• Reproductive and Developmental Biology
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• 제35권3호
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• pp.295-300
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• 2011

The objective of this study was to evaluate efficiency of methyl-beta-cyclodextrin (MBCD) in the sperm preservation of bull. For this study, the freezing of diluted semen were added with Triladyl containing 20% egg-yolk and/or 0, 1, 5, 10 and 20 mM MBCD before freezing process. Analysis of viability in frozen-thawed sperm was estimated by SYBR14/PI double stain, hypoosmotic swelling test(HOST) and acrosome damage with FITC-PNA, and mitochondria activation with Rhodamin123 by flow-cytometry. The sperm viability was significantly in 0 mM and 5 mM concentrations of MBCD than other groups (p<0.05). However, the HOST was significantly lower at 20 mM concentration of MBCD than other concentrations (p<0.05). In addition, acrosome damage and mitochondria activation rates were significantly lower at 20 mM concentration of MBCD than other groups (p<0.05). In conclusion, the viability of sperm was not significantly different among concentrations of MBCD 0, 5 and 10 mM, but MBCD 20 mM was significantly lower than other groups. In addition, as concentrations of MBCD was high, HOST, acrosome damage and mitochondria activation rates had a negative effect in bull sperm.

• Reproductive and Developmental Biology
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• 제35권3호
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• pp.265-271
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• 2011

This study was undertaken to find out the effect of methyl-beta-cyclodextrin (MBCD) on cold shock and membrane cholesterol quantity of sperm during the freezing process in miniature pigs. For this study, semen ejaculated from PWG M-type miniature pig was diluted that freezing solution (with egg yolk group) and m-Modena B (without egg yolk group) treated with 0, 1, 5, 10 and 20 mM MBCD before freezing process. The diluted semen was monitored sperm ability at room temperature, after cooled until $5^{\circ}C$ and after forzen-thawed for cold shock test of spermatozoa. Also, membrane cholesterol of sperm was extracted by folch solution at the same time sperm ability was assessed for viability and acrosomal status. The membrane cholesterol quantity was measured by thin-layer chromatography (TLC) method. The result, viability and acrosome integrity in semen diluted without egg yolk groups were decreased at all temperature range by increasing of MBCD concentration. In particular, sperm of egg yolk group was showed that significantly higher viability and lower acrosome damage when treated with 5 mM MBCD (p<0.05). The results of TLC experiment, cholesterol amounts were increased with MBCD cocentration in egg yolk, and decreased with MBCD concentration in m-Modena B. In cryopreservation efficiency, there was no significant difference at viability, and acrosomal state of sperm in 5 mM MBCD concentration was significantly lower in acrosome damage than other groups (p<0.05). Therefore, the addition MBCD in egg yolk was protected spermatozoa from cold shock injury. This protective effect of MBCD may be due to addition of sperm membrane cholesterol.

• 한국식품과학회지
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• 제23권1호
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• pp.93-97
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• 1991

호알칼리성 Bacillus sp. YC-335가 생산하는 CGTase의 효소학적 특성 및 작용반응을 살펴보았다. ${\alpha}-CD,\;{\beta}-CD$ 및 ${\gamma}-CD$로부터 glucosyl residues를 설탕으로 전이시키는 반응에 대한 효소의 최대 반응속도, Vmax 값은 각각 $16.13,\;21.8,\;9.8{\mu}moles glucose/min/mg\;protein$이었으며 Km 값은 각각 1.68, 0.33, 0.37 mM이었다. 효소의 전분 가수분해활성은 여러 당류에 의해 촉진되었으며 특히 전분 가수분해 산물인 maltose와 glucose에 의한 효과가 가장 좋았다. 이 효소는 ${\beta}CD$에 의해 효소의 전분 분해활성이 저해되었으며 비경쟁적 저해형식을 보였다. 또한 전분으로부터 효소작용에 의해 생성된 산물을 총당량법 및 HPLC 분석을 통해 조사한 결과 이 효소는 cyclization 작용 뿐만 아니라 transglycosylation 작용과 disproportionation 작용을 가지는 것으로 확인하였다.

• 한국미생물·생명공학회지
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• 제26권5호
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• pp.442-449
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• 1998

CGTase를 이용한 당전이 xylitol의 합성과 당전이 xylitol의 비피더스균 생육증식 효과에 대한 연구를 수행하였다. 수종의 세균류가 분비하는 CGTase의 xylitol에 대한 당전이능을 비교하였으며, Thermoanaerobacter sp. 유래의 CGTase가 가장 우수한 당전이능을 보였다. 각종 당공여체를 검토한 결과 압출전분이 가장 우수한 결과를 보였으며, 당전이 효소반응의 최적 조건을 검토하였다. 생성된 당전이 xylitol을 활성탄-셀라이트 칼럼 크로마토그래피를 이용하여 분리하여 두 개의 fraction인 F-I, F-II를 얻었다. 이들의 당쇄결합 양상을 FAB mass spectrometer와 $^{13}$C-NMR spectrometer, 그리고 glucoamylase을 이용한 효소소화법을 이용하여 분석한 결과 xylitol에 glucose와 maltose 분자가 $\alpha$-1,4 결합되어 있는 것으로 유추되었다. 얻어진 당전이 xylitol은 xylitol과는 달리 Bifidobacterium breve에 대한 생육촉진효과를 보였다.

