Oleosins (24 and 18 kDa) Are Hydrolyzed Not Only in Extracted

Jan 9, 2014 - ... oil bodies (oleosomes) using bicarbonate-based soaking and grinding media. Simone De Chirico , Vincenzo di Bari , Tim Foster , David...
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Oleosins (24 and 18 kDa) Are Hydrolyzed Not Only in Extracted Soybean Oil Bodies but Also in Soybean Germination Yeming Chen, Luping Zhao, Yanyun Cao, Xiangzhen Kong, and Yufei Hua* State Key Laboratory of Food Science and Technology, Synergetic Innovation Center of Food Safety and Nutrition, School of Food Science and Technology, Jiangnan University, 1800 Lihu Avenue, Wuxi, Jiangsu Province 214122, PR China ABSTRACT: After oil bodies (OBs) were extracted from ungerminated soybean by pH 6.8 extraction, it was found that 24 and 18 kDa oleosins were hydrolyzed in the extracted OBs, which contained many OB extrinsic proteins (i.e., lipoxygenase, βconglycinin, γ-conglycinin, β-amylase, glycinin, Gly m Bd 30K (Bd 30K), and P34 probable thiol protease (P34)) as well as OB intrinsic proteins. In this study, some properties (specificity, optimal pH and temperature) of the proteases of 24 and 18 kDa oleosins and the oleosin hydrolysis in soybean germination were examined, and the high relationship between Bd 30K/P34 and the proteases was also discussed. The results showed (1) the proteases were OB extrinsic proteins, which had high specificity to hydrolyze 24 and 18 kDa oleosins, and cleaved the specific peptide bonds to form limited hydrolyzed products; (2) 24 and 18 kDa oleosins were not hydrolyzed in the absence of Bd 30K and P34 (or some Tricine-SDS-PAGE undetectable proteins); (3) the protease of 24 kDa oleosin had strong resistance to alkaline pH while that of 18 kDa oleosin had weak resistance to alkaline pH, and Bd 30K and P34, resolved into two spots on two-dimensional electrophoresis gel, also showed the same trend; (4) 16 kDa oleosin as well as 24 and 18 kDa oleosins were hydrolyzed in soybean germination, and Bd 30K and P34 were always contained in the extracted OBs from germinated soybean even when all oleosins were hydrolyzed; (5) the optimal temperature and pH of the proteases were respectively determined as in the ranges of 35−50 °C and pH 6.0−6.5, while 60 °C or pH 11.0 could denature them. KEYWORDS: ungerminated soybean, germinated soybean, extracted oil bodies, Gly m Bd 30K, P34 probable thiol protease, 24 kDa oleosin, 18 kDa oleosin, 16 kDa oleosin

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INTRODUCTION Most of plant seeds store triacylglycerols (TAGs) in organelles termed oil bodies (OBs), also lipid bodies, oleosomes, and spherosomes. It is reported that each OB has a TAGs matrix core coated by one monolayer of phospholipids embedded with OB intrinsic proteins,1 and this structure can keep the integrity of OBs from various environmental stresses, such as freezing and desiccation.2,3 It is widely accepted that plant seed OBs contain at least three classes of intrinsic proteins, oleosin, caleosin (minor), and steroleosin (minor).4 In our previous research,5 seven oleosins, two caleosins (27 and 29 kDa), and one steroleosin (41 kDa) were confirmed in soybean OBs. Two 24 kDa oleosin isoforms (P29530 and P29531) and one 18 kDa oleosin (C3VHQ8) are the three major oleosins, followed by two intermediate ones (C6SZ13, 16 kDa; gi|356576403, 16 kDa), and two minor ones (I1MTE2, 19 kDa; I1MUH0, 18 kDa). Soybean OBs, a natural source of pre-emulsified soybean oil,6 are greatly examined due to its potential utilization in food, cosmetics, pharmaceutical, and other applications requiring highly stable oil-in-water emulsions.7,8 Therefore, many researchers tried to extract soybean OBs and focused on extraction yield,9 dispersion stability,10 oxidative stability,11 and in vitro digestibility.12 Unexpectedly, it was found that the 24 kDa (P29530 and P29531) and 18 kDa (C3VHQ8) oleosins were hydrolyzed by some unknown proteases in the pH 6.8 extracted soybean OBs.5 It was considered that this might be unbeneficial to the utilizations of soybean OBs, but this might be very good news for the enzyme-assisted aqueous extraction © 2014 American Chemical Society

of soybean oil, in which proteases were artificially added into the extraction system to destroy the oleosins of OBs to release oil.13 Generally, the oleosin hydrolysis was reported to happen during the plant seed germination and seedling growth, such as maize,14 rapeseed,15 anise,16 soybean,17 sesame,18 sunflower,19 and cucumber,20 which might be the initial step for the lipaseinduced TAGs mobilization.21 No other researches had ever reported the oleosin hydrolysis in the extracted OBs, and the proteases for the oleosin hydrolysis were also not well-known. In addition to the OB intrinsic proteins, it was found that pH 6.8 extracted OBs contained lipoxygenase, β-conglycinin, γconglycinin, β-amylase, glycinin, Gly m Bd 30K (Bd 30K), and P34 probable thiol protease (P34), but not lectin, Gly m Bd 28K, Kunitz trypsin inhibitor, and Bowman-Birk inhibitor.5 It is considered that Bd 30K and P34 are the most probable proteases for the oleosin hydrolysis in pH 6.8 extracted OBs. In the first research about soybean OB oleosin, P34, a protein comprised of 257 amino acid residues (AARs), was wrongly considered as one kind of oleosin.22 In two later researches,23,24 it was found that P34, having considerable sequence similarity to the thiol proteases of papain family, originated from pro-P34 (47 kDa) during seed maturation and stored in protein storage vacuoles (PSVs). Different from P34, many other seed thiol proteases, which are closely correlated with storage protein Received: Revised: Accepted: Published: 956

