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Coagulation of soy proteins induced by thermolysin and comparison of the coagulation reaction with that induced by subtilisin Carlsberg
Enzyme and Microbial Technology 44 (2009) 229–234
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Coagulation of soy proteins induced by thermolysin and comparison of the coagulation reaction with that induced by subtilisin Carlsberg Kohei Asaoka, Kiyoshi Yasukawa, Kuniyo Inouye ∗ Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan
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Article history: Received 2 September 2008 Received in revised form 1 October 2008 Accepted 2 October 2008 Keywords: Coagulation Metalloproteinase Soy protein Subtilisin Carlsberg Thermolysin Turbidity
a b s t r a c t It is known that coagula are formed when soy proteins are treated by subtilisin Carlsberg. Time-course of the coagulation can be monitored by measuring the turbidity (OD660 ) of soy protein isolates (SPI) solution [Inouye K, Nagai K, Takita T. Coagulation of soy protein isolates induced by subtilisin Carlsberg. J Agric Food Chem 2002;50:1237–42]. In this study, we examined the coagulation of the digests produced in the digestion of SPI with thermolysin. SPI treated at 80 ◦ C coagulated by hydrolysis with either of thermolysin and subtilisin Carlsberg, but that treated at 37 ◦ C did not. The velocities of the coagulation were almost the same between the digestions with thermolysin and subtilisin Carlsberg, while the amount of the coagula in the former was 50–80% of that in the latter. With increasing the thermolysin concentration from 0.1 to 5 M, the velocity of the coagulation increased while the amount of the coagula was constant. With increasing the reaction temperature from 37 to 70 ◦ C, the velocity increased while the amount decreased. The digests from 11S soy proteins coagulated drastically compared to those from either of SPI, 7S soy proteins, and the mixture of 7S and 11S soy proteins (1:2 for w/v), suggesting that the coagulation of the digests from 11S soy proteins might be suppressed by those from 7S soy proteins. © 2008 Elsevier Inc. All rights reserved.
1. Introduction Soybean is a major source of plant oil. About 150 million tons of soybeans are produced every year, and a large amount of defatted soybean meals remain after extraction of oil. They are in part used as feed, but hardly utilized in food industries [1]. Defatted soybean meals contain proteins with high nutritional values and functional properties. In addition, it has been reputed that soy protein diet lowers blood-cholesterol [2] and triglyceride [3,4] levels. The proteolytic digests of soy protein also have bioactive effects such as insulin-mediated antilipolysis [5], insulin-mediated glucose transport and lipogenesis [6], and blood-pressure lowering [7], suggesting that soy protein and its digests could be useful for functional foods. In protease digestion of soy proteins, some of the digests coagulate and remain insoluble, resulting in lower yields of the soluble digests. We developed a convenient method to follow the proteaseinduced coagulation of soy protein isolates (SPI) by measuring continuously the turbidity of the reaction solution [8]. During the hydrolysis, the turbidity decreased in the first phase due to the digestion of SPI to reach the minimum (phase 1), and then increased
due to the coagulation of the digests to reach the maximum (phase 2). After it reached the maximum, it decreased again slowly due to the digestion of the coagula (phase 3) (Fig. 1). On the basis of these observations, we investigated the coagulation of the digests produced in the digestion of SPI with subtilisin Carlsberg [EC 3.4.21.14], and showed that the fragments formed in phase 2, but not in phase 1, were involved in the coagulation through hydrophobic interactions [8,9]. We also reported that the proteolytic digestion of SPI promotes the removal of hexanal, one of the major compounds of the unfavorable soy bean odor, by the treatment with various absorbents [10]. Thermolysin [EC 3.4.24.27] is a thermostable neutral metalloproteinase produced in the culture broth of Bacillus thermoproteolyticus [11–14]. It is widely used for the peptide bond formation through reverse reaction of hydrolysis [15,16]. In this study, we examined the coagulation of the digests produced in the digestion of SPI with thermolysin and compared it with the coagulation induced by subtilisin Carlsberg. 2. Materials and methods 2.1. Materials
∗ Corresponding author. Tel.: +81 75 753 6266; fax: +81 75 753 6265. E-mail address: [email protected] (K. Inouye). 0141-0229/$ – see front matter © 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.enzmictec.2008.10.001
Throughout the experiment, 20 mM phosphate buffer (pH 8.0) containing 0.05% sodium azide was used as a standard buffer. Thermolysin (lot T7LA991) was purchased from Daiwa Kasei (Osaka, Japan) and was dissolved in the standard buffer. Its concentration was determined using the molar absorption coefficient at 277 nm,
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K. Asaoka et al. / Enzyme and Microbial Technology 44 (2009) 229–234 2.4. SDS-PAGE
Fig. 1. Introduced four parameters to characterize the reaction curve of the turbidity. The turbidity was evaluated by OD660 . OD1 and OD2 are the minimum and maximum of OD660 , and T1 and T2 are the times at which OD660 reached OD1 and OD2 , respectively.
ε277 , of 63.3 mM−1 cm−1 [16]. Subtilisin Carlsberg (lot 112k1327) was purchased from Sigma (St. Louis, MO) and was dissolved in the standard buffer. Its concentration was determined using ε280 of 23.0 mM−1 cm−1 [17]. All other chemicals were of reagent grade and purchased form Nacalai Tesque (Kyoto, Japan).
SDS-PAGE was performed under reducing conditions according to the method of Laemmli [21]. In the analysis of undigested SPI, and 7S and 11S soy proteins, a 12.5% polyacrylamide gel (Daiichi Pure Chemicals, Tokyo, Japan) was used. The sample solution was mixed with 4 volumes of the SDS-PAGE sample buffer, and was immediately boiled for 5 min. The solution (10 l) was applied to SDS-PAGE. A constant current of 40 mA was applied for 60 min. In the analysis of digested SPI, and 7S and 11S soy proteins, a 15%/25% gradient gel system (Multigel 15/25, Daiichi Pure Chemicals) was used. Five milliliters of each of SPI, and 7S and 11S fractions (concentrations: >10 mg/ml) was treated at 80 or 96 ◦ C for 30 min. Then, the solution was incubated at 37 ◦ C for 10 min and treated by subtilisin Carlsberg at pH 8.0 and 37 ◦ C. At 5, 10, 20, 30, 60, 90, 120, and 240 min from start of the reaction, a part of the reaction solution was taken out and mixed with 4 volumes of the SDS-PAGE sample buffer. Separately, 1.0 ml of the reaction solution at 120 min was taken out and centrifuged (10,000 × g, 1 min). The supernatant was collected and mixed with 4 volumes of the SDS-PAGE sample buffer. The solution was immediately boiled for 5 min, 10 l of which was applied to SDS-PAGE. A constant current of 40 mA was applied for 60 min. After electrophoresis, proteins were stained with Coomassie brilliant blue R-250. A molecular mass marker kit consisting of phosphorylase b (97 kDa), BSA (66 kDa), rabbit muscle aldolase (42 kDa), bovine erythrocyte carbonic anhydrase (30 kDa), soybean trypsin inhibitor (20 kDa), and hen egg white lysozyme (HEL) (14 kDa) was from Daiichi Pure Chemicals, and heart muscle myoglobin (Mb) (17 kDa), bovine milk ␣-lactalbumin (14 kDa), Mb I+III (11 kDa), Mb I (8.2 kDa), and Mb II (6.2 kDa) were from Sigma.
