혈합육어 Trypsin의 효소적 성질에 대한 반응속도론적 해석

조득문 ( Deuk Moon Cho ),허민수 ( Min Soo Heu ),김형락 ( Hyeung Rak Kim ),김두상 ( Doo Sang Kim ),변재형 ( Jae Hyeung Pyeun ) 한국수산학회 1996 한국수산과학회지 Vol.29 No.1

멸치, 고등어, 황다랭이 및 날개다랭이의 혈합육어에서 정제된 trypsin을 시료로 하여 각각의 BAPNA기질에 대한 반응속도와 그 관련 성질들을 분석 검토하였다. 4종의 혈합육어에서 정제된 trypsin의 Km`와 k(cat)는 멸치 trypsin이 각각 49.3μM과 90.9min 1, 고등어 trypsin A는 53.7μM과 61.2min 1 고등어 trypsin B는 96.5μM과 76.6min 1 황다랭이 trypsin은 62.8μM과 46.4min 1 그리고 날개다랭이 trypsin은 98.3μM과 47.68min 1이었다. TLCK에 대한 K1값은 멸치 trypsin이 20.90μM, 고등어 trypsin A가 2.86μM, 고등어 trypsin B가 3.90μM, 황다랭이 trypsin이 0.96μM, 그리고 날개다랭이 trypsin이 1.82μM이었으며, 황다랭이의 trypsin이 TLCK에 대하여 가장 예민하게 반응하였다. 이들 trypsin의 효소 활성과 촉매효율은 연근해 온 대산 혈합육어인 멸치와 고등어 trypsin이 열대해역에서 온대해역에 걸쳐 널리 회유하는 혈합육어인 황다랭이와 날개다랭이의 trypsin에 비하여 높은 특징을 보였다. Kinetic properties of trypsins purified from dark-fleshed fish (anchovy, mackerel, yellowfin tuna, and albacore) were examined and analyzed on benzoyl-(D,c)-arginine-p-nitroanilide (BAPNA). The values of Km` and k(cat) of the purified trypsins from the four dark-fleshed fish were found to be 49.3μM and 90.9min-1 for anchovy, 53.7μM and 61.2min-1 for mackerel A, 96.5μM and 76.6min-1 for mackerel B, 62.8μM and 46.6min-1 for yellowfin tuna, and 98.3μM and 47.7min-1 for albacore, respectively. The values of K, on tosyl-L-lysine chloromethyl ketone (TLCK) were determined to be 20.90μM for anchovy trypsin, 2.86μM for mackerel trypsin A, 3.90μM for mackerel trypsin B, 0.96μM for yellowfin tuna trypsin, and 1.82μM for albacore trypsin. Thus yellowfin tuna trypsin was the most sensitive to TLCK among all trypsins. The activities and catalytic efficiency of the trypsins purified from the temperate zone fish, anchovy and mackerel, were higher than those of the trypsins purified from yellowfin tuna and albacore which migrate widely from the tropic zone to the temperate zone.

Highly stable trypsin-aggregate coatings on polymer nanofibers for repeated protein digestion

Kim, Byoung Chan,Lopez-Ferrer, Daniel,Lee, Sang-Mok,Ahn, Hye-Kyung,Nair, Sujith,Kim, Seong H.,Kim, Beom Soo,Petritis, Konstantinos,Camp, David G.,Grate, Jay W.,Smith, Richard D.,Koo, Yoon-Mo,Gu, Man B WILEY-VCH Verlag 2009 Proteomics Vol.9 No.7

<P>A stable and robust trypsin-based biocatalytic system was developed and demonstrated for proteomic applications. The system utilizes polymer nanofibers coated with trypsin aggregates for immobilized protease digestions. After covalently attaching an initial layer of trypsin to the polymer nanofibers, highly concentrated trypsin molecules are crosslinked to the layered trypsin by way of a glutaraldehyde treatment. This process produced a 300-fold increase in trypsin activity compared with a conventional method for covalent trypsin immobilization, and proved to be robust in that it still maintained a high level of activity after a year of repeated recycling. This highly stable form of immobilized trypsin was resistant to autolysis, enabling repeated digestions of BSA over 40 days and successful peptide identification by LC-MS/MS. This active and stable form of immobilized trypsin was successfully employed in the digestion of yeast proteome extract with high reproducibility and within shorter time than conventional protein digestion using solution phase trypsin. Finally, the immobilized trypsin was resistant to proteolysis when exposed to other enzymes (i.e., chymotrypsin), which makes it suitable for use in “real-world” proteomic applications. Overall, the biocatalytic nanofibers with trypsin aggregate coatings proved to be an effective approach for repeated and automated protein digestion in proteomic analyses.</P>