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mobilization, are synthesized only after seed germination,24,25 revealing that P34 may possess different biological activity. Structurally, the thiol proteases of the papain family possessed highly conserved catalytic triad (Cys-His-Asn) residues and three disulfide bonds.26 Compared with these thiol proteases, a Gly37 replaced the highly conserved catalytic Cys in P34; of the three disulfides, two were present in P34 (Cys68-Cys108 and Cys171-Cys224). However, one of the three disulfides in other thiol proteases existed as Cys34 and Asn77 in P34, so it was suggested that the Cys34 near Gly37 should be a free thiol and act as the active-site residue.23,24 Until now, only one protein was reported to be similar to P34, which was a 31 kDa thiol protease (SPE31) isolated from the seeds of a legume plant (Pachyrizhus erosus), and they were considered to form a unique subfamily within the papain family.27 However, the substrates of P34 and SPE31 were not clear yet. Several members of the papain protease family have been reported in allergenic reaction, such as fecal dust mite allergen Der 1p28 and archtype papain thiol protease.29 And it was considered that the destructive proteolytic activity of these thiol proteases might be a significant factor in the allergenicity.30 Bd 30K (257 AARs), an allergenic soybean protein, was first identified by Ogawa et al., and considered as similar protein to P34 owing to their identical N-terminal amino acid sequences and amino acid compositions.31,32 However, the Arg46, Ser75, Gln80, and Phe203 of P34 were replaced by the Ser46, Cys75, His80, and Glu203 of Bd 30K (NCBI database). Two studies reported that there were two spots for P34 and Bd 30K on the two-dimensional electrophoresis gel,30,33 revealing that P34 and Bd 30K might be two different proteins with similar biological activities. Many studies reported that P34/Bd 30K had high specificity to interact with soybean OBs,5,12,22−24,34 very similar to the interaction between protease and its substrate. The interaction between P34/Bd 30K and soybean OBs was clarified as the disulfide bond between P34/Bd 30K and 24 kDa oleosin.5 In fact, the best way to confirm one protease is directly mixing it with its substrate. Sewekow et al. reported a method for the purification of P34 by hydrophobic interaction chromatography, and a key buffer of 0.1 M sodium carbonate (pH > 11.0) was used.35 Ogawa et al. reported a method for the purification of Bd 30K, and sodium dodecylsulfate (SDS, 4%) and heat treatment (98 °C, 10 min) were used.32 According to the present study, P34 and Bd 30K (assumed as the proteases) are already denatured at pH 11.0 and also denatured when exposed to SDS and/or high temperature. It is considered that a method for bioactive P34/Bd 30K purification is a great challenge at the present stage. In this study, some properties (specificity, optimal pH, and temperature) of the proteases of 24 and 18 kDa oleosins, and oleosin hydrolysis in soybean germination were examined, and the high relationship between the proteases and P34/Bd 30K was also discussed. It is considered that this study is very meaningful for designing better processing methods for the soybean OB extraction and the enzyme-assisted aqueous extraction of soybean oil.