3. Results 3.1. Preparation of SPI, and 7S and 11S fractions from defatted soybean flakes
2.2. Preparation of SPI, and the 7S and 11S fractions Hexane-defatted soybean flakes (lot 05.10.27) was obtained from Fuji Oil Co. (Osaka, Japan). SPI was prepared according to the method of Tsumura et al. [18]. Briefly, the defatted soybean flakes were dispersed with water (1:10, v/v) with adjusting the pH to 7.0 with 2 M NaOH, and stirred for 1 h, followed by filtration through gauze. The filtrate was centrifuged at 5000 × g for 30 min to collect the supernatant. The pH of the supernatant was adjusted to 4.5 with 2 M HCl, and it was stirred for 1 h and centrifuged at 6500 × g for 20 min to collect the acidprecipitated soy protein. The precipitate was dispersed in distilled water and stored at 4 ◦ C before use as the SPI solution. The 7S and 11S fractions were prepared according to the method of Nagano et al. [19]. Briefly, the defatted soybean flakes were dispersed with water (1:15, v/v) with adjusting the pH to 7.5 with 2 M NaOH, and stirred for 1 h, followed by filtration through gauze and then by centrifugation at 9000 × g for 30 min. NaHSO3 (solid) was added to the supernatant resulted, and was adjusted to 0.98 g/l. After adjusting the pH of the supernatant to 6.4 with 2 M HCl, it was left at 4 ◦ C overnight and then was centrifuged at 6500 × g for 20 min to collect the precipitate and the supernatant. The precipitate was glycinin-rich and dispersed in distilled water. It was stored at 4 ◦ C until use as the 11S fraction. NaCl (solid) was added to the supernatant and was adjusted to 0.25 M. After adjusting the pH of the supernatant to 5.0 with 2 M HCl at 4 ◦ C, it was stirred for 1 h at 4 ◦ C, and centrifuged at 9000 × g for 30 min. The supernatant was mixed with icechilled water (1:1, v/v), and its pH was adjusted to 4.8 with 2 M HCl at 4 ◦ C. After overnight incubation at 4 ◦ C, the precipitate which is -conglycinin-rich was collected by centrifugation at 6500 × g for 20 min, dispersed in distilled water, and stored at 4 ◦ C before use as the 7S fraction. The solutions of SPI, and the 7S and 11S fractions obtained were dialyzed at 4 ◦ C against the standard buffer with the ratio of 1:15 (v/v) three times to adjust their pH to 8.0. After dialysis, the protein concentration was determined by the Lowry method using bovine serum albumin (BSA) as the standard [20]. These solutions were prepared daily and kept at 4 ◦ C before use. The solutions were slightly turbid, and thus, were not solutions but suspensions in a strict meaning, although we called these as solutions in this study for convenience.
We prepared SPI, and 7S and 11S fractions which had not received any thermal treatment. SPI was extracted from defatted soybean flake by alkaline and acidic solutions, and 7S and 11S fractions were extracted from the same material by alkaline and acidic solutions followed by salt precipitation based on the difference in the solubility of -conglycinin and glycinin with sodium hydrogen sulfite, which are described in Section 2. Upon SDS-PAGE under reducing conditions, 7S fraction yielded two bands with molecular masses of 70 and 50 kDa corresponding to ␣ and ␣ subunit and  subunit of -conglycinin, respectively, and 11S fraction yielded three bands with molecular masses of 40, 35, and 22 kDa corresponding to two acidic subunits and one basic subunit of glycinin, respectively (Fig. 2). It is considered that 7S and 11S fractions prepared in our method are exclusively composed of -conglycinin and glycinin, respectively.
2.3. Turbidity measurement The turbidity of the soy protein solution was evaluated by measuring the optical density at 660 nm (OD660 ) with a spectrophotometer DU800 (Beckman Coulter, Tokyo, Japan). SPI, and 7S and 11S fractions were diluted with the standard buffer. After the incubation on ice for 10 min, 5 ml each of the solutions (concentrations: >10 mg/ml) was incubated at 80 or 98 ◦ C, and then immediately incubated at 37 ◦ C for 10 min. The solution (2.4 ml) was transferred to an optical cuvette thermostatted at 37 ◦ C and further incubated for 5 min. The turbidity measurement was performed for 2 min by adding 0.1 ml of the standard buffer to determine OD660 at the reaction time of 0 min. Then, another measurement was performed after adding 0.1 ml of protease solution. The protein concentrations of SPI, 7S and 11S fractions in the reaction solutions were 10 mg/ml.
Fig. 2. SDS-PAGE of soy proteins. Coomassie brilliant blue-stained 12.5% SDSpolyacrylamide gel is shown. Lane 1, molecular mass markers; lane 2, 7S fraction; lane 3, 11S fraction; lane 4, SPI. (a and b) ␣ and ␣ (a) and  (b) subunits in conglycinin. (c–e) Acidic (c and d) and basic (e) subunits in glycinin.