Expression and Role of Trypsin-Like Enzyme Involved in Hatching of Preimplantation Mouse Embryos

김수경,강희규,전진현,최규완,김문규 한국발생생물학회 2001 발생과 생식 Vol.5 No.1

생쥐 초기배아의 체외배양 시 부화에 관련된 단백질 분해효소의 발현 시기와 존재부위를 알아보고 trypsin억제제 benzamidine을 배양액에 첨가하여 부화효소의 역할을 살펴보았다. 부화 효소로 제안되고 있는 trypsin 유사효소의 발현부위를 확인하기 위해 rhodamine이 부착되어 있는 Trypsin subsfrate probe를 이용하여 형광염색하였다. 생쥐 배아의 발생과정에서 trypsin 유사효소의 발현은 후기 상실 배아에서부터 관찰되었으며 This study was conducted to investigate the expression pattern of Trypsin-like enzyme and the effect of a trypsin inhibitor(benzimidine) on hatching process during in-vitro culture of mouse preimplantation embryos. The Trypsin-like enzyme was identified by rhodamine-conjugated Trypsin substrate probe. The expression of trypsin-like enzyme was firstly detected at the late morula stage, and the enzyme was uniformly localized in the trophectoderm of late blastocysts. Especially, intense fluorescence was observed in the blebbing area of hatching blastocysts. Bisbenzamidine, contained in culture media, did not alter embryonic development from 4-cell stage to the expanded blastocyst but decrease the hatching rate in ImM concentration (15.8% vs 89.7%, p<0.02). In the treatment of bisbenzimidine (5mM) for 12 hours according to the embryonic stage of mouse, the hatching rate of control (83.0%) and treatment in late blastocysts (8.7%) were significantly (p<0.01) different. From these results, we suggested that the hatching enzyme having trypsin-like activity was localized from the late morula stage, and the hatching process by this enzyme was activated in the late blastocyst stage of mouse embryos.

해수산 rotifer, Brachionus rotundiformis의 $\alpha$-amylase, total alkaline Protease, trypsin 및 triacylglycerol-lipase 활성 특성

권오남,박흠기,Kwon O-Nam,Park Heum-Gi 한국양식학회 2005 韓國養殖學會誌 Vol.18 No.4

본 연구는 rotifer, B. rotundiformis를 대상으로 소화효소 실험을 하기 위해 이들이 가지고 있는 소화효소의 최고 활성 조건을 확인하기 위해 수행하였다. rotifer, B. rotundiformis의 $\alpha$-amylase, total alkaline Protease, trypsin 및 TG-lipase는 Tris-HCl buffer 보다 phosphate-NaOH buffer 안정적인 효소활성을 보였다. $\alpha$-amylase, trypsin 및 TG-lipase는 pH 8.0에서, total alkaline proteaset pH 7.0에서 높은 효소 활성을 나타내었다. $\alpha$-amylase 활성은 $40^{\circ}C$에서 가장 높은 활성을 보였으며, total alkaline pretense와 trypsin은 $55{\~}60^{\circ}C$의 온도에서 높은 활성을 나타내었다. 반면 TG-lipase 활성은 $25{\~}30^{\circ}C$의 낮은 온도에서 활성이 높았다. $\alpha$-amylase, total alkaline pretense, trypsin 및 TG-lipase의 활성의 적정 기질 농도는 $3.5\%$ starch, $\0.6%$ azo-casein, $87.5{\mu}M$ BApNA and 81.2 mM olive oil이었다. $\alpha$-amylase, total alkaline protease, trypsin 및 TG-lipase의 활성의 적정 반응시간은 40, 60, 30 and 25 min으로 나타났다. 본 연구 결과에서 얻어진 자료는 rotifer, B. rotundiformis의 소화효소 연구를 위한 기초 자료로 이용될 것이다. This study was investigated the condition of their maximum activity to assay the enzymes of rotifer, Brachionus rotundiformis의 $\alpha$-amylase, total alkaline Protease, trypsin and TG-lipase activities of rotifer were higher and more sensitive in phosphate-NaOH buffer than Tris-HCl buffer. $\alpha$-amylase, trypsin and TG-lipase activities were appeared the maximum at pH 8.0, and total alkaline protease activity showed the maximum activity at pH 7.0. $\alpha$-amylase activity showed the highest activity at $40^{\circ}C$, and total alkaline protease and trypsin activities were assayed the highest at $55{\~}60^{\circ}C$. However, TG-lipase activity was appeared the highest at $25{\~}30^{\circ}C$. The optimum substrate concentration of enzyme activity of a-amylase, total alkaline protease, rypsin and TG-lipase were $3.5\%$ starch, $\0.6%$ azo-casein, $87.5{\mu}M$ BApNA and 81.2 mM olive oil, respectively. The optimum reaction time of enzyme activity of $\alpha$-amylase, total alkaline protease, trypsin and TG-lipase were increased up to 40, 60, 30 and 25 min., respectively. The data obtained in this study could be used for the digestive enzyme research of rotifer, B. rotundiformis.

