Evaluation of the Hydrolysis Specificity of an Aminopeptidase from

Dec 5, 2016 - Bacillus licheniformis SWJS33 Using Synthetic Peptides and Soybean ... In the hydrolysis of soy protein isolate, BLAM preferred peptides...
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Evaluation of the Hydrolysis Specificity of an Aminopeptidase from Bacillus licheniformis SWJS33 Using Synthetic Peptides and Soybean Protein Isolate Fenfen Lei,† Qiangzhong Zhao,† Lianzhu Lin,† Baoguo Sun,‡ and Mouming Zhao*,†,# †

School of Food Science and Engineering, South China University of Technology, Guangzhou, China 510640 Beijing Key Laboratory of Flavor Chemistry, Beijing Technology and Business University, Beijing, China 100048 # Advanced Innovation Center for Food Nutrition and Human Health, Beijing Technology and Business University, Beijing, China 100048 ‡

S Supporting Information *

ABSTRACT: The substrate specificity of aminopeptidases has often been determined against aminoacyl-p-nitroanilide; thus, its specificity toward synthetic peptides and complex substrates remained unclear. The hydrolysis specificity of an aminopeptidase from Bacillus licheniformis SWJS33 (BLAM) was evaluated using a series of synthetic peptides and soybean protein isolate. The aminopeptidase showed high specificity for dipeptides with Leu, Val, Ala, Gly, and Phe at the N-terminus, and the specificity was significantly affected by the nature of the penultimate residue. In the hydrolysis of soy protein isolate, BLAM preferred peptides with Leu, Glu, Gly, and Ala at the N-terminus by free amino acid analysis and preferred peptides with Leu, Ala, Ser, Trp, and Tyr at the N-terminus by UPLC-MS/MS. The introduction of complex substrates provides a deeper understanding of the aminopeptidase’s specificity, which can instruct the application of the enzyme in protein hydrolysis. KEYWORDS: aminopeptidase, hydrolysis specificity, synthetic peptides, free amino acid, UPLC-MS/MS

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INTRODUCTION Peptidases account for up to 65% of the global market of enzymes due to their wide application in detergent, leather, pharmaceuticals, and food industries.1 Most of the peptidases studied are endoproteases. In recent decades, exoproteases have attracted much attention due to their different cleavage sites from endoproteases and specific functions.2 Endoproteases often cleave peptide bonds within peptide chains, resulting in peptides of various lengths. Exoproteases attack the peptide chain at the ends (i.e., the N- or C-terminus) through removing a single amino acid (AA) or sometimes a di/tripeptide, which results in the liberation of free amino acids (FAA) and small peptides. Aminopeptidases (EC 3.4.11) are members of the exoprotease family that selectively remove N-terminal AA residues from peptides. The introduction of an aminopeptidase has become an important way to improve the efficiency of protein hydrolysis, especially for plant proteins that could not be completely hydrolyzed by endproteases alone.3−5 Aminopeptidases could debitter protein hydrolysates and increase FAA concentrations because they can remove the hydrophobic AA from the N-terminus of polypeptides that contribute to the bitter taste.6−8 They have also been used in cheese ripening9 and flavor development for different protein substrates.10,11 Aminopeptidases from various sources have been developed and characterized. Most aminopeptidases have specific cleavage cites that often decide their application. A prolyl aminopeptidase (PAP) isolated from the cell extract of Debaryomyces hansenii CECT12487 exclusively hydrolyzed N-terminal-proline-containing substrates.12 Hatanka et al.13 have obtained an X-prolyl dipeptidyl aminopeptidase from thermophilic Streptomyces strain. A novel aminopeptidase from the earthworm © XXXX American Chemical Society

Eisenia fetida has been shown to preferentially hydrolyze the substrate Leu-4-methylcoumaryl-7-amide (Leu-MCA).14 An arginine aminopeptidase from white shrimp muscle has been shown to rapidly hydrolyze Arg-MCA and Lys-MCA.15 Flores et al.16 have purified an methionyl aminopeptidase from porcine skeletal muscle that exhibited maximal activity against Met-, Lys-, Ala-, and Leu-7-amido-4-methyl-coumarin (-AMC), while Pro-AMC was not hydrolyzed. The substrate specificity of an aminopeptidase is a key factor when considering the application in protein hydrolysis because it would directly influence both the hydrolysis efficiency and the nature, composition, and bioactivity of the peptides produced. Most of the studies have evaluated the substrate specificity of aminopeptidases by employing synthetic AA-pNA or AAMCA10−13 as substrates. Some authors have introduced a few synthetic peptides to obtain more information. Bolumar et al.12 found that PAP showed a preference for peptides longer than two residues, and the nature of the AA located at the second position also had an effect on hydrolysis rate. Hatannka et al.17 also pointed out that the substrate specificity of aminopeptidase N from Streptomyces sp. TH-4 toward synthetic peptides was significantly different from its specificity toward AA-pNA derivatives. Arima et al.18 applied AA-pNA and synthetic peptides to evaluate the substrate specificity of an aminopeptidase from Streptomyces griseus and Streptomyces septatus, and found that the hydrolytic activities of bacterial aminoReceived: Revised: Accepted: Published: A

