Preparative Biochemistry & Biotechnology, 44:545–557, 2014 Copyright # Taylor & Francis Group, LLC ISSN: 1082-6068 print/1532-2297 online DOI: 10.1080/10826068.2013.835733
ISOLATION OF A TRYPSIN–CHYMOTRYPSIN INHIBITOR AND ITS FUNCTIONAL PROPERTIES
Shaoyun Wang,1,2 Biao Shao,3 Wei Lu,2 Jing Hong,1,2 and Pingfan Rao2 1 State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi, People’s Republic of China 2 Department of Food Science, Fuzhou University, Fuzhou, People’s Republic of China 3 Nantong Products Quality Supervision and Inspection Institute, Nantong, People’s Republic of China & A novel trypsin inhibitor with thermal and pH stability, designated Merrtine, was isolated from Glycine max L. merr. The procedure involved ammonium sulfate precipitation, ion-exchange chromatography on CM-Sephadex C-50, and affinity chromatography on Affi-gel blue gel. The 20 Nterminal amino acid sequences were determined to be DEYSKPCCDLCMCTRRCPPQ, demonstrating high homology with the sequence of Bowman–Birk type trypsin inhibitors. The molecular mass and isoelectric point of the inhibitor were estimated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and isoelectric focusing to be 20.0 kD and 5.8, respectively. Trypsin could be completely inhibited by Merrtine when the molar ratio was 8.1. The inhibitory activity of Merrtine was unaffected after exposure to temperatures up to 85 C, as well as within the pH range 2–12. Besides inhibiting trypsin–chymotrypsin, the inhibitor demonstrated additional antifungal activity against the species of Alternaria alternate, Fusarium oxysporum, Pythium aphanidermatum, Physalospora piricola, Botrytis cinerea, and Fusarium solani. We herein report not only the trypsin inhibitor’s extraction and isolation for the first time, but also its physiochemical and antifungal properties. Keywords antifungal, Glycine max L.merr, thermostable, trypsin inhibitor
INTRODUCTION Protease inhibitor, as one of components in plant tissue, is usually considered with allergies that have a negative effect on the health of the human body.[1] However, it has drawn more and more attention from a number of investigators for its diverse bioactivities, such as insecticidal, antiviral, and antifungal activities.[2–4] Combined with these bioactivities, protease inhibitors show potential application in fighting against virus (HIV and SARS virus), insects, and fungi.[2,4] Address correspondence to Shaoyun Wang, Department of Food Science, Fuzhou University, Fuzhou 350002, P.R. China. E-mail: [email protected] Color versions of one or more of the figures in the article can be found online at www.tandfonline. com/lpbb.
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Trypsin inhibitors, listed as one type of protease inhibitors, usually show inhibiting activities to trypsin–chymotrypsin.[5] They include Kunitz-type trypsin inhibitors and Bowman–Birk trypsin inhibitors.[5–7] Sometimes a combination of both Bowman–Birk and Kunitz-type trypsin inhibitors is found in a single plant tissue. For example, yellow soybean (Glycine max) concurrently produced two types of trypsin inhibitors.[8] Besides inhibiting trypsin–chymotrypsin, protease inhibitors are also included in the antifungal family with a range of well-known antifungal proteins and peptides such as lysozyme, lectin, chitinase, nonspecific lipid transfer protein, and defense peptide.[9–13] A number of members of the antifungal family have demonstrated their tremendous potential in protecting crops and thus important economic implications due to their defenses against both insects[10] and fungi.[4,11] Chinese Glycine max L. merr (black soybean) is a special cultivar of Glycine max (yellow soybean). It is extensively cultivated in Asian countries and serves as a healthy food, with a function in the traditional Chinese menu different from that of the yellow soybean. However, it has not been studied as extensively as the yellow soybean. In view of the scarcity of literature about the constituents of Glycine max L. merr, we herein report not only the trypsin inhibitor’s extraction and preparation for the first time, but also its physiochemical and antifungal properties. MATERIALS AND METHODS Materials Black soybean (Glycine max L. merr) was purchased from a local supermarket in Fuzhou, China. CM-Sephadex C-50 and Affi-gel blue gel were purchased from Amersham Biosciences (Sweden) and Bio-Rad (USA), respectively. Proteins marker was purchased from Gibco-BRL (Life Tech., USA). All chemicals were of the highest purity available. Sample Preparation One hundred grams of black soybean was soaked in distilled water for 12 hr and homogenized in 0.20 M sodium acetate buffer, pH 5.4. The homogenate was centrifuged at 10,000 g for 20 min at 4 C. The supernatant was collected for further study. Isolation of Merrtine The supernatant was firstly brought to 20% saturation with solid ammonium sulfate. The solution was centrifuged at 10,000 g for 20 min