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3.2. Effects of the thermal treatment on the coagulation of soy proteins induced by thermolysin As we reported previously [8,9], SPI coagulated by the proteolysis with subtilisin Carlsberg. The time-course of the coagulation can be monitored by measuring the turbidity (OD660 ) of the reaction solution (Fig. 1), in which OD1 and OD2 are the minimum and maximum values of OD660 , and T1 and T2 are the times at which OD660 reaches OD1 and OD2 , respectively. In the coagulation of the digests of soy proteins produced in the digestion with subtilisin Carlsberg, thermal treatment of soy proteins at >70 ◦ C is crucial (Inouye, Asaoka, Yasukawa, unpublished data). We first examined if this is true for the digestion with thermolysin (Fig. 3). OD660 of the solutions containing SPI species, namely SPI, and 7S and 11S fractions, decreased in the initial phase of the reaction to reach OD1 at time T1 (phase 1). After that, OD660 did not increase at all with the treatment of the SPI species at 37 ◦ C for 30 min, while it increased drastically to reach OD2 (phase 2) with the treatment at 80 ◦ C for 30 min. This indicates that, like the digestion with subtilisin Carlsberg, the thermal treatment of soy proteins at around 80 ◦ C is required for the coagulation of the digests produced in the digestion with thermolysin. The magnitudes of T2 in the coagulations induced by the two enzymes were almost the same in all SPI species. The magnitudes of OD2 in the thermolysin-induced coagulation of SPI and 11S soy proteins were 50%, and that of 7S soy proteins was 80% of those in the subtilisin Carlsberg-induced coagulation. These results indicate that the velocities of the coagulation are the same between the digestions with thermolysin and subtilisin Carlsberg, while the amount of the coagula in the former is substantially smaller than that in the latter. In the thermolysininduced coagulation of soy proteins treated at 80 ◦ C, OD1 , OD2 , T1 , and T2 for SPI, and 7S and 11S fractions are as follows: OD1 = 0.2, 0.1, and 0.1, respectively; OD2 = 0.4, 0.4, and 1.1, respectively; T1 = 10, 10, and <5 min, respectively; T2 = 40, 80, and 10 min, respectively. The order in the magnitude of T2 was 11S fraction < SPI < 7S fraction, and that in the magnitude of OD2 was 7S fraction, SPI < 11S fraction, both of which are the same as the orders in the coagulation induced by subtilisin Carlsberg. The results indicate that the coagulation induced by either of the two enzymes is the most remarkable in 11S fraction. Similar results were obtained for the thermal treatment of soy proteins at 98 ◦ C (data not shown). 3.3. Effects of the thermolysin concentration and the reaction temperature on the coagulation of soy proteins induced by thermolysin The effects of the thermolysin concentration on the coagulation of the digests produced in the digestion of SPI with thermolysin at 37 ◦ C were examined (Fig. 4). The increase in the turbidity speeded with increasing the thermolysin concentration (Fig. 4A). OD1 and OD2 did not change irrespective of the thermolysin concentration (Fig. 4B). T1 and T2 decreased from 80 to 5 min and from 500 to 20 min, respectively (Fig. 4C) with increasing the thermolysin concentration from 0.1 to 5 M. The result indicates that the velocity of the coagulation depends on the velocity of the hydrolysis reaction, but the amount of the coagula does not. Fig. 5 shows the effects of the reaction temperature on the coagulation of the digests produced in the digestion of SPI with thermolysin (10 M). The turbidity increased rapidly, but the maximum OD660 value decreased with increasing the reaction temperature (Fig. 5A). OD1 was almost constant but OD2 decreased from 0.9 to 0.4 (Fig. 5B), and T1 was almost constant but T2 decreased from 31 to 2 min (Fig. 5C) with increasing the reaction temperature from 37 to 70 ◦ C. The result indicates that the velocity of the coagulation and the amount of the coagula depend on the reaction temperature.
Fig. 3. Effect of the thermal treatment on the coagulation of soy proteins treated by protease. SPI (A), and 7S (B) and 11S (C) fractions (10 mg/ml) were treated at 80 ◦ C (a and b) or 37 ◦ C (c and d) for 30 min, and then treated by subtilisin Carlsberg (a and c) or thermolysin (b and d) (1.0 M) at pH 8.0 and 37 ◦ C. Time-courses of the proteaseinduced turbidity of soy protein solutions as examined by OD660 are shown. Zero minute means start of enzyme reaction.
3.4. Digests produced from soy proteins in the digestion with thermolysin To explore which fragments are involved in the coagulation, SPI, 7S and 11S fractions were treated at 80 ◦ C for 30 min, and then hydrolyzed with thermolysin (1 M) at 37 ◦ C. Aliquots of the reaction mixtures were taken out for specified times, followed by SDS-PAGE (Fig. 6). Protein bands with molecular masses of 70 (␣ and ␣ subunits of -conglycinin) and 50 kDa ( subunit of -conglycinin) and those with molecular masses of 40, 35, and 22 kDa (two acidic subunits and one basic subunit of glycinin,
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Fig. 4. Effect of the thermolysin concentration on the coagulation of soy proteins treated by thermolysin. (A) Time-courses of the turbidity of soy protein solutions treated by thermolysin. SPI (10 mg/ml) was treated at 80 ◦ C for 30 min, and then treated by thermolysin [5.0 M (a), 2.6 M (b), 0.8 M (c), and 0.6 M (d)] at pH 8.0 and 37 ◦ C. (B) Effect of the thermolysin concentration on OD1 and OD2 . OD1 (circle) and OD2 (triangle) of SPI solution are plotted against the thermolysin concentration. (C) Effect of the thermolysin concentration on T1 and T2 . T1 (circle) and T2 (triangle) of SPI solution are plotted against the thermolysin concentration. Zero minute means start of enzyme reaction.
respectively) disappeared completely in the digests at 5 min (lane 3 in Fig. 6A–C). Protein bands with molecular masses of 28 and 24 kDa appeared at 5 min in the digests from SPI and 7S soy proteins, and that with molecular mass of 18 kDa appeared at 5 min in the digests from SPI and 11S soy proteins. In the progress of enzyme reaction, the density of the 28 kDa protein band gradually decreased, that of the 24 kDa one remained constant, and that of the 18-kDa one gradually increased, reached the maximum, and then decreased. Considering that the degree of the coagulation was the most remarkable in 11S soy proteins, it is suggested that the
Fig. 5. Effect of the reaction temperature on the coagulation of soy proteins treated by thermolysin. (A) Time-courses of the turbidity of soy protein solutions treated by thermolysin. SPI (10 mg/ml) was treated at 98 ◦ C for 30 min, and then treated by thermolysin (10 M) at pH 8.0 and 37 ◦ C (a), 50 ◦ C (b), 60 ◦ C (c), and 70 ◦ C (d). (B) Effect of the reaction temperature on OD1 and OD2 . OD1 (circle) and OD2 (triangle) of SPI solution are plotted against the reaction temperature. (C) Effect of the reaction temperature on T1 and T2 . T1 (circle) and T2 (triangle) of SPI solution are plotted against the reaction temperature. Zero minute means start of enzyme reaction.
18 kDa fragment and its digests might play a role in the coagula formation. 3.5. Coagulation of the digests produced from the mixture of 7S and 11S proteins in the digestion of thermolysin The digests produced in the digestion of 11S soy proteins coagulated much more rapidly than those of SPI (Fig. 3). We hypothesized that the digests from 7S soy proteins might inhibit the coagulation of the digests from 11S soy proteins. To test this hypothesis, the mixture of 7S and 11S fractions (10 mg/ml in total, 1:2 for w/v) was treated at 80 ◦ C for 30 min and then hydrolyzed by thermolysin (0.1 M) at 37 ◦ C. It should be noted that the ratio of 7S proteins to 11S proteins in soy proteins is not exactly known, and it is roughly
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Fig. 7. Time-course of the thermolysin-induced turbidity of soy protein solutions. (a) The artificial diagram which is made by combining the time-courses of the turbidity of 7S and 11S fractions alone (10 mg/ml) with the ratio of 1:2 and then reducing the scale to one thirds. (b) Time-course of the turbidity of the mixture of 7S and 11S soy proteins treated by thermolysin. The mixture of 7S and 11S fractions (1:2 for w/v) (10 mg/ml) was treated at 80 ◦ C for 30 min, and then hydrolyzed by thermolysin (0.1 M) at pH 8.0 and 37 ◦ C. Zero minute means start of enzyme reaction.
