• 한국생명과학회:학술대회논문집
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• 한국생명과학회 2001년도 10주년 기념 국제학술 심포지움 및 추계학술대회
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• pp.95-95
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• 2001

• Fisheries and Aquatic Sciences
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• 제1권2호
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• pp.209-215
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• 1998

Trypsin-like enzyme was purified from shrimp hepatopancreas through Q-Sepharose ionic exchange, benzamidine Sepharose-6B affinity, and Superdex 75 gel chromatography. Purity of trypsin-like enzyme was increased 69-fold with $44\%$ yield. The enzyme consisted of a single polypeptide chain with a molecular weight (M.W.) of 32 kDa judged by sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE). The enzyme was completely inactivated by serine enzyme inhibitors such as soybean trypsin inhibitor (SBTI), tosyl-L­lysine chloromethyl ketone (TLCK), and leupeptin. However, the enzyme was not affected by tosyl-L-phenylalanine chloromethyl ketone (TPCK) which is a chymotrypsin specific inhibitor. The enzyme had no activity against benzoyl-tyrosine ethyl ester (BTEE) which is a chymotrypsin specific substrate. The enzyme showed high activity on the carboxyl terminal of Phe, Tyr. Glu, Arg, and Asp. However. no activity was detected against the carboxyl terminal of Pro, Trp, Cys, Gly, Val, and Ala.

• 생명과학회지
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• 제12권2호
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• pp.151-157
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• 2002

한방재료의 하나인 택사(Alismatis Rhizoma, AR)로부터 단백성 trypsin inhibitor(TI)를 분리, 정제하여 특성을 조사하였다. 정제과정은 0-80.% 포화 황산암모늄을 이용한 염석법, DEAE-cellulose ion exchange chromatography, Sep-hadex G-150 chromatography 등을 이용하였다. 정제된 ARTI의 분자량을 gel filtration과 SDS-PAGE 한 결과 모두 약 23,000 Da으로 나타나 monomer로 되어 있는 것으로 나타났다. 온도안정성에 있어 0-6$0^{\circ}C$에서는 안정하였으나 그 이상의 온도에서는 약 35%가지 안정성이 떨어졌다. ARTI와 상품화된 soybean kunitz inhibitor의 저해능을 비교해 본 결과 ARTI 및 soybean inhibitor 각각의 농도가 0.071 $\mu$M, 1.7 $\mu$M일 때 0.025 g/$m\ell$ trypsin활성을 50% 정도 저해하는 것으로 나타났다. ARTI의 trypsin의 가수분해반응에 대한 저해형태는 비경쟁적 저해형인 것으로 나타났으며 km값은 0.81 $\mu$M이었다.

• 한국수산과학회지
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• 제29권6호
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• pp.797-804
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• 1996

Two trypsins were purified from shrimp hepatopancreas through ammonium sulfate fractionation, Q-Sepharose ionic exchange, benzamidine Sepharose-6B affinity, and Sephacryl S-300 gel chromatography. Both enzymes had a single polypeptide chain with a molecular weight (M.W.) of 32 kDa by sodium dodecylsulfate polyacrylamide gel electrophoresis (SOS-PAGE), although trypsin A and B were estimated to be a molecular weight of 27.2 and 22.8 kDa, respectively, using Sephacryl S-300 gel filtration. Both trypsins had similar amino acid compositions and rich in glycine, valine, alanine, aspartic acid, and glutamic acid, but low in methionine and basic amino acids. Both enzymes were completely inactivated by soybean trypsin inhibitor (SBTI), phenylmethylsulfonyl fluoride (PMSF), tosyl-L-lysine chloromethyl ketone (TLCK), benzamidine, leupeptin, however, not affected by tosyl-L-phenylalanine chloromethyl ketone (TPCK) and pepstatin.

• 약학회지
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• 제52권1호
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• pp.79-84
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• 2008

Alismatis Rhizoma has been used as diuretics and antiphlogistics in the Chinese oriental medicine. A trypsin inhibitor was isolated from Alismatis Rhizoma using DEAE ion exchange column, trypsin affinity column, and FPLC chromatography, and its activity and characteristics were studied. The purifed Alismatis Rhizoma trypsin inhibitor (ARTI) was estimated to be about 22 kDa. The sequence determination on N-terminal amino acid residues and 84 amino acid residues has been completed, yet no homology has been found with trypsin inhibitors reported at NCBI. ARTI did not show inhibitory activities on chymotrypsin and elastase, however it exhibited a significant inhibitory activity on bovine trypsin, and formed a complex with rat liver 10-formyltetrahydrofolate dehydrogenase.

• Journal of Microbiology and Biotechnology
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• 제19권10호
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• pp.1191-1196
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• 2009

The production of Streptomyces griseus trypsin (SGT) by S. griseus IFO13350 transformed with the expression vector pWHM3-TR1R2, containing sprT encoding SGT and the two positive regulatory genes sgtR1 and sgtR2, was investigated in various media. Cultivation in Ferm-0 gave 1.4 times more trypsin activity than in C5/L medium. In addition, replacement of 2% glucose and 1% skim milk in Ferm-0 with 2% dextrin and 1% tryptone (designated Ferm-II) enhanced trypsin activity 4.1-fold. To simplify the purification process, the supernatant from the S. griseus transformant cultured in Ferm-II medium was fractionated with ammonium sulfate (25-55%), then subjected to Hitrap Benzamidine FF affinity column chromatography. The specific activity of SGT purified by one-step chromatography was 69,550 unit/mg protein and the overall purification yield was above 8%, indicating that this method is more effective than those previously reported. Purified SGT was most active at pH 8.0 and $50^{\circ}C$, and it maintained activity between pH 7.0 and 9.0 and at temperatures up to $70^{\circ}C$. These enzymatic properties are very similar to those of authentic eukaryotic trypsin purified from bovine pancreas.