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and the resulting supernatant was then adjusted to 80% saturation with ammonium sulfate. The precipitate obtained from centrifugation (10,000 g, 20 min) was collected and dissolved in 300 mL of 0.02 M sodium acetate buffer (pH 5.4). The dissolved solution was sequentially dialyzed against 0.02 M sodium acetate buffer (pH 5.4) overnight at 4 C with several changes. The dialyzed solution was first fractionated by CM-Sephadex C-50 column (2.5 cm 30 cm) equilibrated previously with 0.02 M sodium acetate buffer (pH 5.4). The column was eluted with a 0–1.0 M linear gradient of NaCl. The unadsorbed peak (fraction C1) exhibited trypsin inhibitor activity. The flow rate was 0.5 mL=min, 10 min=tube. The pooled solution was dialyzed against 0.02 M Tris-HCl buffer (pH 7.2) and then applied to affinity chromatography on an Affi-gel blue gel column (2.5 cm 10 cm) previously equilibrated with the Tris-HCl buffer (0.02 M, pH 7.2). Following removal of unadsorbed proteins, the bounded proteins was eluted with a 0–1.0 M linear gradient of NaCl. The flow rate was 1.0 mL=min, 5 min=tube. The homogeneity of the purified sample was identified on a C18 capillary reverse-phase high-performance liquid chromatography (RP-HPLC) column (15 mm 1.0 mm). The running solvent A contained 90% water, 10% acetonitrile, and 0.1% trifluroacetic acid, and solvent B contained 90% acetonitrile, 10% water, and 0.1% trifluroacetic acid. The gradient eluent concentration was 0–50% solvent B in 1 hr. Protein Assay The protein content was measured by the method of Lowry with bovine serum albumin used as the standard.[14] SDS-PAGE and IEF Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was conducted using a 12.5% separating gel and 4.0% stacking gel, according to the method of Laemmli.[15] Polyacrylamide gel isoelectric focusing (IEF) was carried out in a glass column, with the carrier ampholytes (Ampholine, Pharmacia) with pH ranges of 3–10.[16] Mass Spectrometry The Merrtine was analyzed by matrix-assisted laser desorption=ionization time-of-flight (MALDI-TOF) mass spectrometry (Applied Biosystems, Foster City, CA).[4] The sample was mixed with an aromatic organic acid that donates a proton to the analyte and was dried onto a metal sample plate. After the plate was placed in a high-vacuum source chamber in the mass
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spectrometer, a small portion of the sample was vaporized by blasts from a nitrogen laser. The ions produced ‘‘fly’’ up a tube to the mass analyzer and their masses (mass-to-charge ratio) were determined by their time-of-flight. Measurement of Trypsin–Chymotrypsin Inhibitory Activity Ten portions containing 0, 25, 50, 75, 100, 150, 200, 250, 350, and 450 mg of the inhibitor were incubated together with 25 mg trypsin or chymotrypsin in 100 mL of 0.05 M Tris-HCl buffer (pH 8.0) containing 0.20 M CaCl2 for 5 min at 25 C. The reaction was terminated by adding 1 mL of cold 5% trichloroacetic acid after 15 min of incubation. The reactions mixture was centrifuged at 8000 g for 20 min. Residual trypsin or chymotrypsin activity was determined by adding 300 mL of 1% casein substrate at 25 C. The absorbance of the supernatant was determined at 280 nm.[17] N-Terminal Amino Acid Sequence Analysis The N-terminal amino acid sequence of the purified Merrtine was determined by Edman degradation using a protein sequencer (Applied Biosystems model 476A, Perkin Elmer Co., Waltham, MA).[4] Thermostability Assay The purified Merrtine in Tris-HCl buffer containing 0.20 M CaCl2 (0.05 M, pH 8.0) was preheated at different temperatures (from 30 to 100 C) for 30 min and cooled down at 4 C. The residual enzyme activity was determined at 25 C.[17,18] pH Stability Assay The purified Merrtine was incubated in 0.05 M buffers with pH values between 2 to 12 at 25 C for 30 min. Phosphate buffer (pH 2.0 and 3.0), sodium acetate buffer (pH 3.0, 4.0, and 5.0), sodium citrate buffer (pH 6.0), Tris-HCl buffer (pH 7.0, 8.0, and 9.0), and carbonate buffer (pH 10.0, 11.0, and 12.0) were utilized, respectively. The residual enzyme activity was determined.[17] Assay for Antifungal Activity The antifungal activity of the purified Merrtine and the half-maximal inhibitory concentration of antifungal activity (IC50) were determined according to the method introduced by Wang.[4,19]
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Statistical Analyses All experiments were performed in triplicate. All data were presented as means with standard deviations of three independent experiments. Student’s t-test was used for the statistical analysis. A value of p < 0.05 was considered statistically significant. RESULTS AND DISCUSSIONS Isolation of Merrtine The black soybean was homogenized in 0.2 M sodium acetate buffer (pH 5.4), and the homogenate was centrifuged at 12,000 rpm for 20 min
FIGURE 1 Purification of Merrtine. (A) The pretreated sample was applied on a CM-Sephadex C-50 column. (B) The unadsorbed fraction (C1) was subjected to Affi-gel blue gel column.