Fig. 6. SDS-PAGE of soy proteins treated by thermolysin. SPI (A), and 7S (B) and 11S (C) fractions (10 mg/ml) were treated at 80 ◦ C for 30 min, and then treated by thermolysin (1.0 M) at pH 8.0 and 37 ◦ C. SDS-PAGE was performed under reducing conditions. Lane 1, molecular mass markers; lanes 2–9, soy protein digests at 0, 5, 10, 20, 30, 60, 120, and 240 min, respectively. Zero minute means start of enzyme reaction. (a–c) 28-, 24-, and 18-kDa bands, respectively.
estimated to be 1:1–1:2 (w/v). We mixed 7S and 11S fractions with the ratio of 1:2 for (w/v) based on the yields of 7S and 11S proteins in their preparation from soybean flakes. Fig. 7 shows the time-course of its turbidity. The artificial diagram, which is made by combining the time-courses of the turbidity of 7S and 11S fractions alone (10 mg/ml) with the ratio of 1:2 and then reducing the scale to one thirds, is shown for comparison. OD2 and T2 of the mixture of 7S and 11S fractions were 0.5 and 280 min, respectively, and those of the artificial diagram were 0.7 and 40 min, respectively, indicating that the increase in the turbidity of the former was much slower than that of the latter. The result suggests that the coagulation of the digests from 11S soy proteins might be suppressed by those from 7S soy proteins. 4. Discussion There were similarities and differences in the coagulations of the digests produced in the digestion of soy proteins with thermolysin and subtilisin Carlsberg. The similarities are as follows: (1) Thermal
treatment at around 80 ◦ C was crucial for the coagulation (Fig. 3). (2) The coagulation was the most remarkable in 11S soy proteins (Fig. 3). The differences are as follows: (1) The amount of the coagula produced in the digestion with thermolysin was smaller than that induced by subtilisin Carlsberg (Fig. 3). (2) The digests generated by the hydrolysis of SPI with thermolysin (mainly the 28-, 24-, and 18-kDa fragments upon SDS-PAGE under reducing conditions) were almost stable (Fig. 6), while those with subtilisin Carlsberg (mainly the 31-, 27-, and 21-kDa fragments upon SDS-PAGE under reducing conditions) were extensively digested into the smaller fragments (<16 kDa) [8] in the progress of enzyme reaction. The degree of the coagulation induced by thermolysin is smaller than that by subtilisin Carlsberg, suggesting that some of the soluble digests generated by the hydrolysis of SPI with thermolysin might form the coagula if further digested. SPI consists of two main protein components, -conglycinin and glycinin. -Conglycinin has ␣, ␣ , and  subunits and exists as tetramer with a sedimentation coefficient of 7S. Glycinin is composed of an acidic polypeptide (38 kDa) and a basic one (20 kDa) linked by a single disulfide bridge and exists as hexamer with a sedimentation coefficient of 11S. According to the crystallographic analysis [22,23], -conglycinin and glycinin contains core domains (-barrels and ␣-helies) and flexible loops. Assuming that the flexible loops are rapidly digested by each of the two enzymes, the results with SDS-PAGE (Fig. 6) suggest that the structured regions are scarcely digested by thermolysin while they are progressively digested by subtilisin Carlsberg. This might be explained by that thermolysin catalyzes specifically the hydrolysis of peptide bonds with bulky hydrophobic amino acid residues such as Phe or Leu at the P1 position [24,25], while subtilisin has a broad substrate specificity. Thermal treatment around 80 ◦ C is crucial for the coagulation of soy proteins induced by thermolysin and subtilisin Carlsberg (Fig. 3). It should be noted that soy proteins were digested by the enzymes even with the thermal treatment at 37 ◦ C: with the treatment of 11S fraction with subtilisin Carlsberg (1 M) at 37 ◦ C for 10 min, the protein bands corresponding to acidic subunits of glycinin (35 and 40 kDa) completely disappeared upon SDS-PAGE under reducing conditions (Inouye, Asaoka, Yasukawa, unpublished data). Therefore, thermal treatment does not render soy proteins hydrolyzed by proteases, but changes their structure and render them coagulate when hydrolyzed by proteases. We previously reported that, in the progress of the hydrolysis of SPI with subtilisin Carlsberg, the microenvironments
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around the aromatic residues in SPI changed by the measurement of the intrinsic fluorescence of SPI [9]. We also reported that 1-anilinonaphthalene-8-sulfonate (ANS)-binding regions of SPI decreased by the measurement of the externally added ANS [9]. These results suggest that the coagula form by the binding of hydrophobic area of the SPI digests [9]. Recently, the digests produced by the proteolysis of soy [26] and whey [27,28] proteins were characterized for their viscosity [26], amino acid sequence [27], and solubility [28]. The common mechanism for production of insoluble coagula has been provided [29]: unfold peptides with exposed hydrophobic areas released from soy or whey proteins by the proteolysis bind to hydrophobic areas of other molecules to form coagula. This mechanism might be applicable to the coagulation of soy proteins induced by thermolysin. The increases in the thermolysin concentration and the reaction temperature raised the velocity of the coagulation, but the former did not affect and the latter reduced the amount of the coagula (Figs. 4 and 5). The reduced amount of the coagula at high reaction temperature (70 ◦ C) might result from the increase in solubility of the digests rather than thermal inactivation of thermolysin. It was reported that T50 , the temperature required to reduce initial activity by 50% in 30 min, was 70.9 ◦ C [30] or 86.9 ◦ C [31] while T2 was 2 min in the coagulation of SPI treated by thermolysin at 70 ◦ C. The digests from 11S soy proteins coagulated much more rapidly than not only SPI and 7S soy proteins (Fig. 3) but also the mixture of 7S and 11S soy proteins (1:2 for w/v) (Fig. 7). The result suggests that the coagulation of the digests from 11S soy proteins might be suppressed by those from 7S soy proteins. We speculate that unfold peptides with exposed hydrophobic areas released from 11S soy proteins bind to the peptides from 7S soy proteins, and that the complex resulted do not form the coagula. However, there is no direct evidence for the binding of the digests from 7S soy proteins with those from 11S soy proteins. In conclusion, the coagulation of soy proteins induced by proteases is governed by several factors including soy protein species, thermal treatment, protease species, and reaction conditions. Further study is required to fully control the coagulation or completely solubilize the digests. Study of the effects of the solvents and additives on the hydrolysis reaction with thermolysin and subtilisin Carlsberg is currently underway. Acknowledgement
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This study was supported in part (K.I.) by Grants-in-Aid for Scientific Research (Nos. 17380065 and 20380061) from the Japan Society for the Promotion of Science.
