• KSBB Journal
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• 제18권2호
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• pp.155-160
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• 2003

Streptoverticillium morbaraene로부터 미생물 유래 transglutaminase 생산을 위하여 최적의 용존산소 농도를 구명하였다. 용존산소는 용존산소 농도 자동 조절 시스템에 의해 조절되었다. 발효 중 용존산소 농도 조절을 위하여 통기속도는 0.3-3.9 L/min, 교반속도는 260-360 rpm으로 각각 범위를 설정하였다. 용존산소 농도를 조절한 다양한 회분식 배양에서 용존산소가 20%일 때 최대 미생물유래 transgiutaminase 생산이 가능하였다. 최분배양에서 용존산소 농도를 20%로 조절한 경우 미생물유래 transglutaminase 생산은 2.12 U/mL이었고, 용존산소를 조절하지 않은 회분식 배양의 미생물유래 transglutaminase 생산보다 1.1배 향상되었다. 역시 가장 높은 미생물유래 transglutaminase 생산은 용존산소를 20%로 조절한 유가식 배양에서 가능하였으며, 용존산소를 조절하지 않은 회분식 배양의 미생물유래 transglutaminase 생산에 비교해서 1.3배 증가하였다. 최대 건조균체량과 미생물유래 transglutaminase 생산은 각각 13.2 g/L와 2.6 U/mL이었다. 용존산소를 20%로 용존산소 농도 자동 조절 시스템에 의해 조절한 유가식 배양은 미생물유래 transgiutaminase 생산에 적절하였으며 다른 미생물 배양에도 적용할 수 있을 것으로 판단된다.

• 대한화장품학회지
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• 제39권1호
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• pp.25-30
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• 2013

단백질을 연결시키는 효소인 transglutaminase는 모발에서 다양한 효과를 나타낼 수 있는 가능성이 있는데 이중에는 모발의 단단함을 증가시키는 작용도 포함된다. 여러 transglutaminase 효소 중 Streptomyces mobaraensis로부터 분리된 미생물 유래 효소를 손상된 모발에 사용한 후 인장강도를 평가한 결과 초기에 비해 15.64%까지 증가되는 것이 확인되었다. 이러한 효과는 transglutaminas가 샴푸를 사용하여 세정하는 과정에서의 모발 손상을 복구시킬 수 있다는 것을 의미한다. 또한 transglutaminase를 이용해 모발 표면의 특성을 개선 시킴으로써 모발의 윤기를 증가시키고 표면의 마찰력을 감소시키는데 유용하게 이용될 수 있었다.

• 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.

• 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.

• Journal of Microbiology and Biotechnology
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• 제10권2호
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• pp.187-194
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• 2000

An actinomycetes strain, T-2, which produces transglutaminase (EC 2.3.2.13), was isolated from soil and identified as belonging to the Actinomadura sp., based on taxonomc studies. The conditions for the transglutaminase production and its enzymatic properties were investigated. The optimum components for the transglutaminase production were 2% glucose, 1% polypeptone and soytone, and 0.1% MnCl2. The optimum pH and temperature of the enzyme reaction were pH 8.0 and $45^{\circ}C$, respectively. The enzyme was stable within the pH range of 5.0-9.0 and $30^{\circ}C-45^{\circ}C$. The novel enzyme required no calcium ions for its activity. This enzyme polymerized various proteins such as casien, soy protein, hemoglobin, egg white, gelatin, and soybean milk.

• Journal of Microbiology
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• 제41권2호
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• pp.161-164
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• 2003

Recombinant light chain of Clostridium botulinum neurotoxin type B stimulated transglutaminase activity in a dose dependent manner, Compared to native toxin, recombinant light chain showed av greater stimulatory effect on transglutaminase activity. Zn-chelating agents, inhibiting the proteolytic activity of the clostridial toxins, did not interfere with this stimulation. These results suggest that the light chain plays a major stimulatory role, which is not due to its metallopeptidase activity, but is possibly due to specific interaction with transglutaminase. More importantly, this report provides a new insight into the intracellular action of C. botulinum neurotoxins.

