• Journal of Microbiology and Biotechnology
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• 제21권6호
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• pp.617-626
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• 2011

Transglutaminase from Streptomyces mobaraensis is an enzyme of unknown function that cross-links proteins to high molecular weight aggregates. Previously, we characterized two intrinsic transglutaminase substrates with inactivating activities against subtilisin and dispase. This report now describes a novel substrate that inhibits papain, bromelain, and trypsin. Papain was the most sensitive protease; thus, the protein was designated Streptomyces papain inhibitor (SPI). To avoid transglutaminase-mediated glutamine deamidation during culture, SPI was produced by Streptomyces mobaraensis at various growth temperatures. The best results were achieved by culturing for 30-50 h at $42^{\circ}C$, which yielded high SPI concentrations and negligibly small amounts of mature transglutaminase. Transglutaminasespecific biotinylation displayed largely unmodified glutamine and lysine residues. In contrast, purified SPI from the $28^{\circ}C$ culture lost the potential to be cross-linked, but exhibited higher inhibitory activity as indicated by a significantly lower $K_i$ (60 nM vs. 140 nM). Despite similarities in molecular mass (12 kDa) and high thermostability, SPI exhibits clear differences in comparison with all members of the wellknown family of Streptomyces subtilisin inhibitors. The neutral protein (pI of 7.3) shares sequence homology with a putative protein from Streptomyces lavendulae, whose conformation is most likely stabilized by two disulfide bridges. However, cysteine residues are not localized in the typical regions of subtilisin inhibitors. SPI and the formerly characterized dispase-inactivating substrate are unique proteins of distinct Streptomycetes such as Streptomyces mobaraensis. Along with the subtilisin inhibitory protein, they could play a crucial role in the defense of vulnerable protein layers that are solidified by transglutaminase.

• Molecules and Cells
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• 제27권3호
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• pp.337-343
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• 2009

We developed a novel on-chip activity assay using protein arrays for quantitative and rapid analysis of transglutaminase activity in mammalian cells. Transglutaminases are a family of $Ca^{2+}$-dependent enzymes involved in cell regulation as well as human diseases such as neurodegenerative disorders, inflammatory diseases and tumor progression. We fabricated the protein arrays by immobilizing N,N'-dimethylcasein (a substrate) on the amine surface of the arrays. We initiated transamidating reaction on the protein arrays and determined the transglutaminase activity by analyzing the fluorescence intensity of biotinylated casein. The on-chip transglutaminase activity assay was proved to be much more sensitive than the $[^3H]putrescine$-incorporation assay. We successfully applied the on-chip assay to a rapid and quantitative analysis of the transglutaminase activity in all-trans retinoic acid-treated NIH 3T3 and SH-SY5Y cells. In addition, the on-chip transglutaminase activity assay was sufficiently sensitive to determine the transglutaminase activity in eleven mammalian cell lines. Thus, this novel on-chip transglutaminase activity assay was confirmed to be a sensitive and high-throughput approach to investigating the roles of transglutaminase in cellular signaling, and, moreover, it is likely to have a strong potential for monitoring human diseases.

• 한국식품과학회지
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• 제31권5호
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• pp.1262-1267
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• 1999

겔 형성이 어려운 유채단백질의 겔화를 위하여 미생물성 TGase의 촉매작용에 의하여 최적 겔화 반응조건을 겔강도(g)와 겔변형(mm)을 조사하여 검토하였다. 또한 형성된 겔의 고분자화를 SDS-PAGE 및 GL교차결합 함량 측정에 의하여 확인하였다. 단백질-TGase반응에서 효소 첨가량은 $45^{\circ}C$에서 60분 동안 반응 시 단백질농도가 10%에서 효소가 1 : 40([E] : [S])의 비로 첨가되었을 때 겔강도와 변형 값이 가장 높았다. 또한 이 조건에서 기질단백질 농도가 $4{\sim}l2%$로 증가함에 따라 겔강도는 비례적으로 증가함을 보였으나 겔의 변형을 고려할 때 10% 농도가 유채단백질 겔 제조에 적당한 농도로 조사되었다. 최적 활성을 나타내는 반응온도는 $45^{\circ}C$로 관찰되었고, 반응시간은 90분이면 적당한 것으로 조사되었다. 겔강도와 변형은 다른 pH와 비교할 때 pH 7에서 상당히 높은 값을 보여 pH 7이 최적조건이었다. SDS-PAGE분석에서 효소반응 에 따라 유채단백질의 본래 밴드들이 소별되거나 희미해지면서 고분자 밴드들이 형성되고 있음을 보여주었다. 또만 HPLC 분석 결과 GL교차결합 함량이 반응시간에 따라 $0\;{\mu}mol/g$에서 최고 $7.14\;{\mu}mol/g$ gel(반응 90분)까지 증가되고 있어 유채단백질의 겔화가 TGase에 의해 이루어지고 있다는 것을 확인할 수 있었다.

• 한국축산식품학회지
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• 제36권5호
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• pp.671-678
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• 2016

Fractioning and/or preheating treatment on the rheological properties of myofibrillar protein (MP) gels induced by microbial transglutaminase (MTG) has been reported that they may improve the functional properties. However, the optimum condition was varied depending on the experimental factors. This study was to evaluate the effect of red bean protein isolate (RBPI) on the rheological properties of MP gels mediated by MTG as affected by modifications (fractioning: 7S-globulin of RBPI and/or preheat treatment (pre-heating; 95℃/30 min): pre-heating RBPI or pre-heating/7S-globulin). Cooking yields (CY, %) of MP gels was increased with RBPI (p<0.05), while 7S-globulin decreased the effect of RBPI (p<0.05); however, preheating treatments did not affect the CY (p>0.05). Gel strength of MP was decreased when RBPI or 7S-globulin added, while preheat treatments compensated for the negative effects of those in MP. This effect was entirely reversed by MTG treatment. Although the major band of RBPI disappeared, the preheated 7S globulin band was remained. In scanning electron microscopic (SEM) technique, the appearance of more cross-linked structures were observed when RBPI was prepared with preheating at 95℃ to improve the protein-protein interaction during gel setting of MP mixtures. Thus, the effects of RBPI and 7S-globulin as a substrate, and water and meat binder for MTG-mediated MP gels were confirmed to improve the rheological properties. However, preheat treatment of RBPI should be optimized.

• 한국막학회:학술대회논문집
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• 한국막학회 2004년도 추계 총회 및 학술발표회
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• pp.142-145
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• 2004

Anion-exchange porous hollow-fiber membranes with a thickness of about 1.2 mm and a pore size of about $0.30{\mu}m$ were used as a supporting matrix to immobilize cycloisomaltooligosaccharide glucanotransferase (CITase). CITase was immobilized to the membrane via anion-exchange adsorption and by subsequent enzymatic cross-linking with transglutaminase, the amount of which ranged from 3 to 110 mg per g of the membrane. The degree of enzyme multilayer binding was equivalent to 0.3 to 9.8. Dextran, as the substrate, was converted into seven- to nine-glucose-membered cycloisomaltooligosaccharides (CI-7, -8, and -9) at a maxi mum yield of $28\%$ in weight at a space velocity of 10 per hour during the permeation of $2.0(w/w)\%$ dextran solution across the CITase-immobilized porous hollow-fiber membrane.