• 한국어업기술학회:학술대회논문집
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• 한국어업기술학회 1998년도 수산관련공동학술대회발표요지집
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• pp.266-267
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• 1998

• 한국식품영양과학회지
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• 제22권4호
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• pp.448-457
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• 1993

혈합육어 중 연근해 온대산 멸치의 내장과 고등어의 유문수, 그리고 원양 열대산 황다랭이 및 날개다랭이의 유문수를 각각 시료로 하여 trypsin을 분리 정제하였으며, 그 분자량과 아미노산 조성 및 효소의 반응 최적조건에 관하여 비교 분석하였다. 실험결과를 요약하면 다음과 같다. 1. 멸치의 내장과 고등어, 황다랭이 및 날개다랭이의 유문수에서 각각 황산암모늄염석, benzamidine-Sepharose 6B 친화성 크로마토그래피, DEAE-Sephadex A-50 크로마토그래피, Sephadex G-75 겔 여과 크로마토그래피 등의 방법을 혼용하여 고등어로부터는 2종의 trypsin (고등어 trypsin A와 고등어 trypsin B로 명명함)을, 그리고 다른 어류로부터는 각각 1종의 trypsin을 분리 정제하였다. 2. 멸치 trypsin과 고등어 trypsin B는 고등어 trypsin A와 황다랭이 trypsin 및 날개다랭이 trypsin에 비하여 그 고유활성이 월등히 높았다. 3. 이들 혈합육어 trypsin의 분자량은 22kDa-26kDa 범위였다. 4. 이들 trypsin은 공통적으로 glycine, serine, aspartic acid를 많이 함유하고 있었으며, tryptophan, methionine, lysine, tyrosine의 함유량은 적었다. 5. BA-$\rho$-NA기질에 대한 반응 최적조건은 멸치 trypsin은 pH 8.0에서 45$^{\circ}C$, 고등어 trypsin A와 B는 pH 8.0에서 5$0^{\circ}C$, 그리고 황다랭이 trypsin은 PH 9.0에서 55$^{\circ}C$, 날개다랭이 trypsin은 pH 9.0에서 5$0^{\circ}C$였다. 6. 위에 든 실험 결과들은 혈합육 어류의 서식온도가 이들 어류의 trypsin의 반응 최적 온도와는 어느 정도 관계가 있을 것으로 추측되었다.

• Journal of Microbiology and Biotechnology
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• 제8권4호
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• pp.333-340
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• 1998

Streptomyces griseus trypsin (SGT) is an extracellular proteinase produced by S. griseus. The sprT gene, which encodes premature SGT protein, was cloned into the plasmid pWHM3, a Streptomyces-E. coli shuttle vector. When the recombinant plasmid was introduced into Streptomyces lividans TK24, two proteins with molecular weights of 28 kDa and 42 kDa were detected. The 28-kDa protein was a SGT protein while the larger 42-kDa protein is thought to have been a premature form of the SGT protein. The SGT protein was purified to homogeneity via ammonium sulfate fractionation and many column chromatographies, including CM -sepharose chromatography, Mono-S chromatography, and Superose-12 chromatography, from the culture broth of S. lividans TK24 harboring the sprT gene. The N-terminal amino acid sequence, isoelectric points, and stabilities at various conditions of the SGT proteins purified from the Pronase and transformant were almost identical. The amount of the expressed SGT in S. lividans TK 24 was determined to be 5 times more than that of S. griseus based on the enzymatic activity against artificial substrate.

• 한국미생물·생명공학회지
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• 제20권5호
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• pp.534-542
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• 1992

Streptomyces S-217 균주가 생산하는 trypsin 저해물질의 생산 및 정제조건을 검토하였다. 저해물질의 생산은 500ml 후라스크의 배양에서 2 mannitol, 0.9, peptone의 배지와 초기 pH는 7.0 배양시간은 66시간 및 온도30$^{\circ}C$의 조건에서 제일 높았으며, 무기염의 효과는 크게 영향이 없었다. 정제는 column chromatography와 HPLC에 의하여 정제하였다. 정제된 trypsin 저해물질의 초대파장은 $\lambda_{max}$ 215nm이었고, 용해도는 물에는 95, methanol과 dimethylsulfoxide에서는 70% 및 75%이었다.Trypsin 저해물질 복합체 형성시간은 10분 정도였으며, trypsin 효소의 50% 저해농도($IC^{50}$)는 15$\mu$g/ml 이었다. 저해물질의 pH 안정성은 $100^{\circ}C$ 10분간 열처리시 pH 5~9에서 100% 활성이 유지되었으며, 산성측보다 알칼리측이 안정하였고, 열안정성은 $100^{\circ}C$, pH7.0에서 1시간 동안 50% 활성을 유지하였다.