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Coagulation of soy proteins induced by thermolysin and comparison of the coagulation reaction with that induced by subtilisin Carlsberg Kohei Asaoka, Kiyoshi Yasukawa, Kuniyo Inouye ∗ Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan
a r t i c l e
i n f o
Article history: Received 2 September 2008 Received in revised form 1 October 2008 Accepted 2 October 2008 Keywords: Coagulation Metalloproteinase Soy protein Subtilisin Carlsberg Thermolysin Turbidity
a b s t r a c t It is known that coagula are formed when soy proteins are treated by subtilisin Carlsberg. Time-course of the coagulation can be monitored by measuring the turbidity (OD660 ) of soy protein isolates (SPI) solution [Inouye K, Nagai K, Takita T. Coagulation of soy protein isolates induced by subtilisin Carlsberg. J Agric Food Chem 2002;50:1237–42]. In this study, we examined the coagulation of the digests produced in the digestion of SPI with thermolysin. SPI treated at 80 ◦ C coagulated by hydrolysis with either of thermolysin and subtilisin Carlsberg, but that treated at 37 ◦ C did not. The velocities of the coagulation were almost the same between the digestions with thermolysin and subtilisin Carlsberg, while the amount of the coagula in the former was 50–80% of that in the latter. With increasing the thermolysin concentration from 0.1 to 5 M, the velocity of the coagulation increased while the amount of the coagula was constant. With increasing the reaction temperature from 37 to 70 ◦ C, the velocity increased while the amount decreased. The digests from 11S soy proteins coagulated drastically compared to those from either of SPI, 7S soy proteins, and the mixture of 7S and 11S soy proteins (1:2 for w/v), suggesting that the coagulation of the digests from 11S soy proteins might be suppressed by those from 7S soy proteins. © 2008 Elsevier Inc. All rights reserved.
1. Introduction Soybean is a major source of plant oil. About 150 million tons of soybeans are produced every year, and a large amount of defatted soybean meals remain after extraction of oil. They are in part used as feed, but hardly utilized in food industries [1]. Defatted soybean meals contain proteins with high nutritional values and functional properties. In addition, it has been reputed that soy protein diet lowers blood-cholesterol [2] and triglyceride [3,4] levels. The proteolytic digests of soy protein also have bioactive effects such as insulin-mediated antilipolysis [5], insulin-mediated glucose transport and lipogenesis [6], and blood-pressure lowering [7], suggesting that soy protein and its digests could be useful for functional foods. In protease digestion of soy proteins, some of the digests coagulate and remain insoluble, resulting in lower yields of the soluble digests. We developed a convenient method to follow the proteaseinduced coagulation of soy protein isolates (SPI) by measuring continuously the turbidity of the reaction solution [8]. During the hydrolysis, the turbidity decreased in the first phase due to the digestion of SPI to reach the minimum (phase 1), and then increased
due to the coagulation of the digests to reach the maximum (phase 2). After it reached the maximum, it decreased again slowly due to the digestion of the coagula (phase 3) (Fig. 1). On the basis of these observations, we investigated the coagulation of the digests produced in the digestion of SPI with subtilisin Carlsberg [EC 3.4.21.14], and showed that the fragments formed in phase 2, but not in phase 1, were involved in the coagulation through hydrophobic interactions [8,9]. We also reported that the proteolytic digestion of SPI promotes the removal of hexanal, one of the major compounds of the unfavorable soy bean odor, by the treatment with various absorbents [10]. Thermolysin [EC 3.4.24.27] is a thermostable neutral metalloproteinase produced in the culture broth of Bacillus thermoproteolyticus [11–14]. It is widely used for the peptide bond formation through reverse reaction of hydrolysis [15,16]. In this study, we examined the coagulation of the digests produced in the digestion of SPI with thermolysin and compared it with the coagulation induced by subtilisin Carlsberg. 2. Materials and methods 2.1. Materials
∗ Corresponding author. Tel.: +81 75 753 6266; fax: +81 75 753 6265. E-mail address: [email protected] (K. Inouye). 0141-0229/$ – see front matter © 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.enzmictec.2008.10.001
Throughout the experiment, 20 mM phosphate buffer (pH 8.0) containing 0.05% sodium azide was used as a standard buffer. Thermolysin (lot T7LA991) was purchased from Daiwa Kasei (Osaka, Japan) and was dissolved in the standard buffer. Its concentration was determined using the molar absorption coefficient at 277 nm,
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Fig. 1. Introduced four parameters to characterize the reaction curve of the turbidity. The turbidity was evaluated by OD660 . OD1 and OD2 are the minimum and maximum of OD660 , and T1 and T2 are the times at which OD660 reached OD1 and OD2 , respectively.
ε277 , of 63.3 mM−1 cm−1 [16]. Subtilisin Carlsberg (lot 112k1327) was purchased from Sigma (St. Louis, MO) and was dissolved in the standard buffer. Its concentration was determined using ε280 of 23.0 mM−1 cm−1 [17]. All other chemicals were of reagent grade and purchased form Nacalai Tesque (Kyoto, Japan).
SDS-PAGE was performed under reducing conditions according to the method of Laemmli [21]. In the analysis of undigested SPI, and 7S and 11S soy proteins, a 12.5% polyacrylamide gel (Daiichi Pure Chemicals, Tokyo, Japan) was used. The sample solution was mixed with 4 volumes of the SDS-PAGE sample buffer, and was immediately boiled for 5 min. The solution (10 l) was applied to SDS-PAGE. A constant current of 40 mA was applied for 60 min. In the analysis of digested SPI, and 7S and 11S soy proteins, a 15%/25% gradient gel system (Multigel 15/25, Daiichi Pure Chemicals) was used. Five milliliters of each of SPI, and 7S and 11S fractions (concentrations: >10 mg/ml) was treated at 80 or 96 ◦ C for 30 min. Then, the solution was incubated at 37 ◦ C for 10 min and treated by subtilisin Carlsberg at pH 8.0 and 37 ◦ C. At 5, 10, 20, 30, 60, 90, 120, and 240 min from start of the reaction, a part of the reaction solution was taken out and mixed with 4 volumes of the SDS-PAGE sample buffer. Separately, 1.0 ml of the reaction solution at 120 min was taken out and centrifuged (10,000 × g, 1 min). The supernatant was collected and mixed with 4 volumes of the SDS-PAGE sample buffer. The solution was immediately boiled for 5 min, 10 l of which was applied to SDS-PAGE. A constant current of 40 mA was applied for 60 min. After electrophoresis, proteins were stained with Coomassie brilliant blue R-250. A molecular mass marker kit consisting of phosphorylase b (97 kDa), BSA (66 kDa), rabbit muscle aldolase (42 kDa), bovine erythrocyte carbonic anhydrase (30 kDa), soybean trypsin inhibitor (20 kDa), and hen egg white lysozyme (HEL) (14 kDa) was from Daiichi Pure Chemicals, and heart muscle myoglobin (Mb) (17 kDa), bovine milk ␣-lactalbumin (14 kDa), Mb I+III (11 kDa), Mb I (8.2 kDa), and Mb II (6.2 kDa) were from Sigma.