October 4, 2016 December 3, 2016 December 5, 2016 December 5, 2016 DOI: 10.1021/acs.jafc.6b04426 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry

electrophoresis pure BLAM (the enzyme activity units and enzyme grade were defined in Lei et al.5) was added to peptide solutions, and the hydrolysis was conducted in sealed glass conical flask in a thermostatic water bath at 50 °C for 12 h with a stirring speed of 180 rpm. The SPI was prepared from defatted low-heat soybean meal according to the method of Cui et al.19 Ten grams of SPI was mixed thoroughly with 90 g of distilled water. The mixture was adjusted to pH 8.0 with 2 M NaOH and hydrolyzed by Alcalase 2.4L (0.5%, v/w) at 50 °C for 12 h and 24 h, respectively. The SPI hydrolysates catalyzed by Alcalase 2.4L (A-SPHs) were heated in a boiling water bath for 15 min to inactivate Alcalase 2.4 and were further hydrolyzed by 40 U food grade BLAM (the enzyme activity units and enzyme grade were defined in Lei et al.5) from 0 to 12 h with an interval of 3 h. At the end of hydrolysis, the hydrolysates were heated in a boiling water bath for 15 min to inactivate BLAM. The A-SPHs further catalyzed by BLAM were regarded as AB-SPHs. Then, the AB-SPHs were centrifuged in a centrifuge (GL-21M, Xiangyi Instrument Co. Ltd., Changsha, China) at 8000g and 20 °C for 15 min. The supernatants were collected and stored at −20 °C for further analysis. DABS-Cl Derivatization for FAA Analysis of Peptide Hydrolysates by Reversed Phase HPLC. The FAA from single peptide hydrolysates were determined by DABS-Cl derivatization based on Akhlaghi20 with some modifications. Briefly, 200 μL sample and 200 μL derivatization solution (7.5 mM DABS-Cl, dissolved by acetonitrile) were added in a 2 mL vial. The vials were sealed and vortexed thoroughly for 1 min and then heated at 70 °C for 15 min. After placing the vials in the water bath, they were briefly shaken at 5 and 10 min. The vials were then cooled in an ice bath for 2 min, and a 600 μL stop solution (50% ethanol and 50% 50 mM Na2HPO4, pH 7.0) was added to each vial. The mixtures were then centrifuged at 10000g and 4 °C for 10 min in a CR22G high-speed centrifuge (Hitachi Co., Tokyo, Japan), and the supernatant was collected. HPLC analysis was carried out using a Waters 600 HPLC system (Waters, Milford, MA, USA). A Waters (Milford, MA, USA) RP-C18 column (150 × 4.6 mm i.d., 5 μm) was used. Column temperature was kept constant at 25 °C, and the flow rate of 1.0 mL/min was used in the gradient mode for elution. The injection volume was 20 μL. UV detector wavelength was set at 436 nm. Sodium citrate buffer (17 mM, pH 6.4) containing 4% (v/v) DMF was applied as mobile phase A, and acetonitrile was applied as mobile phase B. The mobile phase A was prepared by ultrapure water and filtered through a 0.22-μm filter. The gradient elution program (time/%B) was as follows: 1/25, 13/35, 14/ 42, 18/45, 26/56, 31/56, 35/70, and 42/70. The derivatized samples used for analysis were filtered through a 0.22 μm of nylon membrane. Quantification was accomplished using amino acid standards. FAA release percentage was calculated by the following relationship:

peptidases were affected by the nature of the penultimate residue or flanking moiety and the length of the peptide substrate. From these studies, one could see that the exclusive use of synthetic AA-pNA/AA-MCA to assess the specificity of aminopeptidases is insufficient, as the synthetic AA-pNA/AAMCA fail to address issues of specificity toward natural peptides and the recognition of penultimate residues. In practice, the protein hydrolysis system is very complicated, and the specificity of the evaluated enzyme may not be in accordance with what is found through the analysis of a simple substrate. Therefore, the application of different and complicated substrates is of great importance for a better understanding of the specificity of aminopeptidases. The aminopeptidase from Bacillus licheniformis SWJS33 (BLAM) has been reported to be able to further improve the degree of hydrolysis of soy protein isolate hydrolysates (SPHs) catalyzed by commercial proteases and reduce the bitterness of SPHs.5 The hydrolysis specificity of BLAM was evaluated using a series of synthetic peptides, and it was defined quantitatively by the amount of FAA released during the hydrolysis of a single dipeptide and dipeptide mixtures. The specificity of BLAM toward SPHs was also assessed by FAA analysis and peptide identification using UPLC-MS/MS. The study would provide a deeper understanding of the specificity of BLAM, which can be applied toward the use of BLAM and other aminopeptidases in protein hydrolysis.

