• 한국산학기술학회논문지
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• 제15권8호
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• pp.5412-5418
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• 2014

참돔 이리도바이러스(RSIV)는 이리도바이러스과에 속하며, 많은 아시아 국가에서 감염성 어류 질병을 유발하여 양식산업에 커다란 경제적 손실을 입히는 바이러스이다. 우리는 최근에 효모표면발현(yeast surface display, YSD)를 사용하여 다양한 해양바이러스를 동정하고 검출할 수 있는 새로운 실험시스템을 개발하였다. 이 연구에서 우리는 참돔 이리도 바이러스(RSIV)의 외피단백질을 효모표면 발현 기법을 이용하여 발현시켰다. 바이러스 외피단백질 유전자는 염기서열 데이터베이스에 기초하여 합성되었고, 효모발현벡터인 pCTCON2으로 서브클로닝되었다. 이 벡터는 효모 strain EBY100으로 형질전환 되었다. Flow cytometry와 Western blot analysis를 통해 RSIV 외피단백질의 발현을 확인하였다. ${\beta}$-mercaptoethanol 처리에 의해 발현된 바이러스 외피단백질을 효모 표면로부터 분리하였다. 이 연구의 결과는 YSD 시스템이 해양바이러스 외피단백질을 획득하기 위한 매우 좋은 발현시스템이라는 것을 보여준다.

• 한국식품과학회지
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• 제29권5호
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• pp.1021-1027
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• 1997

Dicyma sp. YCH-37이 생산하는 효모세포벽 용해효소의 성질을 검토한 결과, 각종 환원제와 금속이온에 대체로 안정하였고, guanidine-HCl을 제외한 여러 화학수식제에 대해서도 안정하였다. 배양시간, 전처리 및 배양조건에 따른 영향을 검토한 결과, 정지기 및 사멸기에 있는 효모보다는 대수증식기의 효모, 그리고 생효모에 비해 열처리된 효모가 더 잘 용균되었다. Butanol, acetone 등의 유기용매로 처리된 효모가 그렇지 않은 효모보다 용균도가 좋았으며, 0.5 M ammonium sulfate가 함유된 Yeast extract-Malt extract 배지에서 생육한 효모, 그리고 진탕배양한 효모보다 정치배양한 효모가 용균효소에 의해 더 잘 용균되었다. SDS, Triton X-100, ${\beta}-mercaptoethanol$, potassium chloride, sodium sulfite 등의 화학수식제를 효소반응액에 첨가하였을 때 기질 효모는 더 잘 용균되었다.

• 한국식품과학회지
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• 제22권4호
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• pp.426-433
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• 1990

Bacillus subtilis HP-4의 ${\beta}-galactosidase$를 추출, 정제하여 효소의 고정화조건 및 고정화 효소의 특성을 조사하였다. B. subtilis를 배양하여 얻은 조효소액을 acetone분획 DEAE-cellulose에 의한 ion exchange chromatography, Sephadex G-100에 의한 gel filtration과정을 통하여 정제하였던 바 약 68% 정제되었으며, 이때의 회수율은 19.9%이었다. 본 효소의 최적 고정화조건은 2%(w/v)의 sodium alginate농도, 15%(v/v)의 효소농도, 그리고 2%,(w/v)의 $CaCl_2$ 농도에서 2시간 교반하였을 때 이었다. 고정화 효소의 최적온도와 pH값은 각각 $55^{\circ}C$와 6.5이었다. 고정화 효소의 활성은 Hg이온과 Cu이온에 의하여 저해되었으며, EDTA, 2-mercaptoethanol, KCN 등의 금속이온 그리고 보호제에 의한 영향은 없었다. 고정화 효소의 ONPG와 유당에 대한 $K_m$값과 $V_{max}$값은 각각 $1.82{\times}10^{-2}M$과 $2.94{\times}10^{-2}M$ 그리고 $3.57{\times}10^{-8}mole/min$과 $1.68{\times}10^{-7}mole/min$이었다. 고정화 효소를 $4^{\circ}C$에서 40일간 저장 후 잔존활성과 5회 재사용 후 잔존활성을 조사한 결과는 각각 95%와 81%이었으며, 탈지유(4.8% 유당)와 5% 유당용액에서 $50^{\circ}C$, 9시간 반응시켰을 때 유당분해율은 각각 51%와 43%이었다.

• Asian-Australasian Journal of Animal Sciences
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• 제9권5호
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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.

• 한국식품저장유통학회지
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• 제4권1호
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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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• 제18권5호
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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.

• 한국미생물·생명공학회지
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• 제25권2호
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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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• 제17권3호
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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.

• 한국미생물·생명공학회지
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• 제19권5호
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• pp.456-463
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• 1991

Bif. longum KCTC 3215에 의한 Beta-galactosidase의 최적생성조건은 탄소원으로 lactose 1.0, 초기 pH 7.0, 배양온도 $37^{\circ}C$ 및 배양시간 17시간 후였다. 이 효소는 protamine sulfate, ammonium sulfate, DEAE-Sephadex A-50 ion exchange chromatography 및 Sephadex G-150 gel filtration 등 4단계 정제과정을 거쳐 9.25배 정제되었다.

• Journal of Microbiology and Biotechnology
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• 제19권3호
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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.