• 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.

• Journal of Microbiology and Biotechnology
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• 제8권1호
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• pp.1-7
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• 1998

• Journal of Microbiology and Biotechnology
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• 제15권5호
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• pp.1125-1129
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• 2005

Cloning of a 6.6-kb BamHI digested chromosomal DNA from S. griseus IFO13350 revealed the presence of an additional gene encoding a novel trypsin-like enzyme, named SprU. The SprU protein shows a high homology ($79\%$ identity, $88\%$ similarity) with the SGT protease, which has been reported as a bacterial trypsin in the same strain. The amino acid sequence deduced from the nucleotide sequence of the sprU gene suggests that SprU is produced as a precursor consisting of an amino-terminal presequence (29 amino acid residues), prosequence (4 residues), and mature trypsin consisting of 222 amino acids with a molecular weight of 22.94 kDa and a calculated pI of 4.13. The serine, histidine, and aspartic acid residues composing the catalytic triad of typical serine proteases are also well conserved. When the trypsin activity of the SprU was spectrophotometrically measured by the enzymatic hydrolysis of the artificial chromogenic substrate, N-${alpha}$-benzoyl-DL-arginine-p-nitroanilide, the S. lividans transformant with pWHM3-U gave 3 times higher activity than that of control. When the same recombinant plasmid was introduced into S. griseus, however, the gene dosage effect was not so significant, as in the cases of other genes encoding serine proteases, such as sprA, sprB, and sprD. Although two trypsins, SprU and SGT, have a high degree of homology, the pI values, the gene dosage effect in S. griseus, and the gene arrangement adjacent to the two genes are very different, suggesting that the biochemical and biological function of the SprU might be quite different from that of the SGT.

• Journal of Microbiology
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• 제33권4호
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• pp.307-314
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• 1995

Trypsin like protease (TLP) and metalloprotease (MTP) were induced in associated with the mycelium differentiation in Streptomyces albidoflavus SMF301. TLP and MTP were purified and characterized from the culture. The molecular mass of TLP and MTP were estimated to be 32 kDa and 18 kDa, respectively. The molecular mass of TLP and MTP were estimated to be 32 kDa and 18 kDa, respectively. The optimum pH and temperature of TLP were 10 and 40.$^{\circ}C$ Those of MTP were 8 and 55 $^{\circ}C$ TLP was stable at alkaline pH (6-9) and unstable above 45.$^{\circ}C$and MTP was stable at alkaline pH and unstable above 80.$^{\circ}C$ Km and Vmax values with benzoyl-arginyl p-nitroanilide of TLP were 139 $\mu$M, and 10 nmole of nitroanilide released per min per$\mu\textrm{g}$ protein, respectively. Km, and Vmax values with a synthetic substrate, leucine p-nitroanilide, or MTP were 58.9 $\mu$M, 3.47 nmol of nitroanilide released per min per$\mu\textrm{g}$protein, respectively. TLP was inhibited competitively by leupeptin; the inhibition constant was 0.0031 $\mu$M. MTP was inhibited by EDTA, phenonthroline and bestatin.

• Journal of Life Science
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• 제12권1호
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• pp.14-18
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• 2002

Subpopulations of nucleotides that bind specifically to a variety of proteins have been isolated from a population of random sequence RNA/DNA molecules. Roughly one in $10^{13}$ random sequence RNA/DNA molecules folds in such a way as to create a specific binding site for small ligands. Since the development of in vitro selection procedure, more than 50 nucleic acid ligands (aptamers) have been isolated. These molecules are very useful for the study of molecular recognition between nucleic acid and protein/organic compound. In addition to these basic studies this method gives us a dream to produce new drugs against several diseases. We focused on several aptamers which specifically binds to trypsin-like serine proteases (thrombin, human neutrophil elastase, activated protein C and NS3 protease of human hepatitis C virus) and want to introduce their structural characteristics and some functions.

• 한국수산과학회지
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• 제29권3호
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• pp.296-308
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• 1996

혈합육어 (멸치와 전어)와 백색육어 (농어와 도다리)의 육과 내장에서 추출한 조효소에 대하여 천연기질 및 합성 기질에 대한 효소 활성과 disc-PAG 전기영동에서 분리된 효소 단백질의 분포를 선택적 합성기질을 써서 비교 검토하였다. 육에는 SH-proteinase인 cathepsin L, B, 및 H 유사 효소가 분포하는 것을 확인 할 수 있었으며, 이 밖에도 cathepsin G 및 D도 다소 분포하는 것으로 추정되었다. 그리고 내장에는 강한 활성의 chymotrypsin 및 trypsin 유사 효소가 주로 분포하고 있음이 확인되었다. 육과 내장에서 추출한 조효소들은 공통적으로 멸치, 전어, 도다리 그리고 농어의 순으로 활성이 높은 것을 알 수 있었으며, 전체적으로 혈합육어에 분포되어 있는 효소가 백색육어류에 분포되어 있는 효소에 비하여 약 100배정도 높았다. 육과 내장의 조효소를 방어의 근원섬유단백질에 작용시킨 결과, 육 중의 조효소는 myosin heavy chain의 분해가 두드러졌으며, 특히 멸치의 효소가 근원섬유 단백질의 구성 subunit 모두에 대하여 강한 분해능을 보였다. 내장 조효소의 경우는 반응 초기에 myosin heavy chain과 actin을 현저하게 분해시켰으며, 혈합육어인 멸치와 전어 조효소의 분해능이 백색육어인 농어와 도다리의 조효소보다 강한 것을 확인 할 수 있었다. 육의 조효소는 어종에 따라 서로 다른 분해 양상을 보였으며, 내장 조효소는 혈합육어와 백색육어의 효소 활성뿐만 아니라 효소의 분포상으로도 많은 차이가 있었다.