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MATERIALS AND METHODS

Raw Materials, Reagents, and Chemicals. B. licheniformis SWJS33 was newly isolated in our laboratory from the deep-sea mud of South China Sea and deposited in the China General Microbiological Culture Collection Center (CGMCC No.7388). The following peptides (>95% purity) were synthesized by GL Biochem Ltd. (Shanghai, China) coupled with HPLC reports and MS spectrum graphs: a series of dipeptides with Leu at the N-terminus (Leu-AA), a series of dipetides with Tyr at the second position (AA-Tyr), Leu-PheAsp (LFD), Leu-Phe-Glu (LFG), Leu-Phe-Pro (LFP), Leu-Phe-Ile (LFI), Leu-Phe-Val (LFV), Leu-Phe-Phe (LFF), and Leu-Phe-Ala (LFA). Alcalase 2.4L was purchased from Novozymes (food grade; Beijing, China). Media components were bought from Guangzhou Huankai Microbial Technology Co., Ltd. (Guangzhou, China). DABSCl (4-N,N-dimethylaminoazobenzene-4′-sulfonyl chloride) and amino acid standards were purchased from Sigma (Beijing, China). The ultrapure water that used to prepare all aqueous solutions was obtained from a Milli-Q water purification system (Millipore). Acetonitrile, ethanol, formic acid, and dimethylformamide (DMF) were HPLCgrade. All of the other chemicals and solvents were of analytical grade. Preparation of BLAM. Bacillus licheniformis SWJS33 was grown in a medium (pH 7.0) consisting of 3.0 glycerin, 5.0 glucose, 10.0 yeast extract, 0.5 KH2PO4, 0.3 MgSO4, 1.0 CaCl2, and 10.0 g L−1 sea salt. The culture was incubated at 37 °C with constant shaking at 150 rpm for 48 h to facilitate fermentation. The fermented mixture was then centrifuged at 10000g at 4 °C for 10 min in a CR22G high-speed centrifuge (Hitachi Co., Tokyo, Japan). The supernatant was collected and was precipitated with ammonium sulfate, and the fraction at 60% concentration was used. The resultant precipitate was collected, dialyzed, lyophilized, and stored in a desiccator over silica gel at 4 °C for further use. BLAM purified at this process was regarded as food grade enzyme. Then, it was further purified to electrophoresis purity using the AKTA pure system (GE Healthcare, USA) fitted with DEAE-Sepharose Fast Flow column (2.6 × 20 cm) and Superdex 200 pg column (1.6 × 60 cm). The purification details were described in our previous study.5 Hydrolysis of Synthetic Peptides and SPI. A single peptide was dissolved in 50 mM Tris-HCl buffer (pH 8.5) at a concentration of 20 mM. Peptide mixtures were also dissolved in 50 mM Tris-HCl buffer (pH 8.5) with each peptide at a concentration of 1 mM. 2 U/mL

FAA release percentage (%) = (C t/ C0) × 100 where Ct denotes the concentration of FAA after different hydrolysis time periods, and C0 denotes the initial concentration of the peptide. FAA Analysis of Hydrolysates of Dipeptides Mixture and AB-SPHs by Amino Acid Analyzer. The FAA composition of hydrolysates was determined using an A300 auto amino acid analyzer (Membra Pure, Bodenheim, Germany). An aliquot (4 mL) of a sample was thoroughly mixed with 1 mL of 15% (w/v) sulfosalicylic acid to precipitate proteins, and the resultant mixture was kept at 4 °C for 1 h and centrifuged at 11,000g, 4 °C for 10 min. The supernatant was diluted with a lithium salt sample dilution buffer, filtered through a piece of filter membrane with 0.22 μM pore size (Millipore Co., Bedford, MA), and directly injected into the analyzer for FAA determination. The FAA content was expressed as μmol/mL hydrolysates. The slope of the linear regression analysis of FAA content to hydrolysis time was used to reflect the FAA release rate. Identification of Peptides in A-SPHs and AB-SPHs by UPLCESI-MS/MS. The identification was performed on an Acquity UPLC system (Waters, Milford, MA, USA) connected to a Bruker micrOTOF QII mass spectrometer (Bruker Daltonics, Bremen, Germany) using an Acquity UPLC peptide BEH BEH300 C18 column (1.7 μm, 2.1 mm × 150 mm). The freeze-dried sample was B

DOI: 10.1021/acs.jafc.6b04426 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Figure 1. Hydrolysis specificity of aminopeptidase from Bacillus licheniformis SWJS33 toward individual Leu-AA (A) and AA-Tyr (B). reconstituted at a concentration of 5 mg/mL in deionized water (containing 0.1% formic acid), then centrifuged at 10000g and 4 °C for 10 min. Two microliters of the resulting supernatant was loaded onto the column before elution at a flow-rate of 0.2 mL/min using a mobile phase consisting of water-formic acid (100:0.1, v/v) (eluent A) and acetonitrile (eluent B) under a gradient regime: 0−5 min, isocratic gradient 5% B; 5−10 min, linear gradient 5−10% B; 10−15 min, linear gradient 10−30% B; 15−20 min, linear gradient 30−80%B; 20−25 min, isocratic gradient 80% B. Spectra were recorded over the mass/ charge (m/z) range 200−2000. The positive electrospray ionization (ESI+) mode via the electrospray interface was operated using the following settings: capillary voltage, 30 V; capillary temperature, 250 °C; spray voltage, 4.5 kV; and tube lens voltage, 10 V. High-purity nitrogen was used as nebulizing and drying gas. Peptide sequencing was performed by processing the auto multiple MS (Auto MS/MS) spectra program. Data were analyzed using Bruker Data Analysis software. Mascot 2.3 (Matrix Science, London, UK) was used for peptide identification, which was searched against the SwissProt database. Additional low molecular mass peptides were processed by manual de novo sequencing. Frequency of AA in the PN Position and Cleavage Frequency Percentage. Schechter and Berger21,22 have introduced the subsite nomenclature model to represent enzyme specificity. As aminopeptidases have been defined as enzymes that cut peptide bonds from the N-terminus (the PN position), the frequency of each AA at the PN position of all the identified peptides was calculated. The AA at the PN position was identified from the MS/MS analysis and the AA sequence from SPI. The cleavage frequency percentage was expressed as the ratio of frequency of the AA in the PN position to total the AA residues in the SPI molecule. Statistical Analysis. All experiments and analytical methods were performed in triplicate. Data were expressed as the mean ± standard