Peptic and Tryptic Hydrolysis of Native and Heated Whey Protein to Reduce Its Antigenicity

Kim, S.B.,Ki, K.S.,Khan, M.A.,Lee, W.S.,Lee, H.J.,Ahn, B.S.,Kim, H.S. American Dairy Science Association 2007 Journal of dairy science Vol.90 No.9

This study examined the effects of enzymes on the production and antigenicity of native and heated whey protein concentrate (WPC) hydrolysates. Native and heated (10min at 100<SUP>o</SUP>) WPC (2% protein solution) were incubated at 50<SUP>o</SUP> for 30, 60, 90, and 120min with 0.1, 0.5, and 1% pepsin and then with 0.1, 0.5, and 1% trypsin on a protein-equivalent basis. A greater degree of hydrolysis was achieved and greater nonprotein nitrogen concentrations were obtained in heated WPC than in native WPC at all incubation times. Hydrolysis of WPC was increased with an increasing level of enzymes and higher incubation times. The highest hydrolysis (25.23%) was observed in heated WPC incubated with 1% pepsin and then with 1% trypsin for 120min. High molecular weight bands, such as BSA, were completely eliminated from sodium dodecyl sulfate-PAGE of both native and heated WPC hydrolysates produced with pepsin for the 30-min incubation. The α-lactalbumin in native WPC was slightly degraded when incubated with 0.1% pepsin and then with 0.1% trypsin; however, it was almost completely hydrolyzed within 60min of incubation with 0.5% pepsin and then with 0.5% trypsin. Incubation of native WPC with 1% pepsin and then with 1% trypsin for 30min completely removed the BSA and α-lactalbumin. The β-lactoglobulin in native WPC was not affected by the pepsin and trypsin treatments. The β-lactoglobulin in heated WPC was partially hydrolyzed by the 0.1 and 0.5% pepsin and trypsin treatments and was completely degraded by the 1% pepsin and trypsin treatment. Antigenicity reversibly mimicked the hydrolysis of WPC and the removal of β-lactoglobulin from hydrolysates. Antigenicity in heated and native WPC was reduced with an increasing level of enzymes. A low antigenic response was observed in heated WPC compared with native WPC. The lowest antigenicity was observed when heated WPC was incubated with 1% pepsin and then with 1% trypsin. These results suggested that incubation of heated WPC with 1% pepsin and then with 1% trypsin was the most effective for producing low-antigenic hydrolysates by WPC hydrolysis and obtaining low molecular weight small peptides. Further research is warranted to identify the low molecular weight small peptides in the WPC hydrolysates produced by pepsin and trypsin, which may enhance the use of whey.

택사(Alismatis Rhizoma) trypsin inhibitor의 정제와 특성

박종옥,이인섭 한국생명과학회 2002 생명과학회지 Vol.12 No.2

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. A trypsin inhibitor was isolated and purified from Azismatis Rhizoma which has been used as a galenic for diuretic and antiphlogistic. Purification was carried out by 0-80% saturated ammonium sulfate salting out, DEAE- cellulose ion exchange chromatogrphy, Sephadex G-150 gel filtration. The molecular weight of Alismatis Rhizoma trypsin inhibitor(ARTI) was estimated to be about 23,000 Da by gel filtration and SDS-PAGE, it must be monomer. ARTI was stable at 0~6$0^{\circ}C$, but at higher temperature its activity was decreased about 35%. When benzoyl-dl-arginine p-nitroanilide was used as a substrate of trypsin, half-maximal inhibition of ARTI was observed at 0.071 $\mu$M. ARTI inhibited the hydrolysis of trypsin non-competitively and Km value was 0.81 $\mu$M.