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at 4 C. The solution of ammonium sulfate precipitate (20% to 80% saturation) was dialyzed and applied to a CM-Sephadex C-50 column. The unadsorbed fraction (C1) exhibiting trypsin inhibitor activity was pooled (Figure 1A). The pooled fraction was dialyzed and then applied to the Affi-gel blue gel column; the trypsin inhibitor was separated from other components when the column was eluted with a 0–1.0 M linear gradient of NaCl. The third peak (fraction A3, Merrtine) with trypsin inhibitor activity was collected (Figure 1B). A single peak and a single band were presented on the chromatogram map (Figure 2) and SDS-PAGE (Figure 3), indicating the high homogeneity of the Merrtine. Properties of the Merrtine The molecular mass of the purified Merrtine was calculated by SDS-PAGE pattern to be 20.0 kD, as shown in Figure 3. Mass spectrometric determination on MALDI-TOF showed that the exact molecular mass of the trypsin inhibitor was 19638.451 Da (Figure 4). This offers the precise molecular mass, and provides another form of evidence of the high purity of the purified Merrtine as well. The isoelectric point (pI) of the Merrtine was estimated to be 5.8 according to the profile of isoelectric focusing (Figure 5). The 20 N-terminal amino acid sequence of the trypsin inhibitor Merrtine was determined to be DEYSKPCCDLCMCTRRCPPQ, demonstrating high similarity (between 55% and 90%) to those protease inhibitors and inhibitor precursors from other leguminous plants (Table 1).
FIGURE 2 Capillary reverse-phase HPLC of the purified Merrtine using a C18 column.
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FIGURE 3 SDS-PAGE of the purified Merrtine. Gel was stained by Coomassie blue R-250. From left to right: Lane M was proteins marker; lane S was loaded with 8 mg Merrtine.
The fact that the N-terminal sequence of Merrtine resembles the middle sequence of the compared counterparts suggests that the compared trypsin inhibitors are probably from different varieties=cultivars. From Table 1, the N-terminal sequence of the Merrtine was homologous to those of Bowman– Birk trypsin inhibitors isolated from other plants. The fact that it inhibited both trypsin and chymotrypsin and the inhibition potency toward trypsin was more significant and is comparable with the Bowman–Birk type trypsin inhibitors from Pseudostellaria heterophylla roots[17] and wild soybean.[18] Combined with its high similarity of the N-terminal sequence to other Bowman–Birk type trypsin inhibitors, this therefore suggests that Merrtine should be classified as Bowman–Birk-like trypsin inhibitor. Trypsin and Chymotrypsin Inhibitory Activities The Merrtine inhibited trypsin and chymotrypsin activity as shown in Figure 6. When the molar ratio of inhibitor to chymotrypsin equaled 4.1, 50% of enzyme activity was inhibited. However, the inhibition capacity for trypsin was much stronger than that for chymotrypsin; when the molar ratio of the Merrtine to chymotrypsin equaled 8.0, the residual enzyme activity could hardly be calculated (Figure 6).
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FIGURE 4 Mass spectrometric analysis on MALDI-TOF.
Thermal and pH Stability The purified Merrtine was extremely stable below 85 C; incubation below 85 C for 30 min resulted in almost no loss of activity (Figure 7A). Additionally, it showed excellent pH stability, as its trypsin-inhibitory activity was unaffected with exposure to pH 2–12 (Figure 7B). Although the trypsin inhibitors were extensively reported, the fact that the individuals are often different in both their thermostability and pH stability rpresents the diversity of trypsin inhibitors, even from the same species. The results show that thermostability of the purified Merrtine is similar with to of the trypsin inhibitor reported by Lin and Ng,[18] and
FIGURE 5 A profile of isoelectric focusing electrophoresis result. Gel was stained by Coomassie blue R-250. The carrier ampholytes had pH ranges of 3–10.
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TABLE 1 Comparison of N-Terminal Sequence of Merrtine With Trypsin Protease Inhibitors From Other Leguminous Plants
Source Glycine max Glycine soja Glycinemax Phaseolus microcarpus Phaseolus parvulus Vigna angularis Lens culinaris
Isoinhibitor name
Bowman–Birk type proteinase Bowman–Birk type proteinase Double-headed trypsin inhibitor Double-headed trypsin inhibitor Protease inhibitor Trypsin inhibitor
Isoinhibitor number
N-Terminal sequence
Residue number Identity
1 43
DEYSKPCCDLCMCTRRCPPQ DEYSKPCCDLCMCTRSMPPQ
20 62
100% 90%
37
DEYSKPCCDLCMCTRSMPPQ
56
90%
51
ESSKPCCDQCACTRSIPPQ
69
80%
13
ESSEPCCDLCLCTKSIPPQ
31
60%
8
ESSKPCCDECKCTKSEPPQ
26
60%
1
ESSEPCCDSCICTKSIPPQ
19
55%
Note. The presented N-terminal sequences in this table are from the NCBI database (http://www. ncbi.nlm.nih.gov). Underlined characters, identical amino acids with the purified Merrtine..
higher than those of the trypsin inhibitors reported by Wang et al.[4,19] The pH stability is similar to that of trypsin inhibitor reported by Lin and Ng.[18] The newly purified trypsin inhibitor had both heat-resistant and pH-resistant properties (Figures 7A and 7B) when compared with almost all of previously reported ones. This is of significance for relatively thermostable and pH-stable plant defense mechanisms.
FIGURE 6 Inhibitory activity of the Merrtine on trypsin or chymotrypsin (. and ~ represent trypsin and chymotrypsin, respectively).
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FIGURE 7 (A) Thermal stability of the Merrtine. Data are presented as mean SD (n ¼ 3). (B) pH stability of the Merrtine. Data are presented as mean SD (n ¼ 3).
