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.

어류 알의 Protease Inhibitor 활성 분포

지성준 ( Seong Jun Ji ),이지선 ( Ji Sun Lee ),신준호 ( Joon Ho Shin ),박권현 ( Kwon Hyun Park ),김진수 ( Jin Soo Kim ),김경섭 ( Kyoung Sub Kim ),허민수 ( Min Soo Heu ) 한국수산과학회(구 한국수산학회) 2011 한국수산과학회지 Vol.44 No.1

To identify and examine the distribution of proteolytic inhibitory activity in crude extracts from fish eggs, and to determine the applicability of these protease inhibitors as anti-degradation agents in surimi-based products and fish meat, we compared the inhibitory activities of various extracts from fish eggs to those of commercial proteases, such as trypsin and papain. We used the optimal conditions for the screening of trypsin activity: 30 ug/uL of 0.1 % trypsin and 0.6 mM Na-benzoyl-L-arginine-p-nitroanilide (BAPNA) with a pH of 8.0 at 40℃ for 60 min. The activities of papain and four commercial proteases were investigated after mixing with 100 ug/uL enzymes and 0.3% casein with a pH of 8.0 at 40℃ for 60 min. We performed a screening assay to detect the inhibitory activity (%) of crude extracts from eight species of fish eggs against the target proteases trypsin and papain. The assay revealed a wide distribution of trypsin and papain inhibitors in fish eggs. The specific inhibitory activities (11.6-28.6 U/mg) of crude extracts from fish eggs against trypsin and BAPNA substrate were higher than that (0.64 U/mg) of egg whites, used as a commercial inhibitor. The inhibitory activities of crude extracts from fish eggs against trypsin, and of egg whites against casein substrate (1.94-4.51 U/mg), were higher than those of papain (0.24-1.57 U/mg) and commercial protease (0.04-0.32 U/mg). The extracts from fish eggs were rich in protease inhibitors that exhibited strong inhibitory activity against trypsin, a serine protease, and papain, a cysteine protease.

Co-Expression of a Chimeric Protease Inhibitor Secreted by a Tumor- Targeted Salmonella Protects Therapeutic Proteins from Proteolytic Degradation

( David Quintero ),( Jamie Carrafa ),( Lena Vincent ),( Hee Jong Kim ),( James Wohlschlegel ),( David Bermudes ) 한국미생물 · 생명공학회 2018 Journal of microbiology and biotechnology Vol.28 No.12

Sunflower trypsin inhibitor (SFTI) is a 14-amino-acid bicyclic peptide that contains a single internal disulfide bond. We initially constructed chimeras of SFTI with N-terminal secretion signals from the Escherichia coli OmpA and Pseudomonas aeruginosa ToxA, but only detected small amounts of protease inhibition resulting from these constructs. A substantially higher degree of protease inhibition was detected from a C-terminal SFTI fusion with E. coli YebF, which radiated more than a centimeter from an individual colony of E. coli using a culturebased inhibitor assay. Inhibitory activity was further improved in YebF-SFTI fusions by the addition of a trypsin cleavage signal immediately upstream of SFTI, and resulted in production of a 14-amino-acid, disulfide-bonded SFTI free in the culture supernatant. To assess the potential of the secreted SFTI to protect the ability of a cytotoxic protein to kill tumor cells, we utilized a tumor-selective form of the Pseudomonas ToxA (OTG-PE38K) alone and expressed as a polycistronic construct with YebF-SFTI in the tumor-targeted Salmonella VNP20009. When we assessed the ability of toxin-containing culture supernatants to kill MDA-MB-468 breast cancer cells, the untreated OTG-PE38K was able to eliminate all detectable tumor cells, while pretreatment with trypsin resulted in the complete loss of anticancer cytotoxicity. However, when OTG-PE38K was co-expressed with YebF-SFTI, cytotoxicity was completely retained in the presence of trypsin. These data demonstrate SFTI chimeras are secreted in a functional form and that co-expression of protease inhibitors with therapeutic proteins by tumor-targeted bacteria has the potential to enhance the activity of therapeutic proteins by suppressing their degradation within a proteolytic environment.