• Food Science and Biotechnology
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• 제16권2호
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• pp.223-227
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• 2007

Ground garlic inhibited the cross-linking reaction of myosin and incorporation of monodansylcadaverine (MDC) in salted Alaska pollack surimi catalyzed by transglutaminase (TGase). The component responsible for the inhibition was a thermostable, low molecular weight compound. The component also inhibited microbial transglutaminase (MTGase). The inhibition by garlic was reversibly recovered upon addition of 2-mercaptoethanol. The inhibitory component was therefore hypothesized to contain sulfhydryl groups within its structure. Alliin itself did not inhibit the cross-linking reaction. However, the addition of alliin together with garlic increased the inhibition. This result suggested that compounds derived from alliin was responsible for the inhibition of TGase activity.

• 한국생물공학회:학술대회논문집
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• 한국생물공학회 2000년도 춘계학술발표대회
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• pp.239-242
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• 2000

호기성 균주인 Streptoverticillium mobaraense에 의해 생산되는 Transglutaminase의 고수율을 위한 실험을 하였다. 라틴방격법에 의해 실험을 설계하여 최적 배지를 확정하였고, 호기성 균주인만큼 공기공급을 위한 통기 및 교반이 중요하게 작용됨을 관찰하였다. 이를 위해 임펠러의 형태 및 크기에 관한 실험을 수행하였고, 향후 부피산소공기전달 속도를 vvm과 교반속도에 따라 측정하도록 할 것이다. 또한 미생물의 증식 및 효소생산성에 미치는 온도 및 pH의 조건에 대해 실험한 결과 온도보다는 산성 및 중성에서의 pH가 효소생산성에 높은 영향을 미치고 있다. 플라스크 배양에서 평균적으로 1.3 U/mL 활성의 효소생산이 가능했으며 동일조건의 발효조 배양시 0.7 U/mL로 생산성이 감소하였다.

• 한국식품영양과학회지
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• 제38권6호
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• pp.801-806
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• 2009

본 연구는 transglutaminase 생산능이 우수한 토양유래방선균 strain YK-2를 TGase 최적생산배지에서 $28^{\circ}C$, 5일간 배양하여 얻은 배양여액으로 본 효소의 정제 및 정제된 효소의 효소화학적 특성에 관하여 검토한 것이다. 본 효소의 정제는 50% methanol precipitation, DEAE-Sephadex column chromatography의 정제 절차를 거쳐 56.5%의 수율로 정제되었고, 정제된 효소의 순도는 12.5% SDS-PAGE에서 단일 밴드를 나타내어, 서브유닛트의 분자량이 약 45,000 dalton으로 추정되는 호모형 효소인 것을 알 수 있었다. 정제된 TGase의 생화학적 제 특성을 검토한 결과, 등전점은 pH $6.0{\sim}7.0$ 부근에 있는 것으로 나타났으며, 본 효소의 기질인 CBZ-L-Gln-Gly 농도에 대한 Km치는 18.5 mM으로 추산되었다. 또한 금속이온 및 저해제의 영향으로는 $Hg^{++}$에 의해서 본 효소의 활성이 강하게 저해되었으나, DTT 및 mercaptoethanol에 의해 각각 293% 및 219% 활성이 증가하였다.

• Food Science and Biotechnology
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• 제17권5호
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• pp.904-911
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• 2008

Covalent cross-links between a number of proteins and peptides explain why transglutaminase may be widely used by food processing industries. The objective of this work was optimization of the fermentation process to produce transglutaminase from a new microbial source, the Streptomyces sp. P20. The strategy adopted to modify the usual literature media was: (1) fractional factorial design (FFD) to elucidate the key medium ingredients, (2) central composite design (CCD) to optimise the concentration of the key components. Optimization of the medium resulted in not only an 86% increase in microbial transglutaminase activity as compared to the media cited in the literature, but also a reduction in the production cost. Optimal fermentation conditions - namely temperature and agitation rate - were also studied, using CCD methodology. Usual conditions of $30^{\circ}C$ and 100 rpm were within the optimal area. All other parameters for enzyme production were experimentally proven to be optimum fermentation conditions.