• 한국미생물·생명공학회지
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• 제32권1호
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• pp.29-36
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• 2004

토양으로부터 CGTase를 생산하는 새로운 호열성 세균을 분리하였으며, 형태 및 생리적 특성과 16S ribosomal DNA의 염기서열을 분석한 결과 Geobacillus thermosacchalytycus 로 동정하였다. 호열성 G. thermosacchalytycus가 분비하는 CGTase를 한외여과, 소수성 Phenyl Sephadex CL-4B 클로마토그래피 , 그리고 gel filtration의 순서로 분리 정제하여 정제도 28.2배, 정제수율 12.2%, 그리고 specific activity가 4952.5 units/mg인 CGTase를 얻을 수 있었다. 정제된 CGTase의 분자량은 69,000 dalton이었고, terminal amino sequence는 Asn-Leu-Asn-Lys-Val-Asn-Phe-Thr-Ser-Asp-Val-Val-Tyr-Gln-Ile이었다. 최적 pH 및 온도는 각각 pH 6와$ 50^{\circ}C$였고, pH 6-8과 $60^{\circ}C$에서 장기간 보존할 수 있는 중성 pH의 내열성 효소임을 알 수 있었다. 내열성 CCTase의 cyclization과 coupling 반응의 특정을 검토한 결과 G. thermosacchalytycus가 생산하는 내열성 효소는 $\alpha$-type CGTase이었고, cyclization 반응보다는 coupling반응에 적합한 당전이성이 높은 효소임을 알 수 있었다. 당전이 반응에서 기질 특이성을 검토한 결과 당공여체로는 가용성 전분 그리고 당수용체로는 배당체인 stevioside에 대해 높은 기질 특이성을 보였다. 당전이 반응은 당수용체와 당공여체가 효소와 무작위로 결합하여 진행되는 random ternary complex mechanism으로 반응이 수행되었다.

• 청정기술
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• 제19권3호
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• pp.212-218
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• 2013

초임계 이산화탄소를 역용매로 이용하는 aerosol solvent extraction system (ASES) 방법을 사용하여 HP-${\beta}$-CD 미립자를 제조하였으며, 공정변수가 입자의 크기와 형태에 미치는 영향을 조사하였다. 또한, 초임계 이산화탄소를 이용하여 이부프로펜과 HP-${\beta}$-CD의 포접복합체를 제조하였으며, ASES 공정에 의해 변형된 HP-${\beta}$-CD의 입자 형상이 포접효율에 미치는 영향에 대해 고찰하였다. ASES 공정으로 제조된 HP-${\beta}$-CD 미립자는 50~200 nm 크기의 나노 입자들이 응집된 입자 형상을 나타내었다. 에탄올 수용액을 HP-${\beta}$-CD의 용매로 사용한 경우 구형의 입자가 제조되었으며, 물의 양이 증가함에 따라 입자의 크기가 증가하였다. 초임계 이산화탄소를 이용해 고체상태에서 이부프로펜/HP-${\beta}$-CD 포접복합체를 제조하는 경우 초임계 ASES 방법에 의한 미세입자화 공정을 통해 포접효율을 향상시킬 수 있는 가능성을 확인하였다.

• 한국식품영양과학회지
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• 제43권9호
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• pp.1388-1393
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• 2014

선행연구에서 얻은 alkalophilic Bacillus I-5 유래의 CGTase wild-type과 가수분해능이 강화된 mutant 효소를 활용하여 waxy rice starch로부터 아밀로펙틴 클러스터를 제조하였다. SEC-MALLS-RI 분석법으로 CGTase wild-type과 mutant 효소가 처리된 시료의 평균 분자량을 확인한 결과 10분가량의 효소반응으로 두 반응 모두 평균 분자량은 $10^4{\sim}10^5Da$으로 급격히 감소하였음을 확인하였으며, 일정 반응 시간이 경과한 이후에는 더 이상 분자량의 감소가 일어나지 않음으로 미루어 시료가 아밀로펙틴 클러스터 단위로 분해되었으며 그 분자량은 $10^4{\sim}10^5Da$ 정도임을 알 수 있다. 또한 MALDI-TOF/MS 분석을 통하여 CGTase wild-type은 다양한 종류의 cyclic 형태의 maltodextrin을 생성하고 있으며 mutant 효소는 주로 소량의 maltooligosaccharide 들을 생산함을 확인하였다.