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changes compared to the purified glycinin from soybean stored in a −20 °C freezer.36 These showed that the data in the study should be acceptable. All reagents were purchased from Sigma-Aldrich Trading Co., Ltd. or of analytical reagent grade. Precision Plus Protein All Blue (Bio-Rad, USA), including 250, 150, 100, 75, 50, 37, 25, 15, and 10 kDa proteins, was used as the protein marker. Raw Soymilk Preparation. Soybean (20 g) was soaked in deionized (DI) water at 4 °C for 18 h. The soaking water was poured off, and the soaked soybeans were washed with fresh DI water three times. DI water, precooled in 4 °C refrigerator, was added to make the total weight of 200 g. This mixture was ground in a Waring blender (18 000 rpm, MJ-60BE01B, Midea) for 2 min and filtered through four layers of gauze to obtain raw soymilk (about pH 6.8). Oil Bodies (OBs) Extracted by pH 6.8, 8.0, 9.5, and 11.0 at 4 °C. This was conducted with the method by Chen and Ono34 with the modification that the temperature was controlled at 4 °C. Sucrose was added into raw soymilk to make the concentration of 20% (w/w) and mixed well in 4 °C refrigerator. A total of 40 g of raw soymilk (20% sucrose) was added into the first centrifuge tube. The residual raw soymilk was adjusted to pH 8.0 by 2 M NaOH, and 40 g was added into the second centrifuge tube. The residual raw soymilk was adjusted to pH 9.5, and 40 g was added into the third centrifuge tube. The residual raw soymilk was adjusted to pH 11.0, and 40 g was added into the fourth centrifuge tube. Then they were treated by centrifugation (25,000g, 30 min; 4 °C). The supernatant of pH 6.8 system was collected (termed pH 6.8 supernatant), stored in 4 °C refrigerator and used for the experiment below. The floating fractions were collected, dispersed into 39 g of sucrose solution (20%, w/w), and adjusted to corresponding pH, respectively. They were mixed well and treated by centrifugation (25,000g, 30 min; 4 °C). Then the collection of floating fraction, dispersion into sucrose solution (20%, w/w), pH adjustment, and centrifugation as above was repeated twice. The floating fractions obtained were dispersed into DI water with oil concentrations of 40 mg/mL. The four suspensions were all adjusted to pH 6.5. Each suspension (7 mL) was incubated in 35 °C water bath for 0, 0.5, 1, 3, 6, and 9 h, and 1 mL of suspension was collected and cooled in one ice water bath, respectively. The obtained OB suspensions (1 mL) were defatted by diethyl ether with the method by Tzen et al.37 Briefly, three milliliters of diethyl ether (4 °C) was added, thoroughly mixed for 30 min and centrifuged (15,000g, 5 min; 4 °C). The upper diethyl ether was removed, and the procedure was repeated one more time. The residual diethyl ether was allowed to evaporate in a hood combined with fan blowing, which was conducted in ice water bath. The residual aqueous solution was well mixed. The protein concentration of pH 6.8 extracted OBs was determined by the micro-Kjeldahl method, and 0.5 mL of the defatted sample (well mixed) was diluted with Tricine-SDS-PAGE sample buffer to make the protein concentration of 2 mg/mL. The other three defatted samples (pH 8.0, 9.5 and 11.0), which had lower protein concentrations, were mixed with the same volume of sample buffer above. This method, different from the traditional method, was beneficial for examining the protein release behaviors from OBs. Effect of pH 6.8 Supernatant on pH 11.0 Extracted OBs. A total of 7 mL of the pH 11.0 extracted OB suspension (pH 6.5, 4 °C) was added into 30 mL of pH 6.8 supernatant obtained above. They were stirred in ice water bath for 30 min and centrifuged (25,000g, 30 min; 4 °C). The floating fraction was carefully collected and dispersed well into 7 mL of DI water, and stored in 4 °C refrigerator (termed the first OBs). The supernatant was collected carefully with syringe and mixed in ice water bath for 30 min with another 7 mL of fresh pH 11.0 extracted OB suspension. It was treated by centrifugation (25,000g, 30 min; 4 °C). Then the collection of supernatant fraction, and its mixing with 7 mL of fresh pH 11.0 extracted OB suspension followed by centrifugation was repeated three more times, and floating fraction (OBs) and supernatant (termed the fifth supernatant) were obtained by the last centrifugation. The floating fraction was dispersed into 7 mL of DI water (4 °C) and termed the fifth OBs. The protein concentrations of the fifth supernatant and pH 6.8 supernatant were determined by bicinchoninic acid assay with bovine serum albumin as standard, and diluted with SDS-PAGE sample buffer to make the

MATERIALS AND METHODS

Materials. Soybean Nannong 88−31 (moisture content, 9%), harvested in August, 2011, was used. It was stored at 4 °C until use. This study was conducted from March, 2012 to June, 2013, and the behaviors of oleosin hydrolysis were consistent in this period. One study reported that the structure of glycinin, which was purified from soybean stored at 4 °C for 18 months, did not show significant 957

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concentrations of 20 mg/mL. The first and fifth OBs were incubated in 35 °C water bath for 0, 0.5, 1, 3, 6, and 9 h, and 1 mL of suspension was collected and cooled in an ice water bath. Then they were treated as above for Tricine-SDS-PAGE analysis. Soybean Germination. In order to simulate the natural soybean germination, 20 g of soybeans was washed and soaked in tap water for 9 h at room temperature. A food-grade plastic container, without cover, was used for the soybean germination, and there were many holes on its bottom and walls. The tap water was poured off, and the soybeans were uniformly placed on the container bottom. The boiled tap water (room temperature) was sprayed on the soybean for about 2 min, and a preservative film (with artificially small holes) was used to cover the container for germination (1, 2, 4, 6, and 8 days). Everyday the soybeans was sprayed with boiled tap water (room temperature) four times (at 8:00, 12:00, 16:00, and 20:00). In the soybean germination, the room temperature was within the range of 21−33 °C. DI water, precooled in 4 °C refrigerator, was added into the germinated soybeans to make total weight of 200 g. It was ground for 2 min and filtered through four layers of gauze to obtain raw germinated soymilk. Sucrose was added into raw germinated soymilk to make a concentration of 25% (w/w) and mixed well. Raw germinated soymilk (6.5 mL; 25% sucrose, w/w) was added into the first ultracentrifuge tube. The residual raw germinated soymilk was adjusted to pH 8.0, and 6.5 mL was added into the second ultracentrifuge tube. The residual was adjusted to pH 9.5, and 6.5 mL was added into the third ultracentrifuge tube. The three tubes were all preplaced in the ice water bath. Then 3 mL of 20% (w/w) sucrose solution was carefully layered on the top of the raw germinated soymilk in the three tubes. These were treated by ultracentrifugation (197,000g, 1 h; 4 °C), and solid OB pad was formed on the top of 20% sucrose solution. The OB pad was carefully taken out and rinsed by DI water (4 °C). The residual DI water was carefully absorbed by filter paper. The OB pad was weighed, and 5-fold of DI water (4 °C) was added to disperse it well. Then it was defatted as above. The protein concentration of the defatted sample (pH 6.8 extracted OBs from 1 d germinated soybean) was determined by the micro-Kjeldahl method, and 0.5 mL of the defatted sample was diluted with Tricine-SDSPAGE sample buffer to make the protein concentration of 2 mg/mL. The other defatted samples (pH 8.0, pH 9.5; 2, 4, 6, and 8 d germinated soybean), which had lower protein concentrations, were all diluted with the same volume of sample buffer above and used for Tricine-SDS-PAGE analysis. Effects of Temperature and pH on Oleosin Hydrolysis. pH 6.8 extracted OB suspension (pH 6.5), without repeated washing as above, was used to examine the effect of temperature on oleosin hydrolysis. A total of 15 mL of suspension was incubated in 20, 30, 35, 40, 50, and 60 °C water baths, respectively. After 0, 0.5, 1, 3, 6, and 9 h, 1 mL of suspension was collected and cooled in ice water bath. In addition, 15 mL of pH 6.8 extracted OB suspension was respectively adjusted to pH 4.0, 5.0, 6.0, 6.5, 7.0, 8.0, 9.0, 10.0, and 11.0, and all incubated in 35 °C water bath. After 0, 0.5, 1, 3, 6, and 9 h, 1 mL of suspension was collected and cooled in ice water bath. The obtained OB suspensions (1 mL) were treated as above for Tricine-SDS-PAGE analysis. These experiments were conducted two times and showed the same trend. SDS-PAGE. SDS-PAGE was conducted with the method by Laemmli with the concentrations of the stacking and separating gels being 5% and 12.5%, respectively.38 2-Mercaptoethanol was added into the samples (pH 6.8 supernatant and the fifth supernatant) to the concentration of 2% (v/v) and heated for 3 min in a boiling water bath. Then 10 μL of each sample was loaded into a sample well. SDSPAGE was performed at 15 mA for about 2 h. The gel was stained by using Coomassie Brilliant Blue G-250. Tricine-SDS-PAGE. Tricine-SDS-PAGE was conducted according to the method by Schagger.39 The concentrations of stacking and separating gels were 4% and 16%, respectively. 2-Mercaptoethanol (no 2-mercaptoethanol for nonreducing Tricine-SDS-PAGE) was added into the samples above to the concentration of 2% (v/v), and samples were heated for 3 min in a boiling water bath. Then 20 μL of each sample was loaded into a sample well, and the samples were