• Journal of Microbiology and Biotechnology
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• 제14권6호
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• pp.1350-1355
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• 2004

The sprA and sprB genes, encoding the chymotrypsin-like proteases Streptomyces griseus protease A (SGPA) and Streptomyces griseus protease B (SGPB), and the sprT gene that encodes Streptomyces griseus trypsin (SGT) were cloned from S. griseus and were overexpressed in various strains of S. griseus. When the sprT gene was introduced into S. griseus, trypsin activity increased 2-fold in the A-factor deficient mutant strain, S. griseus HH1, and increased 4-fold in the wild strain, S. grise us IFO 13350. However, there was no detectable increase of chymotrypsin activity in the transformants of S. griseus with either sprA or sprB, in contrast to the results obtained from S. lividans as a heterologous host. To solve the negative gene dosage effects in S. griseus, either the sprA or the sprB genes with their own ribosome binding sites were linked to the downstream of the entire sprT gene, and the coexpression efficiency was examined in S. lividans and S. griseus. The transformants of S. lividans with either pWHM3-TA (sprT+sprA) or pWHM3­TB (sprT+sprB) showed 3-fold increase of trypsin activity over that of the control, however, only the transformant of pWHM3-TB demonstrated 7-fold increase in chymotrypsin activity, indicating that the pWHM3-TB has a successful construction for the overexpression of chymotrypsin in Streptomyces. When the coexpression vectors were introduced into S. griseus IFO 13350, the trypsin level sharply increased by more than 4-fold, however, the chymotrypsin level did not increase. These results strongly suggest that the overexpression of the sprA and sprB genes is tightly regulated in S. griseus.

• 한국동물학회지
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• 제32권1호
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• pp.48-54
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• 1989

인체의 자궁암과 간암조직들이 나타내는 몇 종류의 단백질 분해효소들과 Anti-trypsin의 활성도를 정상조직의 것들과 비교하여서, 암의 종양성 형질과 단백질 분해활성의 변화사이에 어떤 상관관계가 있는지를 조사하였다. Casein과 Insulin의 분해 활성도는, 자궁암에서 2-3배 정도 증가하는 반면, 간암에서는 1/2에서 1/5정도로 감소하였다. 이와는 대조적으로, Anti-trypsin의 활성도는 자궁암에서 약 1/10정도로 감소하였고 간암에서는 2배 가량 증가하였다. 한편, Plamin-like enzyme과 Plasminogen activator의 활성도는 자궁암과 간암조직 모두에서 정상 조직에서보다 10-20% 정도 높게 나타났다. 이러한 결과는, 정상조직 내의 단백질 분해활성도가 단백질 분해효소들과 이들의 활성을 저해하는 단백질들의 균형에 의하여 조절됨을 시사하며, 암조직들에서는 각 암조직들의 종양특이성에 따라 단백질 분해효소와 저해단백질들 사이의 균형이 깨어짐에 따라 단백질 분해활성도가 다르게 나타남을 보여준다.

• 한국수산과학회지
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• 제45권1호
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• pp.50-55
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• 2012

We compared the nutritional requirements of whole larvae of the black seabream Acanthopagrus schlegeli, sweet fish Plecoglossus altivelis, olive flounder Paralichthys olivaeus and rock fish Sebastes schlegeli. The larvae were 20, 30, 14 and 5 DAH (or spawning) of black seabream, sweet fish, olive flounder and rock fish, respectively. Specific ${\alpha}$-amylase activity (mU/mg protein) was highest (8,324.9 mU/mg protein) in rock fish larvae (P<0.05). Specific trypsin-like protease activity was highest (11,330.1 mU/mg protein) in black seabream larvae (P<0.05), which also exhibited the highest activity, 685.5 mU/mg dry weight (P<0.05). The specific activities per mg protein and mg dry weight of black seabream were the highest (187.4 mU/mg protein and 11.3 mU/mg dry weight, respectively) (P<0.05). A/P, P/L and A/L ratios of rock fish were 1.47, 90.3 and 133.1, respectively (P<0.05). We present here basic larval digestive enzymatic nutritional requirement data.

• Journal of Microbiology and Biotechnology
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• 제11권6호
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• pp.1077-1086
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• 2001

The sprA and sprB gene encoding chymotrypsin-like proteases Streptomyces griseus protease A (SGPA) and Streptomyces griseus protease B (SGPB) and the sprT gene that encodes Streptomyces griseus trypsin (SGT) were cloned from Streptomyces griseus ATCC10137 and overexpressed in Streptomyces lividans TK24 as a heterologous host. The chymotrypsin activity of tole culture broth measured with the artificial chromogenic substrate , N-succinyl-ala-ala-pro-phe-p-nitroanilide, was 10, 14 and 14 units/mg in the transformants haboring the sprA, sprB and sprD genes, respectively. The growth of S. lividans reached the maximum cell mass after 4 days of culture, yet SGPA and SGPD production started in the stationary phase of cell growth and kept increasing for up to 10 days of culture in an R2YE medium. The trypsin activity of the culture broth measured with the artificial chromogenic substrate , N-${\alpha}$-benzoyl-DL- arginine-p-nitroanilide , was 16 units/mg and SGT production started in the stationary phase of cell growth and kept increasing for up to 10 days of culture in an R2YE medium. The introduction of the sprA gene into S, lividans TK24 triggered the biosynthesis of pigmented antibiotics, actinorhodin and undecylprodigiosin, and induced significant morphological changes in the colonies in Benedict, R2YE, and R1R2 media. In addition, the introduction of the sprT gene also induced morphological changes in the colony shape without affecting the antibiotic production, thereby implying that certain proteases would appear to play very important and specific roles in secondary-metabolites formation and morphological differentiation in Streptomyces.