Antifungal Activity The antifungal activity of Merrtine against fungal species is illustrated in Figures 8I–8V. It can be seen that the protein showed obvious antifungal activity toward (1) Alternaria alternata (Figure 8I), Fusarium oxysporum (Figure 8II), Pythium aphanidermatum (Figure 8III), Physalospora piricola (Figure 8IV), Botrytis cinerea (Figure 8V), and Fusarium solani (Figure 8VI). In addition, the IC50 toward Alternaria alternata was calculated to be 10.1 lM (Figure 9).
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FIGURE 8 Inhibitory activity of trypsin inhibitor Merrtine. Plate 1, Alternaria alternata; plate 2, Fusarium oxysporum; plate 3, Pythium aphanidermatum; plate 4, Physalospora piricola; plate 5, Botrytis cinerea; and plate 6, Fusarium solani. Disk A, 0.02 M Tris-HCl buffer, pH 7.2, as control; disk B, 50 mg Merrtine; and disk C, 100 mg Merrtine.
Trypsin inhibitor Merrtine displays a spectrum of antifungal activities. It inhibits mycelial growth in a number of fungal species, which is similar with those of the previously reported antifungal proteins.[13,17,20,21] The existence of trypsin inhibitors with antifungal activity might represent a selective advantage toward a wide range of potential pathogens activity. It can be deduced that a combination of antifungal proteins is present in this variety of plants, and that they work together to defend against the attacks from invading pathogens such as disease-causing fungi. Therefore, leguminous protease inhibitors show antifungal activities, even though they are just listed as inhalant allergies and also food allergies.
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FIGURE 9 Determination of the IC50 value of the antifungal activity of trypsin inhibitor Merrtine toward Alternaria alternata. Three doses of the Merrtine (5 lM, 10 lM, and 20 lM) in 0.02 M Tris-HCl buffer pH 7.2 were used. The IC50 was calculated to be 10.1 lM.
CONCLUSIONS Merrtine, a trypsin–chymotrypsin inhibitor with antifungal potency against several fungal species, was first investigated in this study. Results imply the exploitable potential for this class of antifungal protein in the food industry and in the agricultural area as well.
ACKNOWLEDGMENTS This work was supported by the Open Project Program of State Key Laboratory of Food Science and Technology, Jiangnan University, China (No. SKLF-KF-201301), the Fujian Natural Science Foundation, China (No. 2013J01132), the S&T projects of Fujian Provincial Science & Technology Hall (No. 2012N0015 & 2012S0053), and the Project of Science and Technology of Nantong City, China (No. HS2011001).
REFERENCES 1. Birk, Y. Plant Protease Inhibitors. Springer-Verlag: New York, NY, 2003. 2. Ng, T.B.; Huang, B.; Fong, W.P.; Yeung, H.W. Anti-Human Immunodeficiency Virus (anti-HIV) Natural Products with Special Emphasis on HIV Reverse Transcriptase Inhibitors. Life Sci. 1997, 61, 933–949. 3. Mastrolorenzo, A.; Rusconi, S.; Scozzafava, A.; Barbaro, G.; Supuran, C.T. Inhibitors of HIV-1 Protease: Current State of the Art 10 Years After Their Introduction. From Antiretroviral Drugs to Antifungal, Antibacterial and Antitumor Agents Based on Aspartic Protease Inhibitors. Curr. Med. Chem. 2007, 14, 2734–2748.
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4. Wang, S.Y.; Lin, J.; Ye, M.Y.; Ng, T.B.; Rao, P.F.; Ye, X.Y. Isolation and Characterization of a Novel Mung Bean Protease Inhibitor with Antipathogenic and Anti-Proliferative Activities. Peptides 2006, 27, 3129–3136. 5. Zhao, M.; Naude, R.J.; Muramoto, K.; Oelofsen, W. Purification and Characterization of Ostrich Pancreatic Secretory Trypsin Inhibitor. Int. J. Peptide Protein Res. 1996, 48, 174–181. 6. Cavalcanti, M.D.M.; Oliva, M.L.V.; Fritz, H.; Jochum, M.; Mentele, R.; Sampaio, M.; Coelho, L.C.B.B.; Batista, I.F.C.; Sampaio, C.A.M. Characterization of a Kunitz Trypsin Inhibitor with One Disulfide Bridge Purified from Swartzia pickellii. Biochem. Biophys. Res. Commun. 2002, 291, 635–639. 7. Kumar, P.; Rao, A.G.; Hariharaputran, S.; Chandra, N.; Gowda, L.R. Molecular Mechanism of Dimerization of Bowman–Birk Inhibitors. Pivotal Role of ASP76 in the Dimerization. J. Biol. Chem. 2004, 279, 3025–3032. 8. Deshimaru, M.; Hanamoto, R.; Kusano, C.; Yoshimi, S.; Terada, S. Purification and Characterization of Proteinase Inhibitors from Wild Soja (Glycine soja) Seeds. Biosci. Biotechnol. Biochem. 2002, 66, 1897–1903. 9. Wong, R.C.; Fong, W.P.; Ng, T.B. Multiple Trypsin Inhibitors from Momordica cochinchinensis seeds, the Chinese Drug Mubiezhi. Peptides 2004, 25, 163–169. 