electrophoresed at constant voltage of 30 mV until all samples entered into the stacking gel and then at constant voltage of 100 mV until end. The gel was stained using Coomassie Brilliant Blue G-250. Two-Dimensional Electrophoresis. The pH 6.8 and 8.0 extracted OB suspensions obtained without strict temperature control were desalted by dialysis against DI water (4 °C) and lyophilized. The lyophilized OBs were extracted with acetone at 4 °C. After stirred for 30 min, the homogenate was filtered through one layer of filter paper, and the protein fraction was recovered. This was repeated two more times to remove neutral lipids. Then it was further defatted by chloroform/methanol (2/1, v/v) three times. Finally, the protein obtained was placed in the hood to allow the organic solvent evaporated. The two-dimensional electrophoresis was performed according to the Bio-Rad manufacturer’s instructions. One hundred and fifty micrograms of protein obtained above was thoroughly dissolved in 125 μL of 2-D electrophoresis sample preparation solution (7 M urea, 2 M thiourea, 2% (w/v) CHAPS, 75 mM DTT, 0.2% (w/v) ampholyte 3− 10, 0.001% bromophenol blue), assisted by vortexing and ultrasonic treatment. It was loaded onto a linear IPG gel strip (pH 3−10, 7 cm, Bio-Rad, USA) and allowed to hydrate for 16 h at 20 °C. The first dimensional isoelectric focusing (IEF) was conducted at 40 kVh using Protean IEF Cell (Bio-Rad Laboratories). After IEF, the IPG strip was reduced and alkylated with DTT (20 mg/mL) and iodoacetamide (25 mg/mL) in equilibration buffer (6 M urea, 2% (w/v) SDS, 0.375 M Tris-HCl, pH 8.8 and 20% (w/v) glycerol) for 15 min. After rinsed by SDS-PAGE electrode buffer, it was transferred onto the SDS-PAGE separating gel, covered with low melting point agarose and subjected to the second dimensional SDS-PAGE. The gel was stained by Coomassie Brilliant Blue G-250. MALDI-TOF/TOF MS. The protein spots were excised from the Coomassie Brilliant Blue G-250 stained gel, washed in distilled water, and destained completely in a 30% acetonitrile (v/v) containing 100 mM NH4HCO3. After a 100% acetonitrile washing, the destained gel piece was placed into reducing solution (10 μL of 100 mM DTT, 90 μL of 100 mM NH4HCO3) for 30 min at 56 °C. After a 100% acetonitrile washing, 70 μL of 100 mM NH4HCO3 and 30 μL of 200 mM iodoacetic acid were added and incubated for 20 min in dark. After a 100% acetonitrile washing again, 5 μL (10 μg/mL) of trypsin (Promega, V5111) was added and put into 4 °C refrigerator for 30 min. Then 25 mM NH4HCO3 solution was added. The peptides were generated after 20 h incubation at 37 °C. The solution containing peptides was collected and concentrated at 30 °C to get dry powder. A total of 3 μL of 0.1% trifluoroacetic acid (v/v) was used to dissolve dry powder. In the final step before MALDI-TOF/TOF analysis, the sample was prepared by mixing 0.7 μL of the sample and 0.7 μL of 4hydroxy-α-cyanocinnamic acid (matrix) on a MALDI target and airdried. All mass spectra were obtained with an ultrafleXtreme MALDITOF/TOF (Bruker Daltonics, Germany) and were analyzed by flexAnalysis software provided by Bruker Daltonics Corp. Protein identification was performed by searching plant proteins in the latest version of the NCBInr database.