3. Results 3.1. Preparation of SPI, and 7S and 11S fractions from defatted soybean flakes
2.2. Preparation of SPI, and the 7S and 11S fractions Hexane-defatted soybean flakes (lot 05.10.27) was obtained from Fuji Oil Co. (Osaka, Japan). SPI was prepared according to the method of Tsumura et al. [18]. Briefly, the defatted soybean flakes were dispersed with water (1:10, v/v) with adjusting the pH to 7.0 with 2 M NaOH, and stirred for 1 h, followed by filtration through gauze. The filtrate was centrifuged at 5000 × g for 30 min to collect the supernatant. The pH of the supernatant was adjusted to 4.5 with 2 M HCl, and it was stirred for 1 h and centrifuged at 6500 × g for 20 min to collect the acidprecipitated soy protein. The precipitate was dispersed in distilled water and stored at 4 ◦ C before use as the SPI solution. The 7S and 11S fractions were prepared according to the method of Nagano et al. [19]. Briefly, the defatted soybean flakes were dispersed with water (1:15, v/v) with adjusting the pH to 7.5 with 2 M NaOH, and stirred for 1 h, followed by filtration through gauze and then by centrifugation at 9000 × g for 30 min. NaHSO3 (solid) was added to the supernatant resulted, and was adjusted to 0.98 g/l. After adjusting the pH of the supernatant to 6.4 with 2 M HCl, it was left at 4 ◦ C overnight and then was centrifuged at 6500 × g for 20 min to collect the precipitate and the supernatant. The precipitate was glycinin-rich and dispersed in distilled water. It was stored at 4 ◦ C until use as the 11S fraction. NaCl (solid) was added to the supernatant and was adjusted to 0.25 M. After adjusting the pH of the supernatant to 5.0 with 2 M HCl at 4 ◦ C, it was stirred for 1 h at 4 ◦ C, and centrifuged at 9000 × g for 30 min. The supernatant was mixed with icechilled water (1:1, v/v), and its pH was adjusted to 4.8 with 2 M HCl at 4 ◦ C. After overnight incubation at 4 ◦ C, the precipitate which is -conglycinin-rich was collected by centrifugation at 6500 × g for 20 min, dispersed in distilled water, and stored at 4 ◦ C before use as the 7S fraction. The solutions of SPI, and the 7S and 11S fractions obtained were dialyzed at 4 ◦ C against the standard buffer with the ratio of 1:15 (v/v) three times to adjust their pH to 8.0. After dialysis, the protein concentration was determined by the Lowry method using bovine serum albumin (BSA) as the standard [20]. These solutions were prepared daily and kept at 4 ◦ C before use. The solutions were slightly turbid, and thus, were not solutions but suspensions in a strict meaning, although we called these as solutions in this study for convenience.
We prepared SPI, and 7S and 11S fractions which had not received any thermal treatment. SPI was extracted from defatted soybean flake by alkaline and acidic solutions, and 7S and 11S fractions were extracted from the same material by alkaline and acidic solutions followed by salt precipitation based on the difference in the solubility of -conglycinin and glycinin with sodium hydrogen sulfite, which are described in Section 2. Upon SDS-PAGE under reducing conditions, 7S fraction yielded two bands with molecular masses of 70 and 50 kDa corresponding to ␣ and ␣ subunit and  subunit of -conglycinin, respectively, and 11S fraction yielded three bands with molecular masses of 40, 35, and 22 kDa corresponding to two acidic subunits and one basic subunit of glycinin, respectively (Fig. 2). It is considered that 7S and 11S fractions prepared in our method are exclusively composed of -conglycinin and glycinin, respectively.
2.3. Turbidity measurement The turbidity of the soy protein solution was evaluated by measuring the optical density at 660 nm (OD660 ) with a spectrophotometer DU800 (Beckman Coulter, Tokyo, Japan). SPI, and 7S and 11S fractions were diluted with the standard buffer. After the incubation on ice for 10 min, 5 ml each of the solutions (concentrations: >10 mg/ml) was incubated at 80 or 98 ◦ C, and then immediately incubated at 37 ◦ C for 10 min. The solution (2.4 ml) was transferred to an optical cuvette thermostatted at 37 ◦ C and further incubated for 5 min. The turbidity measurement was performed for 2 min by adding 0.1 ml of the standard buffer to determine OD660 at the reaction time of 0 min. Then, another measurement was performed after adding 0.1 ml of protease solution. The protein concentrations of SPI, 7S and 11S fractions in the reaction solutions were 10 mg/ml.
Fig. 2. SDS-PAGE of soy proteins. Coomassie brilliant blue-stained 12.5% SDSpolyacrylamide gel is shown. Lane 1, molecular mass markers; lane 2, 7S fraction; lane 3, 11S fraction; lane 4, SPI. (a and b) ␣ and ␣ (a) and  (b) subunits in conglycinin. (c–e) Acidic (c and d) and basic (e) subunits in glycinin.