10. Murdock, L.L.; Huesing, J.E.; Nielsen, S.S.; Prat, R.C.; Shade, R.E. Biological Effects of Plant Lectins on the Cowpea Weevil. Phytochemistry 1990, 29, 85–89. 11. Wang, S.Y.; Ng, T.B.; Chen, T.; Lin, D.Y.; Rao, P.F.; Ye, X.Y. First Report of a Novel Plant Lysozyme with Both Antifungal and Antibacterial Activities from Phaseolus mungo. Biochem. Biophys. Res. Commun. 2005, 327, 820–827. 12. Ng, T.B. Antifungal Proteins and Peptides of Leguminous and Non-Leguminous Origins. Peptides 2004, 25, 1215–1222. 13. Wang, S.Y.; Wu, J.H.; Ng, T.B.; Ye, X.Y.; Rao, P.F. A Non-Specific Lipid Transfer Protein with Antifungal and Antibacterial Activities from the Mung Bean. Peptides 2004, 25, 1235–1242. 14. Lowry, O.H.; Rosebrough, N.J.; Farr, A.L.; Randall, R.J. Protein Measurement with the Folin Phenol Reagent. J. Biol. Chem. 1951, 193, 265–275. 15. Laemmli, U.K.; Favre, M. Gel Electrophoresis of Proteins. J. Mol. Biol. 1973, 80, 575–599. 16. Tanaka, S.; Takikawa, H.; Wakabayashi, K. Seasonal Variation Ipituitary Gonadotropin in the Adult Male Newt, Cynops pyrrho gaster pyrrhogaster, Revealed by Isoelectric Focusing Technique and Radioreceptor Assay. Endocrinol. Jpn. 1981, 28, 335–345. 17. Wang, H.X.; Ng, T.B. Concurrent Isolation of a Kunitz-Type Trypsin Inhibitor with Antifungal Activity and a Novel Lectin from Pseudostellaria heterophylla Roots. Biochem. Biophys. Res. Commun. 2006, 342, 349–353. 18. Lin, P.; Ng, T.B. A Stable Trypsin Inhibitor from Chinese Dull Black Soybeans with Potentially Exploitable Activities. Process Biochem. 2008, 43, 992–998. 19. Wang, S.Y.; Rao, P.F. A Leguminous Trypsin-Chymotrypsin Inhibitor Limenin with Antifungal Activity from Phaseolus limensis. Eur. Food Res. Technol. 2010, 231, 331–338. 20. Raj, P.A.; Dentino, A.R. Current Status of Defensins and Their Role in Innate and Adaptive Immunity. FEMS Microbiol Lett. 2002, 266, 9–18. 21. Shao, B.; Wang, S.Y.; Zhou, J.W.; Ke, L.L.; Rao, P.F. A Novel Lectin from Fresh Rhizome of Alisma orientale (Sam.) Juzep. Process Biochem. 2011, 46, 1554–1559.
ISOLATION OF A TRYPSIN–CHYMOTRYPSIN INHIBITOR AND ITS FUNCTIONAL PROPERTIES
Shaoyun Wang,1,2 Biao Shao,3 Wei Lu,2 Jing Hong,1,2 and Pingfan Rao2 1 State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi, People’s Republic of China 2 Department of Food Science, Fuzhou University, Fuzhou, People’s Republic of China 3 Nantong Products Quality Supervision and Inspection Institute, Nantong, People’s Republic of China & A novel trypsin inhibitor with thermal and pH stability, designated Merrtine, was isolated from Glycine max L. merr. The procedure involved ammonium sulfate precipitation, ion-exchange chromatography on CM-Sephadex C-50, and affinity chromatography on Affi-gel blue gel. The 20 Nterminal amino acid sequences were determined to be DEYSKPCCDLCMCTRRCPPQ, demonstrating high homology with the sequence of Bowman–Birk type trypsin inhibitors. The molecular mass and isoelectric point of the inhibitor were estimated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and isoelectric focusing to be 20.0 kD and 5.8, respectively. Trypsin could be completely inhibited by Merrtine when the molar ratio was 8.1. The inhibitory activity of Merrtine was unaffected after exposure to temperatures up to 85 C, as well as within the pH range 2–12. Besides inhibiting trypsin–chymotrypsin, the inhibitor demonstrated additional antifungal activity against the species of Alternaria alternate, Fusarium oxysporum, Pythium aphanidermatum, Physalospora piricola, Botrytis cinerea, and Fusarium solani. We herein report not only the trypsin inhibitor’s extraction and isolation for the first time, but also its physiochemical and antifungal properties. Keywords antifungal, Glycine max L.merr, thermostable, trypsin inhibitor
INTRODUCTION Protease inhibitor, as one of components in plant tissue, is usually considered with allergies that have a negative effect on the health of the human body.[1] However, it has drawn more and more attention from a number of investigators for its diverse bioactivities, such as insecticidal, antiviral, and antifungal activities.[2–4] Combined with these bioactivities, protease inhibitors show potential application in fighting against virus (HIV and SARS virus), insects, and fungi.[2,4] Address correspondence to Shaoyun Wang, Department of Food Science, Fuzhou University, Fuzhou 350002, P.R. China. E-mail: [email protected] Color versions of one or more of the figures in the article can be found online at www.tandfonline. com/lpbb.