멸치 육과 내장으로부터 분리한 Cathepsin L, Chymotrypsin 및 Trypsin의 단백질분해 특성

번재형 ( Jae Hyeung Pyeun ),허민수 ( Min Soo Heu ),조득문 ( Deuk Moon Cho ),김형락 ( Hyeung Rak Kim ) 한국수산학회 1995 한국수산과학회지 Vol.28 No.5

어류의 사후 초기의 변화를 육 및 장기조직중에 분포하는 단백질분해효소의 작용과 관련하여 검토할 목적으로 멸치의 육 및 장기에서 분리한 cathepsin L과 chymotrypsin 및 trypsin의 단백질 기질에 대한 특성과 근원섬유단백질에 대한 분해능을 전기영동적으로 분석하여 다음의 결론을 얻었다. 이들 세 효소의 casein에 대한 친화도는 유사하였고, 근원섬유단백질에 대한 친화도는 casein에 대한 친화도보다 높았다. 멸치와 방어의 근원섬유단백질에 대한 cathepsin L과 chymotrypsin의 활성은 trypsin보다 훨씬 높게 나타났다. 0~25%까지의 식염농도에서 세 효소의 단백질분해활성은 식염의 농도에 반비례하였으며, 식염의 공존상태에서 세 효소는 casein 보다 근원섬유단백질에 대하여 높은 활성을 나타내었다. 근원섬유단백질의 효소 분해시에 cathepsin L은 chymotrypsin과 trypsin에 비하여 염농도와 온도에 의한 영향이 적었다. 따라서, 멸치의 사후변화와 젓갈 숙성중의 자가소화는 trypsin보다는 cathepsin L과 chymotrypsin의 단백질분해활성이 더욱 깊이 관여할 것으로 판단된다. Proteolytic properties of enzymes from the muscle and viscera of anchovy have been examined. Cathepsin L, chymotrypsin, and trypsin showed similar Km values for casein. However, they had higher Km values for myofibrillar proteins than those for casein. The k(cat) of cathepsin L and chymotrypsin for myofibrillar proteins were higher than that of trypsin, and also cathepsin L and chymotrypsin caused higher hydrolysis in myofibrillar proteins of anchovy and yellowtail. In the presence of sodium chloride (0~25%), proteolytic activity for myofibrillar proteins from yellowtail was higher than that for casein. Proteolytic activity was decreased with the increase of sodium chloride concentration. Cathepsin L had been less affected by NaCl concentration and temperature on the hydrolysis of myofibrillar proteins than chymotrypsin and trypsin. Cathepsin L and chymotrypsin were more responsible to the autolysis of muscle proteins from fish than trypsin.

개 트립신樣 면역반응성 단클론 항체의 제작

김성수,강지훈,정광면,유재철,정점규,양만표 한국임상수의학회 2008 한국임상수의학회지 Vol.25 No.5

Canine trypsin-like immunoreactivity (cTLI), which is a mirror of the concentration of trypsin and trypsinogen, is a pancreas-specific enzyme and a suitable marker for canine pancreatitis and especially exocrine pancreatic insufficiency (EPI). To develop the immunochromatographic test kit, monoclonal antibodies that recognize cTLI were prepared. Anionic trypsin, cationic trypsin, and chymotrypsin from canine pancreas were successfully purified to homogeneity, using ammonium sulfate fractionation and benzamidine-affinity chromatography. The purification fold for anionic trypsin was 108 times when compared with that of the homogenation of pancreas. The molecular weights by SDS-PAGE analysis were approximately 23 kDa for chymotrypsin and approximately 20 kDa for cationic trypsin and anionic trypsin, respectively. Using the purified trypsin-like proteins, ten hybridomas which secret canine trypsinspecific monoclonal antibody were prepared. Klotz plot indicated that hybridomas, 5G2H10G4 and 2F4A11, have high affinity constant (Ka) of 4.1 × 109 and 1.8 × 109, respectively. Especially, 5F9H3 showed the cationic typsin-specific binding pattern and its Ka was determined to 4.5 × 109. The development of immunochromatographic test kit using these monoclonal antibodies against cTLI will be very useful in the diagnosis of canine EPI or canine pancreatitis.

