• BMB Reports
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• 제44권1호
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• pp.52-57
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• 2011

Termites play an important role in the degradation of dead plant materials and have acquired endogenous and symbiotic cellulose digestion capabilities. The feruloyl esterase enzyme (FAE) gene amplified from the metagenomic DNA of Coptotermes formosanus gut was cloned in the TA cloning vector and subcloned into a pET32a expression vector. The Ft3-7 gene has 84% sequence identity with Clostridium saccharolyticum and shows amino acid sequence identity with predicted xylanase/chitin deacetylase and endo-1,4-beta-xylanase. The sequence analysis reveals that probably Ft3-7 could be a new gene and that its molecular mass was 18.5 kDa. The activity of the recombinant enzyme (Ft3-7) produced in Escherichia coli (E.coli) was 21.4 U with substrate ethyl ferulate and its specific activity was 24.6 U/mg protein. The optimum pH and temperature for enzyme activity were 7.0 and $37^{\circ}C$, respectively. The substrate utilization preferences and sequence similarity of the Ft3-7 place it in the type-D sub-class of FAE.

• KSBB Journal
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• 제14권3호
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• pp.264-272
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• 1999

Mandelate pathway를 거치는 Pseudomonas putida(KCTC 1751)의 전세포 benzoylformate dcarboxylase를 이용하여 benzoylformate를 benzaldehyde로 변환하였고 성장배지의 조성이 cell내부에 축적되는 benzoylformate dcarboxylase의 양에 미치는 영향을 조사하였다. 전세포효소의 재사용을 위하여 calcumalginate 캡슐 고정회법을 이용하여 캡슐고정화 Pseudomonas putida를 제조하였다. 캡슐 고정화 미생물을 M3배지에서 3일간 배양한 후 M1배지에서 1일간 배양한 결과 77.75g/L의 미생물 건조중량을 얻었다. 캡슐 고정화 전세포 benzoyltormate decarboxylase의 비활생도는 자유배양에 의한 전세포효소의 비활성도에 비해 약 1/2값을 나타내었으며 캡슐 고정화 전세포 benzoyltormate decarboxylase를 20회 재사용시 20%의 실활을 보였으며 캡슐 재사용 30회 이후 미생물의 건조중량은 약 10% 감소를 보였다.

• 미생물학회지
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• 제45권3호
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• pp.257-262
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• 2009

Bacillus licheniformis WL-12의 carboxymethyl cellulase (cellulase) 유전자를 함유한 대장균 균체 파쇄상등액으로부터 DEAE-Sepharose와 Q-Sepharose 컬럼 크로마토그래피를 통해 cellulase를 정제하였다. 정제된 효소의 비활성은 163 U/mg이었으며, SDS-PAGE에 의해 측정된 분자량은 약 49.5 kDa으로 나타났다. pH 5.5와 $55^{\circ}C$에서 최대 반응활성을 보였으며, SDS (5mM)에 의해서는 cellulase의 활성이 완전히 저해되었고 $Cu^{2+}$5mM)에 의해서는 약간 증진되었다. 정제된 cellulase는 CMC, konjac, barley $\beta$-glucan과 lichenan을 가수분해하였으나 xylan, locust bean gum 및 p-nitrophenyl-$\beta$-glucopyranoside를 분해하지 못하였다. Cellooligosaccharides를 정제된 WL-12 cellulase로 분해하였을 때 cellobiose와 cellotriose가 주된 최종 반응산물로 관찰되었으며 cellobiose보다는 중합도가 큰 cellotriose, cellotetrasoe와 cellopentaose는 분해하였으나 cellobiose는 분해하지 못하는 것으로 확인되었다.