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Trypsin inhibitors, listed as one type of protease inhibitors, usually show inhibiting activities to trypsin–chymotrypsin.[5] They include Kunitz-type trypsin inhibitors and Bowman–Birk trypsin inhibitors.[5–7] Sometimes a combination of both Bowman–Birk and Kunitz-type trypsin inhibitors is found in a single plant tissue. For example, yellow soybean (Glycine max) concurrently produced two types of trypsin inhibitors.[8] Besides inhibiting trypsin–chymotrypsin, protease inhibitors are also included in the antifungal family with a range of well-known antifungal proteins and peptides such as lysozyme, lectin, chitinase, nonspecific lipid transfer protein, and defense peptide.[9–13] A number of members of the antifungal family have demonstrated their tremendous potential in protecting crops and thus important economic implications due to their defenses against both insects[10] and fungi.[4,11] Chinese Glycine max L. merr (black soybean) is a special cultivar of Glycine max (yellow soybean). It is extensively cultivated in Asian countries and serves as a healthy food, with a function in the traditional Chinese menu different from that of the yellow soybean. However, it has not been studied as extensively as the yellow soybean. In view of the scarcity of literature about the constituents of Glycine max L. merr, we herein report not only the trypsin inhibitor’s extraction and preparation for the first time, but also its physiochemical and antifungal properties. MATERIALS AND METHODS Materials Black soybean (Glycine max L. merr) was purchased from a local supermarket in Fuzhou, China. CM-Sephadex C-50 and Affi-gel blue gel were purchased from Amersham Biosciences (Sweden) and Bio-Rad (USA), respectively. Proteins marker was purchased from Gibco-BRL (Life Tech., USA). All chemicals were of the highest purity available. Sample Preparation One hundred grams of black soybean was soaked in distilled water for 12 hr and homogenized in 0.20 M sodium acetate buffer, pH 5.4. The homogenate was centrifuged at 10,000 g for 20 min at 4 C. The supernatant was collected for further study. Isolation of Merrtine The supernatant was firstly brought to 20% saturation with solid ammonium sulfate. The solution was centrifuged at 10,000 g for 20 min
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and the resulting supernatant was then adjusted to 80% saturation with ammonium sulfate. The precipitate obtained from centrifugation (10,000 g, 20 min) was collected and dissolved in 300 mL of 0.02 M sodium acetate buffer (pH 5.4). The dissolved solution was sequentially dialyzed against 0.02 M sodium acetate buffer (pH 5.4) overnight at 4 C with several changes. The dialyzed solution was first fractionated by CM-Sephadex C-50 column (2.5 cm 30 cm) equilibrated previously with 0.02 M sodium acetate buffer (pH 5.4). The column was eluted with a 0–1.0 M linear gradient of NaCl. The unadsorbed peak (fraction C1) exhibited trypsin inhibitor activity. The flow rate was 0.5 mL=min, 10 min=tube. The pooled solution was dialyzed against 0.02 M Tris-HCl buffer (pH 7.2) and then applied to affinity chromatography on an Affi-gel blue gel column (2.5 cm 10 cm) previously equilibrated with the Tris-HCl buffer (0.02 M, pH 7.2). Following removal of unadsorbed proteins, the bounded proteins was eluted with a 0–1.0 M linear gradient of NaCl. The flow rate was 1.0 mL=min, 5 min=tube. The homogeneity of the purified sample was identified on a C18 capillary reverse-phase high-performance liquid chromatography (RP-HPLC) column (15 mm 1.0 mm). The running solvent A contained 90% water, 10% acetonitrile, and 0.1% trifluroacetic acid, and solvent B contained 90% acetonitrile, 10% water, and 0.1% trifluroacetic acid. The gradient eluent concentration was 0–50% solvent B in 1 hr. Protein Assay The protein content was measured by the method of Lowry with bovine serum albumin used as the standard.[14] SDS-PAGE and IEF Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was conducted using a 12.5% separating gel and 4.0% stacking gel, according to the method of Laemmli.[15] Polyacrylamide gel isoelectric focusing (IEF) was carried out in a glass column, with the carrier ampholytes (Ampholine, Pharmacia) with pH ranges of 3–10.[16] Mass Spectrometry The Merrtine was analyzed by matrix-assisted laser desorption=ionization time-of-flight (MALDI-TOF) mass spectrometry (Applied Biosystems, Foster City, CA).[4] The sample was mixed with an aromatic organic acid that donates a proton to the analyte and was dried onto a metal sample plate. After the plate was placed in a high-vacuum source chamber in the mass