• 한국가금학회:학술대회논문집
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• 한국가금학회 2000년도 춘계 산학협동 심포지움 Proceedings
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• pp.23-36
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• 2000

Hurdle technique was used to remove cholesterol efficiently from liquid egg yolk. The quality of the low cholesterol egg products from the process were evaluated. From the 75 % cholesterol reduced egg yolk through $\beta$-cyclodextrin treatment. 2 times weight of soy bean oil was added to the egg yolk and homogenized followed by centrifuged to be maximized to remove cholesterol. When the pH of the yolk was adjusted to 9, 92 % of cholesterol was removed while 95.4 % of cholesterol was removed when 3 times weight of soy bean oil was added to the egg yolk. As the results of application of supercritical carbon dioxide extraction to the 75 % cholesterol reduced egg yolk through ${\beta}$-cyclodextrin treatment, 92.5 % of the cholesterol was removed from the egg yolk at $35^{\circ}C$, 4,500 psi, for 4 hours under co-solvent. The quality characteristics of the produced low cholesterol egg products were analysed. The cholesterol reduced egg yolk produced from ${\beta}$-cyclodextrin and soy bean oil treatment showed the lower emulsion capacity compared with control. The fatty acid composition of the cholesterol reduced egg yolk produced from ${\bet}a$-cyclodextrin and soy bean oil treatment showed increased C18:2 and C18:3 compared with control while decreased C16:1 and C18: 1 compared with control. The saponification method with extracting solvent of hexane showed that cholesterol concentration was 28.1 %. The quantity of hydrolysis solution(95 % ethanol : 33 % KOH = 94 : 6) was varied from 40 to 80 times of sample weights and the cholesterol concentration of 35.7 % was the highest result in the 60 times(v/w) hydrolysis solution. Cholesterol concentration of 35.7 % was recovered at the first trial with saponification method. but it could be improved up to 95.7 % after 4 times repetitive purification.

• BMB Reports
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• 제36권4호
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• pp.409-416
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• 2003

The isoform 1 of cyclodextrin glycosyltransferase (CGTase, EC 2.4.1.19) from Paenibacillus sp. A11 was purified by a preparative gel electrophoresis. The importance of histidine, tryptophan, tyrosine, and carboxylic amino acids for isoform 1 activity is suggested by the modification of the isoform 1 with various group-specific reagents. Activity loss, when incubated with diethylpyrocarbonate (DEP), a histidine modifying reagent, could be protected by adding 25 mM methyl-$\beta$-cyclodextrin substrate prior to the modification. Inactivation kinetics of isoform 1 with DEP resulted in second-order rate constants ($k_{inactivation}$) of $29.5\;M^{-1}s^{-1}$. The specificity of the DEP-modified reaction for the histidine residue was shown by the correlation between the loss of isoform activity and the increase in the absorbance at 246 nm of N-carbethoxyhistidine. The number of histidines that were modified by DEP in the absence and presence of a protective substrate was estimated from the increase in the absorbance using a specific extinction coefficient of N-carbethoxyhistidine of $3,200\;M^{-1}cm^{-1}$. It was discovered that methyl-$\beta$-CD protected per mole of isoform 1, two histidine residues from the modification by DEP. To localize essential histidines, the native, the DEP-modified, and the protected forms of isoform 1 were digested by trypsin. The resulting peptides were separated by HPLC. The peptides of interest were those with $R_t$ 11.34 and 40.93 min. The molecular masses of the two peptides were 5,732 and 2,540 daltons, respectively. When the data from the peptide analysis were checked with the sequence of CGTase, then His-140 and His-327 were identified as essential histidines in the active site of isoform 1.

• 한국식품과학회지
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• 제16권4호
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• pp.398-402
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• 1984

Rhizopus oryzae 가 생산(生産)하는 glucoamylase 의 각종기질(各種基質)에 대(對)한 분해반응(分解反應)을 검토(檢討)하였다. Glucoamylase I 과 II 는 amylose, amylopectin, glycogen, 가용성 전분, pullulan, maltose, maltotriose, maltotetriose, maltopentaose, maltohexaose, maltoheptaose, maltooctaose를 가수분해(加水分解)하였으나, ${\alpha}-cyclodextrin$, ${\beta}-cyclodextrin$, sucrose, raffinose, 젖당은 가수분해(加水分解)하지 못하였다. $37^{\circ}C$, 32시간(時間)의 반응(反應)에서 glucoamylase I 은 amylopectin, 가용성 전분, amylose를 거의 100% 분해하였고 glycogen 만을 88%정도 분해 하였으나, glucoamylase II 는 공시기질(供試基質) 4 종(種)을 거의 100% 분해하였다. Glucoamylase I 과 II 의 반응생성물질(反應生成物質)은 glucose 만이었고 ${\alpha}-glucosyltransferase$ activity는 없었다. Glucoamylase I 과 II 는 생찹쌀 전분을 가장 잘 분해(分解)시키나 생감자 전분, 생미숙 바나나 전분, 생칡 전분, 생참마 전분, raw high amylose corn starch의 분해능(分解能)은 glucoamyase I 에 비(比)하여 생전분(生澱粉)의 분해력(分解力)이 강(强)하였다.