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RESULTS AND DISCUSSION Oleosin Hydrolysis of pH 6.8 Extracted OBs. The pH 6.8 extracted OB suspension (pH 6.5) was incubated in a 35 °C water bath for 0, 0.5, 1, 3, 6, and 9 h. Figure 1 reveals that 24 kDa oleosin shows an apparent molecular weight (MW) of 24 kDa, 18 kDa oleosin shows an apparent MW of 16 kDa, and 16 kDa oleosins (two bands) show apparent MWs of 13−14 kDa on Tricine-SDS-PAGE gel. The bands of 24 and 18 kDa oleosins gradually became weak with increasing incubation time, while the bands of lipoxygenase, β-conglycinin, γconglycinin, β-amylase, glycinin, Bd 30K, and P34 did not change. The band of 16 kDa oleosin 1 gradually became dense with increasing incubation time and merged with the band of 16 kDa oleosin 2. Band 1 already appeared in the freshly extracted OBs, gradually became dense before 3 h, and became 958

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acyltrasferase and phospholipase activities,40 caleosin could act as peroxygenase to produce peroxidized polyunsaturated fatty acids,41 and steroleosin, also named as sterol dehydrogenase, could dehydrogenate phytosterol into its ketone-containing derivative,42 indicating that OB intrinsic proteins had enzymatic activities on different lipid species, but not on protein. Therefore, it was considered that P34 and Bd 30K (or some Tricine-SDS-PAGE undetectable proteins) might be the proteases of 24 and 18 kDa oleosins. Effect of pH 6.8 Supernatant on pH 11.0 Extracted OBs. It was highly possible that the proteases were the OB extrinsic proteins. As stated above, pH 6.8 extracted OBs were obtained as a floating fraction from raw soymilk by centrifugation. It was considered that some of the proteases might still remain in the pH 6.8 supernatant. In addition, it was reported that soybean OBs without extrinsic proteins could be obtained by pH 11.0 extraction. Therefore, an experiment was designed to eliminate the possibility that OB intrinsic proteins, lipoxygenase, β-conglycinin, γ-conglycinin, β-amylase, and glycinin could hydrolyze 24 and 18 kDa oleosins. The protein in 30 mL of pH 6.8 supernatant was absorbed by the pH 11.0 extracted OB suspension (pH 6.5, 4 °C) five times (7 mL each time), and the first and fifth OBs were obtained. Figure 2 shows the protein compositions of the pH 6.8 supernatant (lane 2) and the fifth supernatant (lane 3). For the

Figure 1. 24 and 18 kDa oleosin hydrolysis in the pH 6.8 extracted OB suspension (pH 6.5) incubated at 35 °C. Lane 1, marker; lanes 2−7, OB suspension incubated for 0, 0.5, 1, 3, 6, and 9 h, respectively. α′, α, and β are the subunits of β-conglycinin; A3, A, and A5 are acidic polypeptides of glycinin; and B is the basic polypeptides of glycinin.

weak after 3 h. Bands 2 and 3 weakly appeared after 0.5−1 h and gradually became dense after 1 h. Bands 4−5 already appeared in the freshly extracted OBs (similar to band 1) and did not show a clear trend with increasing incubation time. The total intensities of 24, 16, and 13−14 kDa band and bands 1−5 on lanes 2−7 were analyzed by Image Lab Software (Bio-Rad, USA), and they were almost the same; 13−14 kDa band on lane 7 was matched to 24 kDa oleosins (P29530 and P29531), 18 kDa oleosin (C3VHQ8), and 16 kDa oleosins (C6SZ13 and gi|35657640) by MALDI-TOF/TOF MS. The apparent MWs of bands 1−3 and bands 4−5 were respectively determined as around 9 and 3−4 kDa by Image Lab Software with the standard on lane 1, indicating that bands 1−3 should be the small hydrolyzed products of 24 kDa oleosins (P29530 and P29531), while bands 4−5 might be the small hydrolyzed products of 18 kDa oleosin (C3VHQ8). Different from bands 1−3, bands 4−5 did not show a clear trend with increasing incubation time, and it was considered that this should result from the smaller MWs of bands 4−5, which were highly possible to be removed in the gel treatment (fixing, staining and destaining). These results clearly showed: (1) the proteases, which had high specificities for 24 and 18 kDa oleosins, were existed in the pH 6.8 extracted OBs; (2) the proteases did not hydrolyze the extrinsic proteins and also were unlikely to hydrolyze 16 kDa oleosins 1 and 2; (3) the large hydrolyzed products of 24 and 18 kDa oleosins contributed to the 13−14 kDa band by the incubation of pH 6.8 extracted OB suspension (pH 6.5); (4) bands 1−3 (around 9 kDa) should be the small hydrolyzed products of 24 kDa oleosin, while bands 4−5 might be the small hydrolyzed products of 18 kDa oleosin; (5) the proteases should cleave specific peptide bonds of 24 and 18 kDa oleosins to form limited hydrolyzed products (13−14 kDa band and bands 1−5, Figure 1). The results above showed that the proteases of 24 and 18 kDa oleosins exactly existed of the pH 6.8 extracted OBs, which contained many OB extrinsic proteins as well as OB intrinsic proteins. On one hand, it was very hard to consider that lipoxygenase (enzyme for lipid), β-conglycinin (storage protein), γ-conglycinin (storage protein), β-amylase (enzyme for amylose), and glycinin (storage protein) could hydrolyze oleosins. On the other hand, it was reported that oleosin could act as a bifunctional enzyme that had both monoacylglycerol