deviation and analyzed by one-way analysis of variances (ANOVA) followed by Duncan’s posthoc test. Statistical significance was considered at p < 0.05, and means presented in tables and figures following different letters are significantly different. Analysis was performed using an SPSS package (SPSS 13.0 for windows, SPSS Inc., Chicago, IL).

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RESULTS AND DISCUSSION Hydrolysis Specificity of BLAM toward Individual LeuAA and AA-Tyr Peptides. The substrate specificity of BLAM toward AA-pNAs had been examined in our previous research, and the results showed that the enzyme preferred Leu-pNA most and that the hydrolysis efficiency of other AA-pNAs was lower than 10% of that of Leu-pNA.5 Considering that the AApNA substrates could not assess the effect of the second AA at the N-terminus, a series of dipeptides with Leu at the Nterminus were introduced as substrates. Figure 1A shows that the nature of the second AA significantly alters the catalytic efficiency of BLAM, and similar results have also been found by Bolumar et al.12 BLAM preferred Leu-Gly over other sequences, and the release rate reached 19.98%. This sequence specificity was followed by Leu-Ala, Leu-Phe, and Leu-Thr (release rate more than 10%). When Pro, Glu, Asp, Asn, and Gln were at the second position, the dipeptides nearly could not be catalyzed by BLAM. No hydrolysis of Leu-Pro, Leu-Glu, and Leu-Asp by an aminopeptidase has also been found by Arima et al.18 When the other AAs (Arg, Trp, Val, Ser, Met, Lys, Cys, Leu, Tyr, His, and Ile) were in the second position, the release rates were lower than 5%. The big differences could C

DOI: 10.1021/acs.jafc.6b04426 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry be due to the structure and constitutive reactive groups of AA at the second position of the N-terminus. Gly and Ala had the simplest R-groups (-H and -CH3, respectively) among the AAs, and the reduced steric hindrance of these smaller side chains may provide an advantage for the attack of the enzyme. The strong preference for Leu-Phe and Leu-Thr might be partly due to the large aromatic rings of the Phe residue and the −OH of Thr residue. The hydrophilic property of some AAs (Glu, Asp, Asn, and Gln) at the second position was adverse for the action of BLAM. The indolic -NH group in Pro restricted its susceptibility to the action of most proteases except for some prolyl aminopeptidases.12 The specificity of BLAM toward dipeptides would be affected by the cooperative effects of the amino acids’ physicochemical properties. For instance, Tyr and Trp both have aromatic rings, but the release rate of Leu-Tyr was much lower than that of Leu-Trp. The concentrations of the two AAs deriving from Leu-AA after being hydrolyzed by BLAM were not always equal, and the content of Leu was higher than that of the other AA for some dipeptides. This phenomenon has not been reported yet as other studies used different methods to assay the activity of aminopeptidase against peptides. Hatanaka et al.13,17 have applied a coupling method using L-amino acid oxidase and peroxidase to investigate the ability of aminopeptidases to hydrolyze synthetic peptides, which was based on the release of total FAA. Bolumar et al.12 introduced a HPLC method to evaluate the degradation of synthetic peptides by quantifying the decrease of substrate peptides. It was a big challenge to explain this phenomenon here. LC-MS analytics is recommended to further demonstrate the hydrolysis procedures catalyzed by BLAM. To evaluate the hydrolysis ability of BLAM toward dipeptides with other AAs at the N-terminus, a series of AATyr were applied to avoid the influence of AA at the second position. As there are too many (20 × 20) combinations to be tested individually, AA-Tyr was selected as representative due to the high antioxidant activity of Tyr (among all AAs) and Tyr containing peptides. Figure 1B shows that BLAM could effectively catalyze Arg-Tyr, Leu-Tyr, His-Tyr, and Asp-Tyr, though the release rates were relatively lower than that of LeuAA (less than 4%). This was different from the specificity of BLAM toward AA-pNA (only catalyze Leu- pNA). AA-pNAs are convenient substrates for assessing the activity of aminopeptidases, but the specificity of BLAM toward them may not reflect an intrinsic preference for peptides. It is necessary to consider the penultimate position in addition to the N-terminus for defining the specificity of aminopeptidases. Specificity of BLAM toward Selected Tripeptides. Since the second AA at the N-terminus would affect the specificity of BLAM, the influence of the third AA at the Nterminus was studied (Figure 2). According to the specificity of BLAM toward dipeptides, seven tripeptides (LFD, LFG, LFP, LFI, LFV, LFF, and LFA) were selected. The rationale for the choice of these tripeptides is due to the results shown in Figure 1A that BLAM had low hydrolysis efficiency toward LD, LG, and LP, moderate hydrolysis efficiency toward LI and LV, and high hydrolysis efficiency toward LF and LA. The dynamic hydrolysis process in the tripeptides was much more complex than that in the dipeptides, as some newly dipeptides produced might be hydrolyzed again. So the final FAAs only showed the relative specificity of BLAM toward the tripeptides. The release rates of Leu in the seven tripeptides were lower than that of LF on the whole. This meant that

Figure 2. Hydrolysis specificity of aminopeptidase from Bacillus licheniformis SWJS33 toward selected tripeptides.