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3.2. Effects of the thermal treatment on the coagulation of soy proteins induced by thermolysin As we reported previously [8,9], SPI coagulated by the proteolysis with subtilisin Carlsberg. The time-course of the coagulation can be monitored by measuring the turbidity (OD660 ) of the reaction solution (Fig. 1), in which OD1 and OD2 are the minimum and maximum values of OD660 , and T1 and T2 are the times at which OD660 reaches OD1 and OD2 , respectively. In the coagulation of the digests of soy proteins produced in the digestion with subtilisin Carlsberg, thermal treatment of soy proteins at >70 ◦ C is crucial (Inouye, Asaoka, Yasukawa, unpublished data). We first examined if this is true for the digestion with thermolysin (Fig. 3). OD660 of the solutions containing SPI species, namely SPI, and 7S and 11S fractions, decreased in the initial phase of the reaction to reach OD1 at time T1 (phase 1). After that, OD660 did not increase at all with the treatment of the SPI species at 37 ◦ C for 30 min, while it increased drastically to reach OD2 (phase 2) with the treatment at 80 ◦ C for 30 min. This indicates that, like the digestion with subtilisin Carlsberg, the thermal treatment of soy proteins at around 80 ◦ C is required for the coagulation of the digests produced in the digestion with thermolysin. The magnitudes of T2 in the coagulations induced by the two enzymes were almost the same in all SPI species. The magnitudes of OD2 in the thermolysin-induced coagulation of SPI and 11S soy proteins were 50%, and that of 7S soy proteins was 80% of those in the subtilisin Carlsberg-induced coagulation. These results indicate that the velocities of the coagulation are the same between the digestions with thermolysin and subtilisin Carlsberg, while the amount of the coagula in the former is substantially smaller than that in the latter. In the thermolysininduced coagulation of soy proteins treated at 80 ◦ C, OD1 , OD2 , T1 , and T2 for SPI, and 7S and 11S fractions are as follows: OD1 = 0.2, 0.1, and 0.1, respectively; OD2 = 0.4, 0.4, and 1.1, respectively; T1 = 10, 10, and <5 min, respectively; T2 = 40, 80, and 10 min, respectively. The order in the magnitude of T2 was 11S fraction < SPI < 7S fraction, and that in the magnitude of OD2 was 7S fraction, SPI < 11S fraction, both of which are the same as the orders in the coagulation induced by subtilisin Carlsberg. The results indicate that the coagulation induced by either of the two enzymes is the most remarkable in 11S fraction. Similar results were obtained for the thermal treatment of soy proteins at 98 ◦ C (data not shown). 3.3. Effects of the thermolysin concentration and the reaction temperature on the coagulation of soy proteins induced by thermolysin The effects of the thermolysin concentration on the coagulation of the digests produced in the digestion of SPI with thermolysin at 37 ◦ C were examined (Fig. 4). The increase in the turbidity speeded with increasing the thermolysin concentration (Fig. 4A). OD1 and OD2 did not change irrespective of the thermolysin concentration (Fig. 4B). T1 and T2 decreased from 80 to 5 min and from 500 to 20 min, respectively (Fig. 4C) with increasing the thermolysin concentration from 0.1 to 5 M. The result indicates that the velocity of the coagulation depends on the velocity of the hydrolysis reaction, but the amount of the coagula does not. Fig. 5 shows the effects of the reaction temperature on the coagulation of the digests produced in the digestion of SPI with thermolysin (10 M). The turbidity increased rapidly, but the maximum OD660 value decreased with increasing the reaction temperature (Fig. 5A). OD1 was almost constant but OD2 decreased from 0.9 to 0.4 (Fig. 5B), and T1 was almost constant but T2 decreased from 31 to 2 min (Fig. 5C) with increasing the reaction temperature from 37 to 70 ◦ C. The result indicates that the velocity of the coagulation and the amount of the coagula depend on the reaction temperature.
Fig. 3. Effect of the thermal treatment on the coagulation of soy proteins treated by protease. SPI (A), and 7S (B) and 11S (C) fractions (10 mg/ml) were treated at 80 ◦ C (a and b) or 37 ◦ C (c and d) for 30 min, and then treated by subtilisin Carlsberg (a and c) or thermolysin (b and d) (1.0 M) at pH 8.0 and 37 ◦ C. Time-courses of the proteaseinduced turbidity of soy protein solutions as examined by OD660 are shown. Zero minute means start of enzyme reaction.
3.4. Digests produced from soy proteins in the digestion with thermolysin To explore which fragments are involved in the coagulation, SPI, 7S and 11S fractions were treated at 80 ◦ C for 30 min, and then hydrolyzed with thermolysin (1 M) at 37 ◦ C. Aliquots of the reaction mixtures were taken out for specified times, followed by SDS-PAGE (Fig. 6). Protein bands with molecular masses of 70 (␣ and ␣ subunits of -conglycinin) and 50 kDa ( subunit of -conglycinin) and those with molecular masses of 40, 35, and 22 kDa (two acidic subunits and one basic subunit of glycinin,
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Fig. 4. Effect of the thermolysin concentration on the coagulation of soy proteins treated by thermolysin. (A) Time-courses of the turbidity of soy protein solutions treated by thermolysin. SPI (10 mg/ml) was treated at 80 ◦ C for 30 min, and then treated by thermolysin [5.0 M (a), 2.6 M (b), 0.8 M (c), and 0.6 M (d)] at pH 8.0 and 37 ◦ C. (B) Effect of the thermolysin concentration on OD1 and OD2 . OD1 (circle) and OD2 (triangle) of SPI solution are plotted against the thermolysin concentration. (C) Effect of the thermolysin concentration on T1 and T2 . T1 (circle) and T2 (triangle) of SPI solution are plotted against the thermolysin concentration. Zero minute means start of enzyme reaction.
respectively) disappeared completely in the digests at 5 min (lane 3 in Fig. 6A–C). Protein bands with molecular masses of 28 and 24 kDa appeared at 5 min in the digests from SPI and 7S soy proteins, and that with molecular mass of 18 kDa appeared at 5 min in the digests from SPI and 11S soy proteins. In the progress of enzyme reaction, the density of the 28 kDa protein band gradually decreased, that of the 24 kDa one remained constant, and that of the 18-kDa one gradually increased, reached the maximum, and then decreased. Considering that the degree of the coagulation was the most remarkable in 11S soy proteins, it is suggested that the
Fig. 5. Effect of the reaction temperature on the coagulation of soy proteins treated by thermolysin. (A) Time-courses of the turbidity of soy protein solutions treated by thermolysin. SPI (10 mg/ml) was treated at 98 ◦ C for 30 min, and then treated by thermolysin (10 M) at pH 8.0 and 37 ◦ C (a), 50 ◦ C (b), 60 ◦ C (c), and 70 ◦ C (d). (B) Effect of the reaction temperature on OD1 and OD2 . OD1 (circle) and OD2 (triangle) of SPI solution are plotted against the reaction temperature. (C) Effect of the reaction temperature on T1 and T2 . T1 (circle) and T2 (triangle) of SPI solution are plotted against the reaction temperature. Zero minute means start of enzyme reaction.
18 kDa fragment and its digests might play a role in the coagula formation. 3.5. Coagulation of the digests produced from the mixture of 7S and 11S proteins in the digestion of thermolysin The digests produced in the digestion of 11S soy proteins coagulated much more rapidly than those of SPI (Fig. 3). We hypothesized that the digests from 7S soy proteins might inhibit the coagulation of the digests from 11S soy proteins. To test this hypothesis, the mixture of 7S and 11S fractions (10 mg/ml in total, 1:2 for w/v) was treated at 80 ◦ C for 30 min and then hydrolyzed by thermolysin (0.1 M) at 37 ◦ C. It should be noted that the ratio of 7S proteins to 11S proteins in soy proteins is not exactly known, and it is roughly
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Fig. 7. Time-course of the thermolysin-induced turbidity of soy protein solutions. (a) The artificial diagram which is made by combining the time-courses of the turbidity of 7S and 11S fractions alone (10 mg/ml) with the ratio of 1:2 and then reducing the scale to one thirds. (b) Time-course of the turbidity of the mixture of 7S and 11S soy proteins treated by thermolysin. The mixture of 7S and 11S fractions (1:2 for w/v) (10 mg/ml) was treated at 80 ◦ C for 30 min, and then hydrolyzed by thermolysin (0.1 M) at pH 8.0 and 37 ◦ C. Zero minute means start of enzyme reaction.