Figure 2. SDS-PAGE profiles of the pH 6.8 supernatant and the fifth supernatant. Lane 1, marker; lane 2, the pH 6.8 supernatant; lane 3, the fifth supernatant.

detectable protein bands, it is found that the band indicated by an arrow (lane 2), identified as P34 and Bd 30K by MALDITOF/TOF MS, is almost disappeared for the fifth supernatant (lane 3). For the first OBs, 24 and 18 kDa oleosins are respectively hydrolyzed to about 48% and 64% by 9 h incubation (Figure 3a,c). For the fifth OBs, 24 and 18 kDa oleosins are not hydrolyzed (Figure 3b,d). The detectable protein composition of the fifth OBs (Figure 3b) is almost the same as that of the first OBs (Figure 3a). However, the band indicated by an arrow in Figure 3a is obviously denser than the corresponding one in Figure 3b. The band in Figure 3a is matched to P34, Bd 30K, and caleosin, while the one in Figure 3b is matched to caleosin by MALDI-TOF/TOF MS. These results clearly revealed: (1) the proteases of 24 and 18 kDa oleosins were the OB extrinsic proteins; (2) 24 and 18 kDa oleosins still could be hydrolyzed even after exposed to pH 11.0; (3) the hydrolysis of 24 and 18 kDa oleosins was not happened in the absence of P34 and Bd 30K (or some TricineSDS-PAGE undetectable proteins). These results also made us 959

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Figure 3. (a) Tricine-SDS-PAGE profile of the first OBs incubated at 35 °C. (b) The Tricine-SDS-PAGE profile of the fifth OBs incubated at 35 °C. Lane 1, marker; lanes 2−7, the fifth OBs incubated for 0, 0.5, 1, 3, 6, and 9 h, respectively. (c) The effect of incubation time on the hydrolysis of 24 and 18 kDa oleosins in the first OBs. (d) The effect of incubation time on the hydrolysis of 24 and 18 kDa oleosins in the fifth OBs.

realize that it was the OB extrinsic proteins, not the microbial spoilage, that was responsible for the oleosin hydrolysis. Oleosin Hydrolysis in the pH 6.8, 8.0, 9.5, and 11.0 Extracted OBs. The results above exactly revealed that the proteases were the OB extrinsic proteins. Chen and Ono reported that more and more extrinsic proteins were released from OBs with increasing alkaline pH.34 It was considered that the proteases might also show the similar trend. And it is wellknown that protease must keep its natural conformation to possess its proteolytic activity and must contact its substrate to express its proteolytic activity. Therefore, OBs were obtained by pH 6.8, 8.0, 9.5, and 11.0 extractions, respectively. Lanes 2 in Figure 4a−d show the protein compositions of pH 6.8, 8.0, 9.5, and 11.0 extracted OBs before incubation. Different from the other extrinsic proteins, considerable amounts of Bd 30 K/P34 are remained in the pH 8.0 (lane 2, Figure 4b) and pH 9.5 (lane 2, Figure 4c) extracted OBs. There are two bands near the position of P34 and Bd 30K for the pH 11.0 extracted OBs (lane 2, Figure 4d), and they are matched to caleosins by MALDI-TOF/TOF MS.5 pH 6.8, 8.0, 9.5, and 11.0 extracted OBs were dispersed into DI water, respectively. All of them were adjusted to pH 6.5, and incubated in 35 °C water bath for 0, 0.5, 1, 3, 6, and 9 h (Figure 4a−d). The band intensities of 24 and 18 kDa oleosins were analyzed by Image Lab Software (Bio-Rad, USA). Figure 4e shows that the residual 24 kDa oleosin of pH 6.8 extracted OBs is decreased to 43% by 9 h incubation, followed by pH 8.0 (53%) and 9.5 (74%) extracted OBs, and the 24 kDa oleosin of pH 11.0 extracted OBs is unlikely to be hydrolyzed. Figure 4f shows that the residual 18 kDa oleosin of pH 6.8 extracted OBs is decreased to 45% by 9 h incubation, while the 18 kDa oleosin

of pH 8.0, 9.5, and 11.0 extracted OBs is slightly or unlikely to be hydrolyzed. It was interesting that high proteolytic activity of 24 kDa oleosin-hydrolyzing protease was remained, but very low proteolytic activity of 18 kDa oleosin-hydrolyzing protease was remained in the suspension (pH 6.5) of pH 8.0 extracted OBs. In addition, obvious proteolytic activity of 24 kDa oleosinhydrolyzing protease was still remained in the suspension (pH 6.5) of pH 9.5 extracted OBs. These clearly revealed that the interaction between 24 kDa oleosin and its protease was strong to alkaline pH. In our previous research, it was confirmed that all of P34 and Bd 30K were bound to 24 kDa oleosin by disulfide bonds (SS).5 Together with the P34 and Bd 30K behavior in the pH 6.8, 8.0, 9.5, and 11.0 extracted OBs, it showed: (1) some of the disulfide bonds were weak to alkaline pH, but some of the disulfide bonds were strong to alkaline pH; (2) P34 and Bd 30K had high relationship with the proteases of 24 kDa and 18 kDa oleosins. In addition, it was considered that P34 and Bd 30K were different proteins owing to their four different amino acid residues (NCBI database) and their behaviors on the two-dimensional electrophoresis gel.30,33 Therefore, it was considered that P34 and Bd 30K might be bound to 24 kDa oleosin through disulfide bonds with different bond energies. In order to confirm this hypothesis, twodimensional electrophoresis was conducted for the proteins of pH 6.8 and 8.0 extracted OBs. Figure 5 shows that there are two spots (PX1 and PX2) resolved for the P34 and Bd 30K.30,33 This further made us realize that P34 and Bd 30K should be different proteins. Unluckily, both of PX1 and PX2 were matched to P34 and Bd 30K by MALDI-TOF/TOF MS, which should be resulted from their high homologies with each other. According to their amino acid sequences (NCBI 960