BLAM showed a lower preference for peptides longer than two residues. Asp, Glu, and Pro at the second position of a dipeptide could not be effectively hydrolyzed by BLAM, while their adverse influence disappeared when at the third position of a tripeptide. Phe and Ala at the second position of a dipeptide were highly preferred but when in the third position, the hydrolysis specificity was even lower than that of other tripeptides. The above results suggested that the AA at the third position also affected the substrate specificity of BLAM, but the effects were not the same as that of AA at the second position of the N-terminus. The effect of the AA at the third position might be caused by its steric hindrance and its R-groups that might affect the rupture of the peptide bond. In spite of the release of Leu from the N-terminus, the release percentages of the other two AAs also had obvious differences. The second AA in LFD and the third AA in LFP had rather high release rates, and that of other AAs at the second and third position was very low. All of these could possibly be due to the hydrolysis specificity of BLAM and the competition among substrates. Hydrolysis Specificity of BLAM toward Mixtures of Dipeptides. Synthetic peptides have often been used as substrates solely to evaluate the specificity of aminopeptidase.10,16 The performance of BLAM toward complicated substrates was tested by using Leu-AA mixtures and AAs-Tyr mixtures as substrates. After being hydrolyzed by BLAM, the concentration of FAA was measured (Table 1). The total FAA from Leu-AA hydrolysates was 4 times greater than that from AA-Tyr hydrolysates. This result was consistent with the former conclusion that BLAM had a high specificity toward peptides with Leu at the N-terminus. The sum of the concentration of AA at the second position or the N-terminus was approximately equal to that of Leu or Tyr, respectively. For Leu-AA mixtures, BLAM had better specificity for Leu-Val, Leu-Ala, Leu-Gly, and Leu-Phe, whereas Leu-Pro, Leu-Met and Leu-Ser were poorly preferred. These results were not the same as the specificity obtained from a single dipeptide, which might be caused by the competition among substrates. When used to catalyze AA-Tyr mixtures, BLAM had better specificity for Glu-Tyr, Arg-Tyr, His-Tyr, and Leu-Tyr. This was in accordance with the results obtained from the single dipeptide hydrolysis experiments, with the exception of Glu-Tyr. In general, the substrate specificity of BLAM toward the dipeptide mixtures had some similarity to its specificity for individual dipeptides; the differences happened most often when acidic residues (Glu and Asp) were at the Nterminus or the second position. Hydrolysis Specificity of BLAM in SPI Hydrolysis by FAA Analysis. BLAM (in combination with endoprotease) has D

DOI: 10.1021/acs.jafc.6b04426 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry Table 1. Hydrolysis Specificity of an Aminopeptidase from Bacillus licheniformis SWJS33 toward Mixtures of Synthetic Dipeptides Leu-AA mixtures

a b

AA-Tyr mixtures

amino acid

concentration (μmol/mL)

amino acid

Leu Val Ala Gly Phe Tyr Lys Glu Asp Arg Trp Ile Cys Thr His Pro Met Ser Gln Asn total

11.38 ± 0.52 1.28 ± 0.06 ga 1.22 ± 0.05 g 1.19 ± 0.06 g 1.07 ± 0.10 f 0.96 ± 0.03 e 0.90 ± 0.05 de 0.85 ± 0.06 d 0.85 ± 0.04 d 0.83 ± 0.03 d 0.81 ± 0.05 d 0.79 ± 0.04 d 0.62 ± 0.03 c 0.56 ± 0.05 c 0.53 ± 0.03 c 0.38 ± 0.02 b 0.23 ± 0.03 a 0.22 ± 0.02 a -b 24.65 ± 0.58

Tyr Glu Arg His Leu Lys Asp Pro Met Trp Cys Gly Thr Ser Phe Ile Ala Val Gln Asn total

concentration (μmol/mL) 2.84 0.70 0.26 0.25 0.24 0.22 0.20 0.19 0.15 0.12 0.11 0.11 0.11 0.10 0.09 0.08 0.08 0.06 5.92

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.17 0.08 0.01 0.04 0.02 0.01 0.03 0.01 0.04 0.02 0.01 0.02 0.01 0.01 0.01 0.01 0.01 0.01

j i i hi h g g f e de de cde cde bcd abc ab a

Figure 3. Free amino acid content (μmol/mL hydrolysates) and release rate (data above the bar) of each amino acid in the hydrolysates using different substrates: (A) SPI hydrolyzed by Alcalase 2.4L for 12 h; (B) SPI hydrolyzed by Alcalase 2.4L for 24 h.