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spectrometer, a small portion of the sample was vaporized by blasts from a nitrogen laser. The ions produced ‘‘fly’’ up a tube to the mass analyzer and their masses (mass-to-charge ratio) were determined by their time-of-flight. Measurement of Trypsin–Chymotrypsin Inhibitory Activity Ten portions containing 0, 25, 50, 75, 100, 150, 200, 250, 350, and 450 mg of the inhibitor were incubated together with 25 mg trypsin or chymotrypsin in 100 mL of 0.05 M Tris-HCl buffer (pH 8.0) containing 0.20 M CaCl2 for 5 min at 25 C. The reaction was terminated by adding 1 mL of cold 5% trichloroacetic acid after 15 min of incubation. The reactions mixture was centrifuged at 8000 g for 20 min. Residual trypsin or chymotrypsin activity was determined by adding 300 mL of 1% casein substrate at 25 C. The absorbance of the supernatant was determined at 280 nm.[17] N-Terminal Amino Acid Sequence Analysis The N-terminal amino acid sequence of the purified Merrtine was determined by Edman degradation using a protein sequencer (Applied Biosystems model 476A, Perkin Elmer Co., Waltham, MA).[4] Thermostability Assay The purified Merrtine in Tris-HCl buffer containing 0.20 M CaCl2 (0.05 M, pH 8.0) was preheated at different temperatures (from 30 to 100 C) for 30 min and cooled down at 4 C. The residual enzyme activity was determined at 25 C.[17,18] pH Stability Assay The purified Merrtine was incubated in 0.05 M buffers with pH values between 2 to 12 at 25 C for 30 min. Phosphate buffer (pH 2.0 and 3.0), sodium acetate buffer (pH 3.0, 4.0, and 5.0), sodium citrate buffer (pH 6.0), Tris-HCl buffer (pH 7.0, 8.0, and 9.0), and carbonate buffer (pH 10.0, 11.0, and 12.0) were utilized, respectively. The residual enzyme activity was determined.[17] Assay for Antifungal Activity The antifungal activity of the purified Merrtine and the half-maximal inhibitory concentration of antifungal activity (IC50) were determined according to the method introduced by Wang.[4,19]
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Statistical Analyses All experiments were performed in triplicate. All data were presented as means with standard deviations of three independent experiments. Student’s t-test was used for the statistical analysis. A value of p < 0.05 was considered statistically significant. RESULTS AND DISCUSSIONS Isolation of Merrtine The black soybean was homogenized in 0.2 M sodium acetate buffer (pH 5.4), and the homogenate was centrifuged at 12,000 rpm for 20 min
FIGURE 1 Purification of Merrtine. (A) The pretreated sample was applied on a CM-Sephadex C-50 column. (B) The unadsorbed fraction (C1) was subjected to Affi-gel blue gel column.
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at 4 C. The solution of ammonium sulfate precipitate (20% to 80% saturation) was dialyzed and applied to a CM-Sephadex C-50 column. The unadsorbed fraction (C1) exhibiting trypsin inhibitor activity was pooled (Figure 1A). The pooled fraction was dialyzed and then applied to the Affi-gel blue gel column; the trypsin inhibitor was separated from other components when the column was eluted with a 0–1.0 M linear gradient of NaCl. The third peak (fraction A3, Merrtine) with trypsin inhibitor activity was collected (Figure 1B). A single peak and a single band were presented on the chromatogram map (Figure 2) and SDS-PAGE (Figure 3), indicating the high homogeneity of the Merrtine. Properties of the Merrtine The molecular mass of the purified Merrtine was calculated by SDS-PAGE pattern to be 20.0 kD, as shown in Figure 3. Mass spectrometric determination on MALDI-TOF showed that the exact molecular mass of the trypsin inhibitor was 19638.451 Da (Figure 4). This offers the precise molecular mass, and provides another form of evidence of the high purity of the purified Merrtine as well. The isoelectric point (pI) of the Merrtine was estimated to be 5.8 according to the profile of isoelectric focusing (Figure 5). The 20 N-terminal amino acid sequence of the trypsin inhibitor Merrtine was determined to be DEYSKPCCDLCMCTRRCPPQ, demonstrating high similarity (between 55% and 90%) to those protease inhibitors and inhibitor precursors from other leguminous plants (Table 1).
FIGURE 2 Capillary reverse-phase HPLC of the purified Merrtine using a C18 column.
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FIGURE 3 SDS-PAGE of the purified Merrtine. Gel was stained by Coomassie blue R-250. From left to right: Lane M was proteins marker; lane S was loaded with 8 mg Merrtine.
The fact that the N-terminal sequence of Merrtine resembles the middle sequence of the compared counterparts suggests that the compared trypsin inhibitors are probably from different varieties=cultivars. From Table 1, the N-terminal sequence of the Merrtine was homologous to those of Bowman– Birk trypsin inhibitors isolated from other plants. The fact that it inhibited both trypsin and chymotrypsin and the inhibition potency toward trypsin was more significant and is comparable with the Bowman–Birk type trypsin inhibitors from Pseudostellaria heterophylla roots[17] and wild soybean.[18] Combined with its high similarity of the N-terminal sequence to other Bowman–Birk type trypsin inhibitors, this therefore suggests that Merrtine should be classified as Bowman–Birk-like trypsin inhibitor. Trypsin and Chymotrypsin Inhibitory Activities The Merrtine inhibited trypsin and chymotrypsin activity as shown in Figure 6. When the molar ratio of inhibitor to chymotrypsin equaled 4.1, 50% of enzyme activity was inhibited. However, the inhibition capacity for trypsin was much stronger than that for chymotrypsin; when the molar ratio of the Merrtine to chymotrypsin equaled 8.0, the residual enzyme activity could hardly be calculated (Figure 6).
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FIGURE 4 Mass spectrometric analysis on MALDI-TOF.