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Figure 4. Tricine-SDS-PAGE profile of pH 6.8 (a), 8.0 (b), 9.5 (c), and 11.0 (d) extracted OB suspensions (pH 6.5) incubated at 35 °C. Lane 1, marker; lanes 2−7, OB suspension incubated for 0, 0.5, 1, 3, 6, and 9 h, respectively. (e) The 24 kDa oleosin hydrolysis in pH 6.8, 8.0, 9.5, and 11.0 extracted OB suspensions (pH 6.5) incubated for 0, 0.5, 1, 3, 6, and 9 h. (f) The 18 kDa oleosin hydrolysis in pH 6.8, 8.0, 9.5, and 11.0 extracted OB suspensions (pH 6.5) incubated for 0, 0.5, 1, 3, 6, and 9 h.

Because both P34 and Bd 30K were interacted with 24 kDa oleosin by disulfide bonds, the behavior of the interaction in the incubation of pH 6.8 extracted OBs could be examined by nonreducing Tricine-SDS-PAGE (Figure 6a) combined with its corresponding reducing Tricine-SDS-PAGE (Figure 6b). The band intensities (24 kDa oleosin; 18 kDa oleosin; 16 kDa oleosin 1 + 16 kDa oleosin 2 + large hydrolyzed products of 24 and 18 kDa oleosins; band 1) were analyzed by Image Lab Software (Bio-Rad, USA). It was found that 24 kDa oleosin

database), Bd 30K was more acidic than P34. Therefore, PX1 and PX2 could be preliminarily matched to P34 and Bd 30K, respectively. It is found that the PX2/PX1 intensity ratio of pH 8.0 extracted OBs is obviously larger than that of pH 6.8 extracted OBs (Figure 5), exactly revealing that PX1 (possibly P34) has weak resistance to alkaline pH, while PX2 (possibly Bd 30K) has strong resistance to alkaline pH. This is greatly consistent with the protease behaviors on the hydrolysis of 24 and 18 kDa oleosins. 961

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Figure 5. Protein profiles of pH 6.8 (a) and 8.0 (b) extracted OBs by two-dimensional electrophoresis. Both of PX1 and PX2 were matched to Bd 30K and P34 by MALDI-TOF/TOF MS.

kDa oleosin. As a result, PX1 was easily removed from OBs, while PX2 was difficult to remove by alkaline pH. According to the UniProtKB database, 24 kDa oleosin (P29530) is divided into three parts: N-terminal (76 AARs), hydrophobic central domain (54 AARs) and C-terminal (96 AARs); 24 kDa oleosin (P29531) is also divided into three parts: N-terminal (57 AARs), hydrophobic central domain (72 AARs), and C-terminal (94 AARs). The hydrophobic central domain is buried in the TAGs matrix of OBs, while N- and Cterminals are exposed to the exterior. Both of P34 and Bd 30K have 257 AARs, so it is highly possible that P34/Bd 30K can interact with 18 kDa oleosin by noncovalent molecular interactions in addition to the disulfide bond with 24 kDa oleosin. Therefore, it was considered that the hypothesis above was reasonable and acceptable. Oleosin in Germinated Soybean. If 24 kDa oleosin was hydrolyzed in soybean germination, it meant that its protease must be bound to OBs for the 24 kDa oleosin hydrolysis. In addition, the results above clearly showed that 24 kDa oleosinhydrolyzing protease was still bound to OBs even at pH 8.0 and 9.5. Therefore, it was considered that the pH 6.8, 8.0, and 9.5 extracted OBs from germinated soybean should contain the 24 kDa oleosin-hydrolyzing protease if the assumption above was correct. Soybean was naturally germinated for 1, 2, 4, 6, and 8 d, and OBs of each germinated soybean were extracted by pH 6.8, 8.0, and 9.5, respectively. Figure 7 shows that almost all of the 24 kDa oleosin is hydrolyzed by 4 d germination. If 24 kDa oleosin-hydrolyzing protease was detectable protein by TricineSDS-PAGE, it should appear not only in Figure 7, but also in Figures 1−6. As a result, P34/Bd 30K should be the most possible protein as the protease of 24 kDa oleosin. Figure 7e shows that four protein bands (6−9) remained for the OBs of 8 d germinated soybean. By MALDI-TOF/TOF MS, band 6 was matched to several lipoxygenase isoforms, band 7 was not matched to any proteins, band 8 was matched to P34, Bd 30K, and caleosin, and band 9 was matched to 24 kDa oleosins (P29530 and P29531). These results revealed (1) 24, 18, and 16 kDa oleosins were all hydrolyzed in soybean germination, showing a different trend from the extracted OBs of ungerminated soybean; (2) Bd 30K and P34 were always bound to the extracted OBs from germinated soybean even when all oleosins were hydrolyzed. The mechanism for the 16