Fig. 6. SDS-PAGE of soy proteins treated by thermolysin. SPI (A), and 7S (B) and 11S (C) fractions (10 mg/ml) were treated at 80 ◦ C for 30 min, and then treated by thermolysin (1.0 M) at pH 8.0 and 37 ◦ C. SDS-PAGE was performed under reducing conditions. Lane 1, molecular mass markers; lanes 2–9, soy protein digests at 0, 5, 10, 20, 30, 60, 120, and 240 min, respectively. Zero minute means start of enzyme reaction. (a–c) 28-, 24-, and 18-kDa bands, respectively.
estimated to be 1:1–1:2 (w/v). We mixed 7S and 11S fractions with the ratio of 1:2 for (w/v) based on the yields of 7S and 11S proteins in their preparation from soybean flakes. Fig. 7 shows the time-course of its turbidity. The artificial diagram, which is made by combining the time-courses of the turbidity of 7S and 11S fractions alone (10 mg/ml) with the ratio of 1:2 and then reducing the scale to one thirds, is shown for comparison. OD2 and T2 of the mixture of 7S and 11S fractions were 0.5 and 280 min, respectively, and those of the artificial diagram were 0.7 and 40 min, respectively, indicating that the increase in the turbidity of the former was much slower than that of the latter. The result suggests that the coagulation of the digests from 11S soy proteins might be suppressed by those from 7S soy proteins. 4. Discussion There were similarities and differences in the coagulations of the digests produced in the digestion of soy proteins with thermolysin and subtilisin Carlsberg. The similarities are as follows: (1) Thermal
treatment at around 80 ◦ C was crucial for the coagulation (Fig. 3). (2) The coagulation was the most remarkable in 11S soy proteins (Fig. 3). The differences are as follows: (1) The amount of the coagula produced in the digestion with thermolysin was smaller than that induced by subtilisin Carlsberg (Fig. 3). (2) The digests generated by the hydrolysis of SPI with thermolysin (mainly the 28-, 24-, and 18-kDa fragments upon SDS-PAGE under reducing conditions) were almost stable (Fig. 6), while those with subtilisin Carlsberg (mainly the 31-, 27-, and 21-kDa fragments upon SDS-PAGE under reducing conditions) were extensively digested into the smaller fragments (<16 kDa) [8] in the progress of enzyme reaction. The degree of the coagulation induced by thermolysin is smaller than that by subtilisin Carlsberg, suggesting that some of the soluble digests generated by the hydrolysis of SPI with thermolysin might form the coagula if further digested. SPI consists of two main protein components, -conglycinin and glycinin. -Conglycinin has ␣, ␣ , and  subunits and exists as tetramer with a sedimentation coefficient of 7S. Glycinin is composed of an acidic polypeptide (38 kDa) and a basic one (20 kDa) linked by a single disulfide bridge and exists as hexamer with a sedimentation coefficient of 11S. According to the crystallographic analysis [22,23], -conglycinin and glycinin contains core domains (-barrels and ␣-helies) and flexible loops. Assuming that the flexible loops are rapidly digested by each of the two enzymes, the results with SDS-PAGE (Fig. 6) suggest that the structured regions are scarcely digested by thermolysin while they are progressively digested by subtilisin Carlsberg. This might be explained by that thermolysin catalyzes specifically the hydrolysis of peptide bonds with bulky hydrophobic amino acid residues such as Phe or Leu at the P1 position [24,25], while subtilisin has a broad substrate specificity. Thermal treatment around 80 ◦ C is crucial for the coagulation of soy proteins induced by thermolysin and subtilisin Carlsberg (Fig. 3). It should be noted that soy proteins were digested by the enzymes even with the thermal treatment at 37 ◦ C: with the treatment of 11S fraction with subtilisin Carlsberg (1 M) at 37 ◦ C for 10 min, the protein bands corresponding to acidic subunits of glycinin (35 and 40 kDa) completely disappeared upon SDS-PAGE under reducing conditions (Inouye, Asaoka, Yasukawa, unpublished data). Therefore, thermal treatment does not render soy proteins hydrolyzed by proteases, but changes their structure and render them coagulate when hydrolyzed by proteases. We previously reported that, in the progress of the hydrolysis of SPI with subtilisin Carlsberg, the microenvironments
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around the aromatic residues in SPI changed by the measurement of the intrinsic fluorescence of SPI [9]. We also reported that 1-anilinonaphthalene-8-sulfonate (ANS)-binding regions of SPI decreased by the measurement of the externally added ANS [9]. These results suggest that the coagula form by the binding of hydrophobic area of the SPI digests [9]. Recently, the digests produced by the proteolysis of soy [26] and whey [27,28] proteins were characterized for their viscosity [26], amino acid sequence [27], and solubility [28]. The common mechanism for production of insoluble coagula has been provided [29]: unfold peptides with exposed hydrophobic areas released from soy or whey proteins by the proteolysis bind to hydrophobic areas of other molecules to form coagula. This mechanism might be applicable to the coagulation of soy proteins induced by thermolysin. The increases in the thermolysin concentration and the reaction temperature raised the velocity of the coagulation, but the former did not affect and the latter reduced the amount of the coagula (Figs. 4 and 5). The reduced amount of the coagula at high reaction temperature (70 ◦ C) might result from the increase in solubility of the digests rather than thermal inactivation of thermolysin. It was reported that T50 , the temperature required to reduce initial activity by 50% in 30 min, was 70.9 ◦ C [30] or 86.9 ◦ C [31] while T2 was 2 min in the coagulation of SPI treated by thermolysin at 70 ◦ C. The digests from 11S soy proteins coagulated much more rapidly than not only SPI and 7S soy proteins (Fig. 3) but also the mixture of 7S and 11S soy proteins (1:2 for w/v) (Fig. 7). The result suggests that the coagulation of the digests from 11S soy proteins might be suppressed by those from 7S soy proteins. We speculate that unfold peptides with exposed hydrophobic areas released from 11S soy proteins bind to the peptides from 7S soy proteins, and that the complex resulted do not form the coagula. However, there is no direct evidence for the binding of the digests from 7S soy proteins with those from 11S soy proteins. In conclusion, the coagulation of soy proteins induced by proteases is governed by several factors including soy protein species, thermal treatment, protease species, and reaction conditions. Further study is required to fully control the coagulation or completely solubilize the digests. Study of the effects of the solvents and additives on the hydrolysis reaction with thermolysin and subtilisin Carlsberg is currently underway. Acknowledgement
[5] [6]
[7] [8] [9]
[10] [11] [12] [13]
[14]
[15]
[16]
[17]
[18]
[19]
[20] [21] [22]
[23] [24] [25]
This study was supported in part (K.I.) by Grants-in-Aid for Scientific Research (Nos. 17380065 and 20380061) from the Japan Society for the Promotion of Science.
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