Lipoxygenase 및 Kunitz Trypsin Inhibitor 단백질 결핍 콩 계통 육성

김명식 ( Myung Sik Kim ),성미경 ( Mi Kyung Sung ),서상배 ( Sang Bae Seo ),김경록 ( Kyung Roc Kim ),이경자 ( Kyoung Ja Lee ),박모세 ( Mo Se Park ),정종일 ( Jong Il Chung ) 한국콩연구회 2008 韓國콩硏究會誌 Vol.25 No.1

성숙한 콩 종실에 존재하는 성분 중 비린내를 일으키는 lipoxygenase와 단백질의 분해를 방해하는 효소인 Kunitz trypsin inhibitor (KTI) 단백질이 결핍된 녹색자엽 검정종피 및 노란종피 계통을 육성하기 위해여 진행된 연구에서 얻어진 결과는 다음과 같다. P1 x P2 및 P3 x P4의 교배로부터 얻어진 F2 종자에서 lipoxygenase-2, 3 및 KTI 단백질이 결핍된 종자가 선발되었다. 계통육종법으로 세대를 진전시킨 결과 녹색자엽이고 검정 종피색을 가진 lipoxygenase 및 KTI 단백질 결핍 계통은 생육습성이 직립형이고 신육형은 유한신육형이며 경장은 약 50cm로 나타났다. 종실 크기는 백립중이 약 23.2 g 정도이고 종실모양은 편구형이며 6월 초순 파종시 성숙기가 10월 초순경이었다. 노란종피를 가진 lipoxygenase 및 KTI 단백질 결핍 계통은 배축색이 중간 정도의 자색이며 신육형은 유한신육형이었다. 생육습성은 직립형이고 경장은 약 54cm로 중간 정도이며, 화색은 담자색이며 백립중이 약 24g 정도이고 종실모양은 구형이며 제색은 흑색이었다. Lipoxygenase and Kunitz trypsin inhibitor proteins in mature soybean seed are well known as major anti-nutritional factors. Lipoxygenase is responsible for the beany flavor and soybean Kunitz trypsin inhibitor (KTI) protein is responsible for the inferior nutritional quality of unheated or incompletely heated soybean meal. Anthocyanins from black soybean seed coat are known to have many pharmaceutical effects. The objective of this study was to breed soybean genotype of black coated-seed lacking lipoxygenase-2,3 and KTI protein and to breed soybean genotype of yellow coated-seed lacking lipoxygenase-2,3 and KTI protein. Two breeding populations were developed using four different parents. In F2 seed generations of both populations, F2 seeds deficient in lipoxygenase-2,3 and KTI protein were selected by SDS-PAGE. Selected F2 seeds were planted to advance F2 plant. Traits for seed coat and mature embryo colors were observed on the F3 seeds harvested from each F2 plant. F3 lines were advanced from F3 seeds selected. Plant type, height, and maturity date for each F3 plant within line were observed. After single plant harvesting, seed color and absence of lipoxygenase-2,3 and KTI protein were confirmed on random F4 seed of each F3 plant. Selected lines were advanced to the F6 generation. Maturity date, stem type, seed coat and cotyledon color, seed weight were observed on the new soybean lines with absence of lipoxygenase-2,3 and KTI protein.

A Simple and Rapid Method to Isolate Low Molecular Weight Proteinase Inhibitors from Soybean

Hari B. Krishnan 韓國作物學會 2004 Korean journal of crop science Vol.49 No.4

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the 60~% isopropanol extract of soybean(Glycine max [L.] Merr.) seed revealed two abundant proteins with molecular masses of 19 and 10 kDa. Amino acid analysis revealed that the isopropanol-extractable protein fraction was rich in cysteine. Two-dimensional gel electro-phoretic analysis indicated that the 19kDa and 10kDa proteins had pI of 4.2 and 4.0 respectively. Peptide mass fingerprints of trypsin digests of the two proteins obtained using matrix-assisted, laser desorption/ionization-time of flight (MALDI-TOF) mass spectroscopy revealed the 19kDa protein was Kunitz trypsin inhibitor and the 10kDa protein was Bowman-Birk proteinase inhibitor. When resolved under non-denaturing conditions, the isopropanol-extracted proteins inhibited trypsin and chymotrypsin activity. Results presented in this study demonstrate that isopropanol extraction of soybean seed could be used as a simple and rapid method to obtain a protein fraction enriched in Kunitz trypsin and Bowman-Birk proteinase inhibitors. Since proteinase inhibitors are rich in sulfur amino acids and are putative anticarcinogens, this rapid and inexpensive isolation procedure could facilitate efforts in nutrition and cancer research.

A Simple and Rapid Method to Isolate Low Molecular Weight Proteinase Inhibitors from Soybean

Krishnan Bari B. The Korean Society of Crop Science 2004 Korean journal of crop science Vol.49 No.4

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the $60\%$ isopropanol extract of soybean(Glycine max [L.] Merr.) seed revealed two abundant proteins with molecular masses of 19 and 10 kDa. Amino acid analysis revealed that the isopropanol-extractable protein fraction was rich in cysteine. Two-dimensional gel electro-phoretic analysis indicated that the 19kDa and 10kDa proteins had pI of 4.2 and 4.0 respectively. Peptide mass fingerprints of trypsin digests of the two proteins obtained using matrix-assisted, laser desorption/ionization-time of flight (MALDI-TOF) mass spectroscopy revealed the 19kDa protein was Kunitz trypsin inhibitor and the 10kDa protein was Bowman-Birk proteinase inhibitor. When resolved under non-denaturing conditions, the isopropanol-extracted proteins inhibited trypsin and chymotrypsin activity. Results presented in this study demonstrate that isopropanol extraction of soybean seed could be used as a simple and rapid method to obtain a protein fraction enriched in Kunitz trypsin and Bowman-Birk proteinase inhibitors. Since proteinase inhibitors are rich in sulfur amino acids and are putative anticarcinogens, this rapid and inexpensive isolation procedure could facilitate efforts in nutrition and cancer research.