± 0.33

Data with different letters indicate significant differences (p < 0.05). -: the concentration is lower than the detection limit.

that in A-SPHs-12h. These results revealed that BLAM had a high specificity for peptides with Leu, Glu, Gly, and Ala at the N-terminus. Hydrolysis specificity of BLAM in SPI hydrolysis by identifying peptides from AB-SPHs and A-SPHs by UPLC-MS/MS. The application of LC−MS/MS to study the hydrolytic specificity of an enzyme by identifying peptides from the hydrolysates has been reported by several researchers,21,25,26 but its use in predicting the specificity of an aminopeptidase has not yet been attempted. Peptides in the substrate (A-SPHs) and the hydrolysates after BLAM hydrolysis (AB-SPHs) were identified by UPLC-MS/MS. Following the database search, peptides from AB-SPHs and A-SPHs were mainly derived from the primary sequence of βconglycinin (α-chain) and glycinin G1. A total of 55 and 62 larger peptides (more than 6 amino acids) were derived from these two subunits, respectively. Other subunits with few matched peptides were not considered here because different subunits of SPI have many similar sequences, and these made it hard to estimate from where the small peptides were derived. Additional small peptides (no more than 6 amino acids) were also found in the hydrolysates by manual analysis of the mass spectra, and 192 and 167 sequences were manually identified from AB-SPHs and A-SPHs, respectively. 157 peptides were found both in AB-SPHs and A-SPHs (Table S1), and 86 and 69 peptides existed only in AB-SPHs and A-SPHs, respectively. Not all the peptides identified matched with the sequence of β-conglycinin and glycinin G1; some were partially matched the sequences of these two subunits, while some were totally different. Some of the unmatched sequences might come from the other subunits of SPI as we just chose the two subunits with the highest scores. Others could be due to the generation of new peptides as large

great potential to be used in food protein hydrolysis, and the specificity of BLAM in a protein-hydrolysis system was evaluated. As an exoprotease, BLAM effectively removes AA from the N-terminus, and the release of FAA could be used to evaluate the specificity of the enzyme. Since the hydrolysis activity of BLAM toward macromolecular proteins and polypeptides was not very effective, A-SPHs with different hydrolysis times (12 h and 24 h) were introduced as the substrates of BLAM. Alcalase 2.4L was selected due to its wide application in SPI hydrolysis and higher hydrolysis efficiency than pepsin, papain, and chymotrypsin.23 The FAA release rates of AB-SPHs are shown in Figure 3, and Asn and Gln were not included as their contents were lower than the detection limit. A good linear relationship was found between the concentration of FAA and the hydrolysis time (R2 > 0.9). For both substrates, BLAM had high preference for peptides with Leu, Glu, Gly, and Ala at the N-terminus. This was similar to the results obtained from Leu-AA mixtures. In addition, the especially high release rate of Leu further confirmed that BLAM belongs to the leucine aminopeptidase. When A-SPHs24h was used as a substrate (Figure 3B), most AAs had higher release rates than that of the others, except for Ile, Leu, Tyr, Phe, and Arg (Figure 3A). This could be owing to the fact that A-SPHs-24h had a higher hydrolysis degree and more small peptides than A-SPHs-12h, which might be more easily cleaved by aminopeptidase. The cleavage sites for Alcalase 2.4L were reported to be Ala-, Leu-, Val-, Tyr-, Phe-, and Trp-,24 which led to a greater exposure of Ile, Leu, Tyr, Phe, and Arg residuals in A-SPHs-12h, so high release rates of these residues were observed when BLAM was applied. In A-SPHs-24h, some Ile, Leu, Tyr, Phe, and Arg residues had been further hydrolyzed and released as FAA, which resulted in lower release rates than E

DOI: 10.1021/acs.jafc.6b04426 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry

present. Therefore, various assays were recommended when studying the specificity of an enzyme in a complicated system. In conclusion, BLAM preferred peptides with Leu, Val, Ala, Gly, and Phe at the N-terminus when dipeptide mixtures were used as substrates. When A-SPHs were used as a substrate, BLAM preferred peptides with Leu, Glu, Gly, and Ala at the Nterminus by FAA analysis and preferred peptides with Leu, Ala, Ser, Trp, and Tyr at the N-terminus by UPLC-MS/MS. These results show that BLAM has broad substrate specificity. The specificity of other reported leucine aminopeptidases had some similarity to what we observed with BLAM, regardless of the different evaluation methods. Leucine aminopeptidase from the skeletal muscle of the common carp (Cyprinus carpio) preferred Leu, Arg, Ala, and Tyr residuals at the N-terminus.33 Recombinant leucine aminopeptidase II from Bacillus stearothermophilus (rLAP II) was active against Leu-p-NA, Met-pNA, and Gly-p-NA.34 An aminopeptidase from Bacillus subtilis Zj016 was most active toward p-nitroaniline derivatives of Leu, Arg, and Lys.35 Leucine aminopeptidase from red sea bream (Pagrus major) skeletal muscle rapidly hydrolyzed Leu-MCA, Arg-MCA, Ala-MCA, and Tyr-MCA.2 This study evaluated the hydrolysis specificity of aminopeptidases toward individual dipeptides, dipeptides mixtures, and protein hydrolysates for the first time, and achieved much more useful information compared with that obtained against AA-pNA. The study confirmed that the influence of the second AA at the N-terminus could not be ignored in the evaluation of the specificity of aminopeptidases, and the specificity of BLAM toward complex substrates was not totally in accordance with that toward a single substrate. BLAM has been proved to be able to improve hydrolysis efficiency of SPI and reduce the bitterness of hydrolysates, and it still has great potential to be used in other food protein hydrolysis for raising protein recovery, improving hydrolysis degree, changing functional characteristics, and producing bioactive peptides. The hydrolysis specificity against A-SPHs could provide a reference for choosing a protein substrate and predicting the properties of the hydrolysates.