Inhibition of Trypsin-Induced Mast Cell Activation by Water Fraction of Lonicera japonica

Kang, Ok-Hwa,Choi, Yeon-A,Park, Hye-Jung,Lee, Joo-Young,Kim, Dae-Ki,Choi, Suck-Chei,Kim, Tae-Hyun,Nah, Yong-Ho,Yun, Ki-Jung,Choi, Suck-Jun,Kim, Young-Ho,Bae, Ki-Hwan,Lee, Young-Ml The Pharmaceutical Society of Korea 2004 Archives of Pharmacal Research Vol.27 No.11

Lonicera japonica Thunb.(Caprifoliaceae) has long been known as an anti-inflammatory. In the present study, the effect of water fraction of Lonicera japonica (LJ) on trypsin-induced mast cell activation was examined. HMC-1 cells were stimulated with trypsin (100 nM) in the presence or absence of LJ (10, 100, and 1000 $\mu$ g/mL). TNF-$\alpha$ and tryptase production were measured by enzyme-linked immunosorbent assay (ELISA) and reverse transcription-PCR. Extracellular signal-regulated kinase (ERK) phosphorylation was assessed by Western blot. Trypsin activity was measured by using Bz-DL-Arg-p-nitroanilide (BAPNA) as substrate. LJ (10, 100, and 1000 $\mu$g/mL) inhibited TNF-$\alpha$ secretion in a dose-dependent manner. LJ (10, 100, and 1000 $\mu$g/mL) also inhibited TNF-$\alpha$ and tryptase mRNA expression in trypsin-stimulated HMC-1. Furthermore, LJ inhibited trypsin-induced ERK phosphorylation. However, LJ did not affect the trypsin activity even 1000 $\mu$g/mL. These results indicate that LJ may inhibit trypsin-induced mast cell activation through the inhibition of ERK phosphorylation than the inhibition of trypsin activity.

Trypsin 반응에 대한 용매의 유전상수 및 압력의 영향

박현,지영민 한국산업미생물학회 2000 한국미생물·생명공학회지 Vol.28 No.1

Trypsin 촉매반응의 반응효율에 대하여 용매의 유전상수 및 압력이 어떠한 영향을 미치는가에 대하여 조사하였다. Trypsin 반응의 촉매효율은 용매의 유전상수가 감소함에 따라 대수적으로 증가하였다. 또한 용매의 유전상수의 감소에 대하여 활성화 체적은 직선적으로 감소하는 것을 보였는데, 이는 용매의 유전상수의 감소에 따라 반응중간과정에서 형성되는 극성 쌍극자에 대한 용매의 전축이 증가하여, 극성 쌍극자의 형성을 위한 정전기적 에너지가 감소되었음을 의미하는 것으로 해석되었다. Trypsin 촉매 반응에 대한 용매의 유전상수의 영향에 대하여 열역학 변수를 통하여 고찰해 본 결과 용매의 유전상수가 4.7 만큼 감소하였을 때 활성화 에너지는 2.262 KJ/mol이 감소하였고, 활성화 자유에너지는 3.169 KJ/mol이 감소되는 결과를 보였다. 이와 같은 결과로부터 용매의 유전상수를 조절함으로서 효소반응과정의 천이 상태를 안정화시킴으로서 반응 속도를 증가시킬 수 있음을 시사하고 있다. 따라서 효소를 이용하여 유용물질을 생산하고자 할 때 효소반응의 반응기작에 대한 더 많은 정보를 가지고 용매의 유전상수 및 압력을 조절함으로서 반응효율을 개선시킬 수 있을 것으로 사료된다. Electrostatic forces contribute to the high degree of enzyme transition state complementarity in enzyme catalyzed reaction and such forces are modified by the solvent through its dielectric constant and polar properties. The contributions of electrostatic interaction the formation of ES complex and the stabilization of transition state of the trypsin catalyzed reaction were probed by kinetic studies with high-pressure and solvent dielectric constant. A good correlation has been observed between the increase of catalytic efficiency of trypsin and the decrease of solvent dielectric constant. Activation volume linearly decreased as the dielectric constant of solvent decreased, which means the increase in the reaction rate. Moreover, the decrease of activation volume by lowering the solvent dielectric constant implies a solvent penetration of the active site and a reduction of electrostatic energy for the formation of dipole of the active site oxyanion hole. When the dielectric constant of the solvents was lowered to 4.7 unit, the loss of activation energy and that of free energy of activation were 2.262 KJ/mol and 3.169 KJ/mol, respectively. The results of this study indicate that the high pressure kinetics combined with solvent effects can provide unique information on enzyme reaction mechanisms, and the controlling the solvent dielectric constant can stabilize the transition state of the trypsin-catalyzed reaction.