Figure 6. Nonreducing (a) and reducing (b) tricine-SDS-PAGE profiles of pH 6.8 extracted OB suspension (pH 6.5) incubated at 40 °C. Lane 1, marker; lane 2, raw soymilk; lanes 3−8, OB suspension incubated for 0, 0.5, 1, 3, 6, and 9 h, respectively.

bands in the nonreducing condition were weaker than their corresponding ones in the reducing condition, while the other three were almost the same as each other. Figure 6a shows that a thin band is located at the position of P34 and Bd 30K, and it is matched to one subunit of glycinin and caleosin by MALDITOF/TOF MS. These revealed that P34 and Bd 30K were always bound to 24 kDa oleosin by disulfide bonds in the incubation, very similar to the interaction between enzyme and its substrate in the reaction system that enzyme was less than its substrate. In addition, if Bd 30K and P34 were not the proteases, the Bd 30K/P34-SS-polypeptides (13−14 kDa) or the Bd 30K/P34-SS-polypeptides (around 9 kDa) might be produced in the incubation, meaning that the 13−14 kDa band or the band around 9 kDa in the nonreducing condition (Figure 6a) should be less than the corresponding one in the reducing condition (Figure 6b). However, this is unlikely happened in Figure 6. Therefore, one hypothesis was suggested as below: PX1 (the protease of 18 kDa oleosin) was not only bound to 24 kDa oleosin by disulfide bond, but also interacted with 18 kDa oleosin by noncovalent molecular interactions for hydrolyzing 18 kDa oleosin, which might be able to weaken bond energy of the disulfide bond above; PX2 (the protease of 24 kDa oleosin) was just bound to 24 kDa oleosin without interaction with 18 962

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clearly revealing that 35−50 °C is the optimal temperature range for the hydrolysis of 24 and 18 kDa oleosins, while 60 °C can denature the proteases. This showed that the proteases of 24 and 18 kDa oleosins had the similar resistances to temperature. The pH 6.8 extracted OB suspensions were respectively adjusted to pH 4.0, 5.0, 6.0, 6.5, 7.0, 8.0, 9.0, 10.0, and 11.0, and incubated in 35 °C water bath for 0, 0.5, 1, 3, 6, and 9 h. Figure 8c,d show that pH 6.0−6.5 is the optimal pH range for the hydrolysis of 24 and 18 kDa oleosins. The hydrolysis of 24 kDa oleosin was gradually slowed down from pH 6.5 to 10.0 and almost completely inhibit at pH 11.0. Different from 24 kDa oleosin, the hydrolysis of 18 kDa oleosin was greatly inhibited at pH ≥8.0, agreeing with the results in Figure 4. Therefore, it was considered that pH ≥11.0 or ≥60 °C might be better for the extraction of soybean OBs, while the combination of pH 6.0−6.5 and 35−50 °C might be better for the enzyme-assisted aqueous extraction of soybean oil. In all, this study showed that the proteases (OB extrinsic proteins) in the pH 6.8 extracted OBs had high specificity to hydrolyze 24 and 18 kDa oleosins, and they should cleave specific peptide bonds to form limited hydrolyzed products (13−14 kDa band and bands 1−5, Figure 1). In addition, the optimal temperature and pH of the proteases were respectively

Figure 7. Protein profiles of pH 6.8, 8.0, and 9.5 extracted OBs from germinated soybean by Tricine-SDS-PAGE. (a) 1 d germinated soybean. (b) 2 d germinated soybean. (c) 4 d germinated soybean. (d) 6 d germinated soybean. (e) 8 d germinated soybean. Lane 1, marker; lanes 2−4, pH 6.8, 8.0, and 9.5 extracted OBs, respectively.

kDa oleosin hydrolysis needed further researches, which should be closely correlated with the germination. Effects of Temperature and pH on Oleosin Hydrolysis. The pH 6.8 extracted OB suspensions (pH 6.5) were respectively put into 20, 30, 35, 40, 50, and 60 °C water baths and incubated for 0, 0.5, 1, 3, 6, and 9 h. The hydrolysis behaviors of 24 and 18 kDa oleosins (analyzed by Image Lab Software) at different temperatures are shown in Figure 8a,b,

Figure 8. Effects of temperature (a, b) and pH (c, d) on the hydrolysis of 24 (a, c) and 18 kDa (b, d) oleosins in the pH 6.8 extracted OB suspensions. 963

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clarified as in the ranges of 35−50 °C and 6.0−6.5, and 60 °C or pH 11.0 could denature them. Different from the extracted OBs from ungerminated soybean, 16 kDa oleosin was also hydrolyzed in soybean germination in addition to 24 and 18 kDa oleosins. Although this study did not show direct evidence for confirming the proteases, all results in this study showed a high possibility that P34 and Bd 30K were the proteases of 24 and 18 kDa oleosins. For further research, it is considered that the respective purification of the bioactive Bd 30K and P34 (a great challenge at present stage) and their effects on oleosin hydrolysis should be examined. At last, it is suggested that some Tricine-SDS-PAGE undetectable proteins should be also considered in further studies.

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AUTHOR INFORMATION

Corresponding Author

*Phone: 0510-85917812. Fax: 0510-85329091. E-mail: yfhua@ jiangnan.edu.cn. Funding

We gratefully acknowledge the financial support received from Natural Science Foundation of China (31301496), Fundamental Research Fund of Ministry of Education (2050205), Natural Science Foundation of China (31201380), Natural Science Foundation of Jiangsu Province, PR China (BK2011151), and a Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions. Notes

The authors declare no competing financial interest.

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