감태 물 추출물의 Trypsin 저해활성에 대한 열 및 pH 안정성

정슬아(Seul-A Jung),김꽃봉우리(Koth-Bong-Woo-Ri Kim),김민지(Min-Ji Kim),김동현(Dong-Hyun Kim),선우찬(Chan Sunwoo),김현지(Hyun-Jee Kim),정다현(Da-Hyun Jeong),정희예(Hee-Ye Jeong),김태완(Tae-Wan Kim),조영제(Young-Je Cho),안동현(Dong-Hyun 한국식품영양과학회 2012 한국식품영양과학회지 Vol.41 No.6

본 연구는 감태 물 추출물의 일반특성과 trypsin 저해활성을 알아보고 산업적으로 이용가능성을 확인하기 위하여 열 및 pH에 대한 안정성을 실시하였다. 감태 물 추출물의 색도 및 pH를 측정한 결과, 색도는 명도가 86.21로 높고 적색도 및 황색도가 각각 0.38 및 15.49로 낮게 나타났으며 pH는 6.17로 약산성으로 나타났다. 감태 물 추출물의 trypsin 저해활성은 5, 2.5 및 1 ㎎/㎖ 농도에서 각각 76.21%, 62.41% 및 60.41%를 나타냈으며 IC50값은 0.83 ㎎/㎖이었다. 열 및 pH에 대한 안정성을 측정한 결과, 80oC까지 열처리에 안정하였고 pH 2~8 범위에서 안정하였으나 100℃와 121℃ 열처리와 pH 10에서 활성이 약간 감소하였으나 전체적으로 높은 활성을 유지하여 열 및 pH에 안정한 것을 확인하였다. 이상의 결과를 통해 감태 물 추출물이 지니는 trypsin 저해활성이 열 및 pH에 대해 안정성을 지녀 식품산업에 응용가능할 것으로 사료된다. This research was done to verify the inhibitory activity of water extracts from Ecklonia cava(WE-EC) against trypsin and the effects on various temperature and pH conditions. The WE-EC showed high trypsin inhibitory activity of 76, 62 and 60% at concentrations of 5, 2.5 and 1 ㎎/㎖, respectively. In all heat treatments excepted for two conditions, such as 100℃ for 20 min and 121℃ for 15 min, the inhibitory activity was stable compared with the untreated group. With regard to pH stability, the WE-EC showed no significant changes at pH 2~8, but somewhat decreased inhibitory activity was revealed at pH 10. Therefore, the WE-EC could be used in the food industry as a natural trypsin inhibitor.

Characterization of the AlTI13 protein from Indian siris (Albizia lebbeck) that inhibits the growth of cotton bollworm (Helicoverpa armigera)

Faiyaz K. Shaikh,Prafull P. Gadge,Ashok A. Shinde,Manohar V. Padul,Manvendra S. Kachole 한국응용곤충학회 2014 Journal of Asia-Pacific Entomology Vol.17 No.3

Here, we report multiple molecular forms of Albizia lebbeck trypsin inhibitors (AlTIs) by using a simple and sensitivegel X-ray film contact print technique. About 17 AlTIs were detected in the seed extracts of A. lebbeck. Twogroups of AlTIs—1 major (10 AlTIs; slow migration on the gel) and 1 minor (7 AlTIs; fast migration on the gel)were identified. The formerwas specific only toward trypsin. However, the latter was specific toward both trypsinand Helicoverpa armigera gut proteinases (HaGPs). The most potent AlTI (AlTI13) was purified to assess itsin vivo bioefficacy toward HaGPs. Purification was achieved using (NH4)2SO4 fractionation, Sephadex G-100 columnchromatography, and preparative native-polyacrylamide gel electrophoresis (PAGE). The dose dependentbioefficacies of AlTIs in the (NH4)2SO4 F3 fractions (0.1%, 0.5%, and 1%) were approximately 79%, 83%, and 90%,respectively, resulting in reductions in the average larval weight of H. armigera. Artificial diet containing a singledose of AlTI13 (5 μg/g diet) reduced the larval weight by about 76%, with 60% mortality. The half-maximal inhibitoryconcentrations (IC50) of AlTI13 for trypsin and HaGPs were 0.14 and 0.17 μmol/ml, respectively. The optimumconditions for AlTI13 were pH 8 and temperatures ranging from 35 to 40 °C. Reducing sodium dodecylsulfate-PAGE analysis indicated that ~28 kDa Kunitz-like trypsin inhibitor was present. Thus, we showed thatAlTIs, particularly, AlTI13 of A. lebbeck could be used as a transgene macromolecule to markedly increase insectresistance in genetically engineered plants.