• Journal of Microbiology and Biotechnology
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• 제29권3호
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• pp.410-418
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• 2019

To investigate a novel ${\beta}$-glucosidase from Bifidobacterium breve ATCC 15700 (BbBgl) to produce compound K (CK) via ginsenoside $F_2$ by highly selective and efficient hydrolysis of the C-3 glycoside from ginsenoside Rd, the BbBgl gene was cloned and expressed in E. coli BL21. The recombinant BbBgl was purified by Ni-NTA magnetic beads to obtain an enzyme with specific activity of 37 U/mg protein using pNP-Glc as substrate. The enzyme activity was optimized at pH 5.0, $35^{\circ}C$, 2 or 6 U/ml, and its activity was enhanced by $Mn^{2+}$ significantly. Under the optimal conditions, the half-life of the BbBgl is 180 h, much longer than the characterized ${\beta}$-glycosidases, and the $K_m$ and $V_{max}$ values are 2.7 mM and $39.8{\mu}mol/mg/min$ for ginsenoside Rd. Moreover, the enzyme exhibits strong tolerance against high substrate concentration (up to 40 g/l ginsenoside Rd) with a molar biotransformation rate of 96% within 12 h. The good enzymatic properties and gram-scale conversion capacity of BbBgl provide an attractive method for large-scale production of rare ginsenoside CK using a single enzyme or a combination of enzymes.

• BMB Reports
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• 제48권6호
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• pp.313-318
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• 2015

Matrix metalloproteinases (MMPs) are zinc-dependent endopeptidases that degrade the extracellular matrix (ECM) and regulate the extracellular microenvironment. Despite the significant role that MMP activity plays in cell-cell and cell-ECM interactions, migration, and differentiation, analyses of MMPs in vitro and in vivo have relied upon their abundance using conventional immunoassays, rather than their enzymatic activities. To resolve this issue, diverse nanoprobes have emerged and proven useful as effective activity-based detection tools. Here, we review the recent advances in luminescent nanoprobes and their applications in in vitro diagnosis and in vivo imaging of MMP activity. Nanoprobes with the purpose of sensing MMP activity consist of recognition and detection units, which include MMP-specific substrates and luminescent (fluorescent or bioluminescent) nanoparticles, respectively. With further research into improvement of the optical performance, it is anticipated that luminescent nanoprobes will have great potential for the study of the functional roles of proteases in cancer biology and nanomedicine. [BMB Reports 2015; 48(6): 313-318]

• 약학회지
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• 제19권4호
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• pp.234-239
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• 1975

Protocollagen proline hydroxylase (PPH) activity in the course of rebound of rat carrageenin granuloma was assyed to investigate its relationship with collagen blosynthesis. The specific activity of PPH which was inhibited significantly by treatments with hydrocortisone acetate 93mg/kg/day, 3days) was recovered near to the normal level by 48hr after the cessation of the corticoid treatments. The total enzyme activity of the granuloma of treated group was not yet recovered even on the 3rd day, still showing a significant difference from control. PPH activity expressed in terms of granuloma cells between control and the drug treated group, however, was changed showing no apparent difference throughout the experimental period. These results suggested that the synthesis of protocollagen(collagen precursor) rather than PPH activity was directly affected by administration and withdrawal of corticoid.

• Applied Biological Chemistry
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• 제36권3호
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• pp.191-196
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• 1993

본 연구는 합성기질인 $X-{\alpha}-Gal$를 이용하여 발효유 및 유제품내의 Bifidobacteria 생균수를 측정할 목적으로 하였다. 젖산균과 Bifidobacteria의 ${\alpha}-galactosidase$ specific activity를 측정한 결과 Bifidobacteria 균주에서는 높은 ${\alpha}-galactosidase$ activity를 가지고 있었으며, 그 중 Bif. longum KCTC 3215의 specific activity는 8.57 unit/mg protein으로 가장 높게 나타났다. Lactobacillus, Streptococcus, Pediococcus와 Leuconostoc 균주에서는 활성이 미약하거나 없었다. 합성기질인 $X-{\alpha}-Gal$을 MRS agar 배지에 $100{\;}{\mu}M$ 첨가한 결과 Bifidobacteria는 blue colony로, Lac. bulgaricus, Lac. casei와 Leu. mescenteroides 균주는 light blue colony로, 그 외 젖산균에서는 white colony로 나타났다.