Thermal and pH Stability The purified Merrtine was extremely stable below 85 C; incubation below 85 C for 30 min resulted in almost no loss of activity (Figure 7A). Additionally, it showed excellent pH stability, as its trypsin-inhibitory activity was unaffected with exposure to pH 2–12 (Figure 7B). Although the trypsin inhibitors were extensively reported, the fact that the individuals are often different in both their thermostability and pH stability rpresents the diversity of trypsin inhibitors, even from the same species. The results show that thermostability of the purified Merrtine is similar with to of the trypsin inhibitor reported by Lin and Ng,[18] and
FIGURE 5 A profile of isoelectric focusing electrophoresis result. Gel was stained by Coomassie blue R-250. The carrier ampholytes had pH ranges of 3–10.
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TABLE 1 Comparison of N-Terminal Sequence of Merrtine With Trypsin Protease Inhibitors From Other Leguminous Plants
Source Glycine max Glycine soja Glycinemax Phaseolus microcarpus Phaseolus parvulus Vigna angularis Lens culinaris
Isoinhibitor name
Bowman–Birk type proteinase Bowman–Birk type proteinase Double-headed trypsin inhibitor Double-headed trypsin inhibitor Protease inhibitor Trypsin inhibitor
Isoinhibitor number
N-Terminal sequence
Residue number Identity
1 43
DEYSKPCCDLCMCTRRCPPQ DEYSKPCCDLCMCTRSMPPQ
20 62
100% 90%
37
DEYSKPCCDLCMCTRSMPPQ
56
90%
51
ESSKPCCDQCACTRSIPPQ
69
80%
13
ESSEPCCDLCLCTKSIPPQ
31
60%
8
ESSKPCCDECKCTKSEPPQ
26
60%
1
ESSEPCCDSCICTKSIPPQ
19
55%
Note. The presented N-terminal sequences in this table are from the NCBI database (http://www. ncbi.nlm.nih.gov). Underlined characters, identical amino acids with the purified Merrtine..
higher than those of the trypsin inhibitors reported by Wang et al.[4,19] The pH stability is similar to that of trypsin inhibitor reported by Lin and Ng.[18] The newly purified trypsin inhibitor had both heat-resistant and pH-resistant properties (Figures 7A and 7B) when compared with almost all of previously reported ones. This is of significance for relatively thermostable and pH-stable plant defense mechanisms.
FIGURE 6 Inhibitory activity of the Merrtine on trypsin or chymotrypsin (. and ~ represent trypsin and chymotrypsin, respectively).
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FIGURE 7 (A) Thermal stability of the Merrtine. Data are presented as mean SD (n ¼ 3). (B) pH stability of the Merrtine. Data are presented as mean SD (n ¼ 3).
Antifungal Activity The antifungal activity of Merrtine against fungal species is illustrated in Figures 8I–8V. It can be seen that the protein showed obvious antifungal activity toward (1) Alternaria alternata (Figure 8I), Fusarium oxysporum (Figure 8II), Pythium aphanidermatum (Figure 8III), Physalospora piricola (Figure 8IV), Botrytis cinerea (Figure 8V), and Fusarium solani (Figure 8VI). In addition, the IC50 toward Alternaria alternata was calculated to be 10.1 lM (Figure 9).
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FIGURE 8 Inhibitory activity of trypsin inhibitor Merrtine. Plate 1, Alternaria alternata; plate 2, Fusarium oxysporum; plate 3, Pythium aphanidermatum; plate 4, Physalospora piricola; plate 5, Botrytis cinerea; and plate 6, Fusarium solani. Disk A, 0.02 M Tris-HCl buffer, pH 7.2, as control; disk B, 50 mg Merrtine; and disk C, 100 mg Merrtine.
Trypsin inhibitor Merrtine displays a spectrum of antifungal activities. It inhibits mycelial growth in a number of fungal species, which is similar with those of the previously reported antifungal proteins.[13,17,20,21] The existence of trypsin inhibitors with antifungal activity might represent a selective advantage toward a wide range of potential pathogens activity. It can be deduced that a combination of antifungal proteins is present in this variety of plants, and that they work together to defend against the attacks from invading pathogens such as disease-causing fungi. Therefore, leguminous protease inhibitors show antifungal activities, even though they are just listed as inhalant allergies and also food allergies.
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FIGURE 9 Determination of the IC50 value of the antifungal activity of trypsin inhibitor Merrtine toward Alternaria alternata. Three doses of the Merrtine (5 lM, 10 lM, and 20 lM) in 0.02 M Tris-HCl buffer pH 7.2 were used. The IC50 was calculated to be 10.1 lM.
CONCLUSIONS Merrtine, a trypsin–chymotrypsin inhibitor with antifungal potency against several fungal species, was first investigated in this study. Results imply the exploitable potential for this class of antifungal protein in the food industry and in the agricultural area as well.
ACKNOWLEDGMENTS This work was supported by the Open Project Program of State Key Laboratory of Food Science and Technology, Jiangnan University, China (No. SKLF-KF-201301), the Fujian Natural Science Foundation, China (No. 2013J01132), the S&T projects of Fujian Provincial Science & Technology Hall (No. 2012N0015 & 2012S0053), and the Project of Science and Technology of Nantong City, China (No. HS2011001).
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