Characterization and Potent Application of Pleurotus floridanus Trypsin Inhibitor (PfTI)

Manzur Ali Pannippara,Sapna Kesav,Rekha Mol Kollakal Naduvil Raghavan,Abraham Mathew,Sarita Ganapathy Bhat,Elyas Kothanan Kozhiyil 한국생약학회 2020 Natural Product Sciences Vol.26 No.3

Characterization and in vitro inhibition studies of protease inhibitor from the mushroom Pleurotus floridanus (PfTI) towards the pest Papilio demoleus is studied. The addition of 1 mM Mn2+, Na2+, Ba2+ and Ni 2+ enhanced the PfTI activity. The ICP-atomic emission spectrum showed the presence of Ca2+, Mg2+ and Zn2+ in the PfTI. Surfactants SDS and CTAB at a concentration of 1% reduced the PfTI activity whereas, the nonionic detergents Triton X and Tween 80 increased the activity. The inhibitory activity gradually decreased with increase in concentration of DMSO and H2O2. The activity was increased by dithiothreitol up to a concentration of 80 µM and inactivated at 140 µM. The activity of PMSF modified PfTI was drastically reduced to 0.234 U/mL at 4 mM concentration and similar results were obtained for modification of cysteine by N-Ethylmaleimide at slightly higher concentrations. The complex of trypsin and PfTI showed complete loss in fluorescence intensity at 343 nm compared with control. In vitro inhibition studies of PfTI with midgut proteases isolated from citrus pest P. demoleus with protease activity of 1.236 U was decreased to 0.613 U by 50 µL (0.1 mg/mL) of the inhibitor. Inhibitor was stable up to 0.04 M concentration of HCl.

Effects of Heat Treatment on Soybeans With and Without the Gene Expression for the Kunitz Trypsin Inhibitor: Chick Growth Assays

Burnham, L.L.,Kim, I.H.,Hancock, J.D.,Lewis, A.J. Asian Australasian Association of Animal Productio 2000 Animal Bioscience Vol.13 No.12

A total of 864 broiler chicks were used at Kansas State University and the University of Nebraska to determine the effects of heat treatment of two soybean genotypes on the growth performance. The soybeans were Williams 82 variety with (+K) and without (-K) gene expression for the Kunitz trypsin inhibitor. Heat treatment (autoclaving at $121^{\circ}C$ and $1.1kg/cm^2$) was applied for 0, 3, 6, 12, 18, and 24 min, resulting in a $2{\times}6$ factorial arrangement of treatments. Station and station treatment effects occurred, indicating that response in nutritional value of the soybean genotypes to heat treatment varied from year to year and location to location. However, the interactions were in magnitude of response rather than direction of response, with greater reductions in trypsin inhibitor concentrations for the soybeans heat processed at the Nebraska location. Pooled data indicated that -K supported greater (p<0.001) ADG, ADFI and gain/feed than the +K genotype. As the length of heat treatment increased, the ADG, ADFI, and the gain/feed ratio increased for chicks fed both soybean genotypes (p<0.0001). However, heating the -K soybeans resulted in a greater response in ADG, ADFI, and gain/feed than heating the +K soybeans (genotype heat treatment interaction, p<0.001). Pancreatic weights (mg pancreas/g of BW) of chicks fed -K soybeans were reduced compared to those from chicks fed +K (p<0.001). Increasing heat treatment decreased pancreas weights in chicks fed both soybean genotypes (p<0.001). Chicks fed heated soybeans in the Nebraska experiment had lower pancreatic weights than chicks fed heated soybeans in the Kansas experiment (station heat treatment interaction, p<0.0001). Chick growth performance was improved and pancreatic weights decreased by feeding raw -K soybeans versus raw +K soybeans, and by increasing heat treatment of both soybean genotypes. However, the response to heat treatment was not independent of genotype. Both +K and -K soybeans heated for 24 min supported similar ADG, ADFI, gain/feed, and pancreas weights, although chicks fed raw +K soybeans had lower growth performance than chicks fed -K soybeans. In conclusion, raw -K soybeans supported greater growth performance in broiler chicks than raw +K soybeans, although this advantage was lost when both soybean genotypes were heated for 24 min. Heat treatment of +K soybeans supported similar growth performance to heated -K soybeans, even though +K soybeans supported lower rates and efficiencies of gain than -K soybeans when fed raw.