• 한국미생물·생명공학회지
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• 제24권1호
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• pp.9-18
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• 1996

Streptococcus mutans has been implicated as primary causative agents of dental caries by insoluble glucan (IG) in human and experimental animals. An attempt was made to search for the ${\alpha}$-1,3 glucanase that degrades IG produced by S. mutans. ${\alpha}$-1,3 glucanase was detected in the culture supernatant of microorganisms, which are isolated from soils on agar medium containing IG as a sole carbon source. This Streptomyces sp. hydrolysed IG produced by immobilized S. mutans and was named as Y9373. This enzyme required ${\alpha}$-1,3 glucan (IG) as an inducer. The optimum conditions for enzyme production were studied. The enzyme was purified by 30~70% $(NH_4)_2SO_4$ precipitation, anion exchange chroma tography on DEAE-cellulose and gel filtration on Sepadex G-75. The purified enzyme has a specific activity of 7840.0 U/mg protein giving 32.1-fold purification and final yield of 0.53%. The molecular weight was estimated to be about 22.5 kDa by SDS-PAGE. The optimum pH and temperature for enzyme reaction were 6.5 and 37$^{\circ}C$, respectively and the enzyme was relatively stable at the temperature below 60$^{\circ}C$. The activity of purified enzyme was enhanced by adding $Co^{2+},\;Mn^{2+}\;and\;Mg^{2+}$ into the medium, whereas inhibited by adding $Hg^{2+},\;Zn^{2+}$ and SDS. The $K_m\;and\;V_{max}$ value of ${\alpha}$-1,3 glucanase for IG were estimated to be 2.50 mM and 0.0431 mM/min, respectively. The thin layer chromatographic analysis of hydrolysates from IG with ${\alpha}$-1,3 glucanase showed that glucose was the main product of reaction. This enzyme activity was about 14 times higher than marketing dextranase as preventive agent against artificial dental caries by S. mutans in TH medium including 5% sucrose after 30 minutes.

• 한국균학회지
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• 제15권3호
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• pp.175-182
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• 1987

1. $(NH_4)_2SO_4$침 전, DEAE-cellulose column chromatography, CM-Sephadex C-50 및 Sephadex G-75 gel filtration에 의하여 정제된 효소는 조효소보다 18배가 높았으며 회수율은 13.40%였다. 2. 겉보기 분자량은 약 40,000dalton이었다. 3. 최적 pH와 온도는 4.0, $40^{\circ}C$일 때이며, 안정범위는 pH2.0에서 pH5.0, 온도는 $60^{\circ}C$ 이하이었다. 4. 금속이온 첨가에 의한 영향은 $10^{-2}M$ $BaCl_2$첨가시 효소활성이 증가되었으며, $10^{-2}M$ $Pb(NO_3)_2$, $K_3Fe(CN)_6$, $AgSO_4$와 $ZnSO_4$첨가시는 효소활성이 완전히 저해되었다. 5. 각종 유기화합물 첨가에 의한 효소의 영향은 citric acid 첨가시 효소활성이 현저히 저해되었다. 6. 기질 특이성을 보면 dextrin과 glycogen을 기질로 하였을 때는 효소활성이 증가하였으나 saccharose 첨가시에는 현저히 저하되었다. 7. COD 제거율을 측정한 결과는 67에서 68% 정도이었다.

• 한국식품과학회지
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• 제30권1호
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• pp.82-89
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• 1998

새우젓에서 protease를 추출, 정제하여 이의 효소학적 특성을 조사하였다. 새우젓 protease를 추출, 전기투석으로 탈염, ammonium sulfate로 단백질을 분획 시킨 후 Sephadex G-100 column chromatography와 DEAE-cellulose column chromatography를 행하여 정제하였으며 정제 효소는 전기영동상 단일 band로 나타났으며 분자량은 24 kDa, 수율은 14%, 비활성역가는 8.4 unit/mg, 정제배수는 9.8이었다. 정제효소의 최적 pH는 8.0이었고 $pH\;7.0{\sim}10.0$의 범위에서 안정하였다. 단백질 분해 효소 활성의 최적 온도는 $40^{\circ}C$이었고 $30{\sim}60^{\circ}C$의 온도 범위에서 안정성을 나타내었다. 기질에 대한 특이성으로 합성기질인 BAPNA, TAME에는 활성을 보였으며 hammersten casein을 기질로 사용하였을때의 Km값은 $5.1{\times}10^{-7}\;M$이었다. 금속 이온의 영향으로는 $M^{++}$만이 활성이 증가되었고 다른 금속 이온들에 의해서는 저해되었다. 저해제의 효과에서는 STI차 TLCK에 의해 현저하게 저해되었다. 이상의 성질에서 새우젓에서 분리된 protease는 serine계의 trypsin-like enzyme으로 추정되었다.