was devised which employed treatment of fruit with CO2 gas to diminish astringency prior to RNA extraction, followed by extraction of tissue powders with Proteinase K extraction buffer containing PVP and ascorbate at an alkaline pH. This procedure resulted in the removal of tannins and other polyphenolics and extraction of relatively large amount of high-quality RNA suitable for cDNA library construction and polymerase chain reaction(PCR). Futhermore, the procedure does not use the toxic and corrosive chemical guanidinium isothiocyanate or require ultracentrifugation.

• Journal of Microbiology and Biotechnology
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• v.18 no.5
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• pp.933-941
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• 2008

An extracellular $\beta$-glucosidase produced by Monascus purpureus NRRL1992 in submerged cultivation was purified by acetone precipitation, gel filtration, and hydrophobic interaction chromatography, resulting in a purification factor of 92-fold. A $2^2$ central-composite design (CCD) was performed to find the best temperature and pH conditions for enzyme activity. Maximum activity was observed in a wide range of temperature and pH values, with optimal conditions set at $50^{\circ}C$ and pH 5.5. The $\beta$-glucosidase showed moderate thermostability, was inhibited by $HgCl_2$, $K_2Cr_O_4$, and $K_2Cr_2O_7$, whereas other reagents including $\beta$-mercaptoethanol, SDS, and EDTA showed no effect. Activity was slightly stimulated by low concentrations of ethanol and methanol. Hydrolysis of p-nitrophenyl-$\beta$-D-glucopyranoside (pNPG), cellobiose, salicin, n-octyl-$\beta$-D-glucopyranoside, and maltose indicates that the $\beta$-glucosidase has broad substrate specificity. Apparently, glucosyl residues were removed from the nonreducing end of p-nitrophenyl-$\beta$-D-cellobiose. $\beta$-Glucosidase affinity and hydrolytic efficiency were higher for pNPG, followed by maltose and cellobiose. Glucose and cellobiose competitively inhibited pNPG hydrolysis.

• Microbiology and Biotechnology Letters
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• v.25 no.2
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• pp.109-114
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• 1997

The Oomycete Saprolegnia ferax produces an extracellular $\beta$-amylase, Maximum enzyme yield was attained after 7 days of growth in YNB starch medium (pH 6.5) at 25$\circ$C. The amylase was pu- rified 24-fold by ultrafitration, HPLC DEAE column and HPLC gel filtration. The purfied enzyme was a monomeric glycoprotein with a molecular weight of about 44,000 dalton. The pH and temperature optima were 6.5 and 50$\circ$C, respectively. The enzyme was fairly stable up to 50$\circ$C and at acidic pH region (pH 4.0-7.0). The apparent Km and Vmax values of the enzyme against soluble starch were 0.77 mg/ml and 2,174 $\mu$moles/mg protein, respectively. Amino acid analysis indicated that the enzyme was enriched in alanine, glycine, leucine and acidic amino acid. Starch hydrolysis with the enzyme released maltose but not glucose, whereas maltotriose, Schardinger dextrin ($\alpha$-cyclodextrin) and pullulan were not hydrolysed by the enzyme. The enzyme was inhibited by Schardinger dextrin, p-chloromercuribenzoate(PCMB), CU$^{2+}$' and Hg$^{2+}$. Inhibition of the enzyme by PCMB could be reversed by the addition of cysteine and mercaptoethanol.

• 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.

• Microbiology and Biotechnology Letters
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• v.19 no.5
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• pp.456-463
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• 1991

$\beta$-Galactosidase of Bifidobacterium longum KCTC 3215 was studied on the production, purification, and characterization. Optimum conditions for the enzyme production were in the medium of 1.0% lactose as carbon source, initial pH 7.0 and in 17 hours of cultivation at $37^{\circ}C$. The enzyme was purified 9.25 folds by protamine sulfate precipitation, ammonium sulfate fractionation, DEAE-Sephadex A-50 ion exchange chromatography and Sephadex G-150 gel filtration. The maximal P-galactosidase activity was observed at pH 6.5 and at the temperature of $40^{\circ}C$ This enzyme was stable at pH 6.0-8.5. Metal ions such as $Ca^{2+} \;and \; Co^{2+}$, 2-mercaptoethanol, cysteine, and glutathione stimulated B-galactosidase activity. The enzyme activity was inhibited by addition of $Mg^{2+}, Fe^{2+}, Cs^{1+}, Li^{1+}$, DETA, galactose, and $\rho$-chloromercuribenzoic acid. The kinetics of o-nitrophenyl-$\beta$-D-galactopyranoside and lactose were $K_m$ = 1.66 mM, $V_{max}= 0.30 mM/min\cdot mg\cdot protein$ and $KK_m = 3.18 mM, \; V_{max}= 0.42 mM/min \cdot mg\cdot$ protein, respectively. The molecular weight of native enzyme was about 360, 000 dalton and the enzyme consisted of 2 identical subunits with a molecular weight of 180, 000.

• Journal of Microbiology and Biotechnology
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• v.19 no.3
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• pp.257-264
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• 2009

A $\beta$-agarase gene, agaB34, was functionally cloned from the genomic DNA of a marine bacterium, Agarivorans albus YKW-34. The open reading frame of agaB34 consisted of 1,362 bp encoding 453 amino acids. The deduced amino acid sequence, consisting of a typical N-terminal signal peptide followed by a catalytic domain of glycoside hydrolase family 16 (GH-16) and a carbohydrate-binding module (CBM), showed 37-86% identity to those of agarases belonging to family GH-16. The recombinant enzyme (rAgaB34) with a molecular mass of 49 kDa was produced extracellularly using Escherichia coli $DH5{\alpha}$ as a host. The purified rAgaB34 was a $\beta$-agarase yielding neoagarotetraose (NA4) as the main product. It acted on neoagarohexaose to produce NA4 and neoagarobiose, but it could not further degrade NA4. The maximal activity of rAgaB34 was observed at $30^{\circ}C$ and pH 7.0. It was stable over pH 5.0-9.0 and at temperatures up to $50^{\circ}C$. Its specific activity and $k_{cat}/K_m$ value for agarose were 242 U/mg and $1.7{\times}10^6/sM$, respectively. The activity of rAgaB34 was not affected by metal ions commonly existing in seawater. It was resistant to chelating reagents (EDTA, EGTA), reducing reagents (DTT, $\beta$-mercaptoethanol), and denaturing reagents (SDS and urea). The E. coli cell harboring the pUC18-derived agarase expression vector was able to efficiently excrete agarase into the culture medium. Hence, this expression system might be used to express secretory proteins.