FAA and small peptides existing within the system might promote the synthesis of peptide bonds. Proteases have been shown to mediate the formation of peptide bonds between αamino acid monomers both in organic and aqueous conditions.27,28 Aminopeptidases have also been exploited for peptide synthesis under appropriate conditions, and high conversion ratios were achieved.29,30 The peptides synthesized in the hydrolysis system might be rather few but still could be detected by UPLC-MS/MS. As this study is not able to evaluate the concentration of every peptide, the influence of BLAM on the content of peptides needs further exploration. Some peptides existed exclusively in AB-SPHs or A-SPHs (Table S2). This showed that the application of BLAM can further catalyze the polypeptide chains to be small peptides, as more small peptides than longer peptides were identified from AB-SPHs than from A-SPHs. Figure 4 presents the distribution

Figure 4. Distribution of amino acid in the PN position for the peptides identified in the soy protein isolate hydrolysates catalyzed by Alcalase 2.4L and aminopeptidase from Bacillus licheniformis SWJS33 (AB-SPHs). The cleavage frequency percentage (%) for each amino acid according to their respective proportion in the β-conglycinin and glycinin G1 is presented above the bar.

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of each AA in the PN position for the 143 peptides derived from AB-SPHs. Accounting for the number of AA residues in a particular protein was very important in describing the specificity of an enzyme.22 The cleavage frequency percentages for all of the AAs in the PN position, according to their respective proportion in the β-conglycinin and glycinin G1 molecules, were also calculated and indicated above each bar. Leu appeared most frequently at the PN position of the peptides (48) and had the highest frequency (52.7%). This was in accordance with prior results from the synthetic peptide hydrolysis and FAA analysis. Ala (49.1%) and Ser residues (32.9%) also had high cleavage frequency at the PN position, followed by Typ and Tyr residues. Asn, Glu, and Gln residues at the PN position had high appearance, but their cleavage frequency percentages were not as high as that of Leu, Ala, and Ser. Breddam and Svendsen have pointed out that nonspecific behavior with respect to the amino acid residue of the enzyme would be exhibited during prolonged hydrolysis and high enzyme concentration.31,32 These results had some similarity to the results obtained from FAA release (Leu, Glu, Gly, and Ala), but some differences were observed. It might be due to the fact that FAA analysis and UPLC-MS/MS analytics evaluated the hydrolysis specificity from different aspects. The protein hydrolysis system was rather complicated, and no method have been able indicate its dynamic changes comprehensively at

ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jafc.6b04426. Peptides identified both in A-SPHs and AB-SPHs by UPLC-MS/MS and peptides identified only in A-SPHs and AB-SPHs by UPLC-MS/MS (PDF)

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AUTHOR INFORMATION

Corresponding Author

*Tel/Fax: +86 20 87113914. E-mail: [email protected]. ORCID

Mouming Zhao: 0000-0003-0221-3838 Funding

This work was kindly supported by Guangdong Marine Economic Development and Innovation of Regional Demonstration Project (GD2012-D01-002) and the Public Science and Technology Research Funds Projects of Ocean (No. 201305018-7). Notes

The authors declare no competing financial interest. F

DOI: 10.1021/acs.jafc.6b04426 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry

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(20) Akhlaghi, Y.; Ghaffari, S.; Attar, H.; Hoor, A. A. A rapid hydrolysis method and DABS-Cl derivatization for complete amino acid analysis of octreotide acetate by reversed phase HPLC. Amino Acids 2015, 47, 2255−2263. (21) Schechter, I.; Berger, A. On the size of the active site in proteases.I. Papain. Biochem. Biophys. Res. Commun. 1967, 27, 157− 162. (22) Doucet, D.; Otter, D. E.; Gauthier, S. F.; Foegeding, E. A. Enzyme-induced gelation of extensively hydrolyzed whey proteins by Alcalase: peptide identification and determination of enzyme specificity. J. Agric. Food Chem. 2003, 51, 6300−6308. (23) Sukan, G.; Andrews, A. T. Application of the plastein reaction to caseins and to skim milk powder. I. Protein hydrolysis and plastein formation. J. Dairy Res. 1982, 49, 265−278. (24) Adler-Nissen, J. A Review of Food Protein Hydrolysis. In Enzymic Hydrolysis of Food Proteins; Elsevier: New York, 1986; pp 435−438. (25) Norris, R.; Poyarkov, A.; O’Keeffe, M. B.; FitzGerald, R. J. Characterisation of the hydrolytic specificity of Aspergillus niger derived prolyl endoproteinase on bovine β-casein and determination of ACE inhibitory activity. Food Chem. 2014, 156, 29−36. (26) Kalyankar, P.; Zhu, Y.; O’Keeffe, M.; O’Cuinn, G.; FitzGerald, R. J. Substrate specificity of glutamyl endopeptidase (GE): hydrolysis studies with a bovine α-casein preparation. Food Chem. 2013, 136, 501−512. (27) Yazawa, K.; Numata, K. Recent advances in chemoenzymatic peptide syntheses. Molecules 2014, 19, 13755−13774. (28) Baker, P. J.; Numata, K. Chemoenzymatic synthesis of poly (Lalanine) in aqueous environment. Biomacromolecules 2012, 13, 947− 951. (29) Arima, J.; Morimoto, M.; Usuki, H.; Mori, N.; Hatanaka, T. The aminolysis reaction of Streptomyces S9 aminopeptidase promotes the synthesis of diverse prolyl dipeptides. Appl. Environ. Microb. 2010, 76, 4109−4112. (30) Arima, J.; Uesugi, Y.; Uraji, M.; Iwabuchi, M.; Hatanaka, T. Dipeptide synthesis by an aminopeptidase from Streptomyces septatus TH-2 and its application to synthesis of biologically active peptides. Appl. Environ. Microb. 2006, 72, 4225−4231. (31) Breddam, K.; Meldal, M. Substrate preferences of glutamic-acidspecific endopeptidases assessed by synthetic peptide substrates based on intramolecular fluorescence quenching. Eur. J. Biochem. 1992, 206, 103−107. (32) Svendsen, I.; Breddam, K. Isolation and amino acid sequence of a glutamic acid specific endopeptidase from Bacillus licheniformis. Eur. J. Biochem. 1992, 204, 165−171. (33) Liu, B. X.; Du, X. L.; Zhou, L. G.; Hara, K.; Su, W. J.; Cao, M. J. Purification and characterization of a leucine aminopeptidase from the skeletal muscle of common carp (Cyprinus carpio). Food Chem. 2008, 108, 140−147. (34) Wang, F.; Ning, Z.; Lan, D.; Liu, Y.; Yang, B.; Wang, Y. Biochemical properties of recombinant leucine aminopeptidase II from Bacillus stearothermophilus and potential applications in the hydrolysis of Chinese anchovy (Engraulis japonicus) proteins. J. Agric. Food Chem. 2012, 60, 165−172. (35) Gao, X.; Cui, W.; Tian, Y.; Zhou, Z. Over-expression, secretion, biochemical characterisation, and structure analysis of Bacillus subtilis aminopeptidase. J. Sci. Food Agric. 2013, 93, 2810−2815.

REFERENCES

(1) Gupta, R.; Beg, Q.; Lorenz, P. Bacterial alkaline proteases: molecular approaches and industrial applications. Appl. Microbiol. Biotechnol. 2002, 59, 15−32. (2) Wu, G. P.; Cao, M. J.; Chen, Y.; Liu, B. X.; Su, W. J. Leucine aminopeptidase from red sea bream (Pagrus major) skeletal muscle: purification, characterization, cellular location, and tissue distribution. J. Agric. Food Chem. 2008, 56, 9653−9660. (3) Lee, J. Y.; Lee, H. D.; Lee, C. H. Characterization of hydrolysates produced by mild-acid treatment and enzymatic hydrolysis of defatted soybean flour. Food Res. Int. 2001, 34, 217−222. (4) Giesler, L.; Linke, D.; Rabe, S.; Appel, D.; Berger, R. G. Hydrolysis of wheat gluten by combining peptidases of Flammulina velutipes and electrodialysis. J. Agric. Food Chem. 2013, 61, 8641−8649. (5) Lei, F.; Zhao, Q.; Sun-Waterhouse, D.; Zhao, M. Characterization of a salt-tolerant aminopeptidase from marine Bacillus licheniformis SWJS33 that improves hydrolysis and debittering efficiency for soy protein isolate. Food Chem. 2017, 214, 347−353. (6) Huang, W. Q.; Zhong, L. F.; Meng, Z. Z.; You, Z. J.; Li, J. Z.; Luo, X. C. The structure and enzyme characteristics of a recombinant leucine aminopeptidase rLap1 from Aspergillus sojae and its application in debittering. Appl. Biochem. Biotechnol. 2015, 177, 190−206. (7) Stressler, T.; Eisele, T.; Schlayer, M.; Lutz-Wahl, S.; Fischer, L. Characterization of the recombinant exopeptidases PepX and PepN from Lactobacillus helveticus ATCC 12046 important for food protein hydrolysis. PLoS One 2013, 8, e70055. (8) Selamassakul, O.; Laohakunjit, N.; Kerdchoechuen, O.; Ratanakhanokchai, K. A novel multi-biofunctional protein from brown rice hydrolysed by endo/endo-exoproteases. Food Funct. 2016, 7, 2635−2644. (9) Wilkinson, M. G.; Guinee, T. P.; O’Callaghan, D. M.; Fox, P. F. Autolysis and proteolysis in different strains of starter bacteria during Cheddar cheese ripening. J. Dairy Res. 1994, 61, 249−262. (10) Li, S. W.; Lin, L. P.; Chen, S. H.; Fu, M. Y.; Wu, G. P. Purification and biochemical characterization of a puromycin-sensitive aminopeptidase from black carp muscle. Process Biochem. 2015, 50, 1061−1067. (11) Lin, S. J.; Chen, Y. H.; Chen, L. L.; Feng, H. H.; Chen, C. C.; Chu, W. S. Large-scale production and application of leucine aminopeptidase produced by Aspergillus oryzae LL1 for hydrolysis of chicken breast meat. Eur. Food Res. Technol. 2008, 227, 159−165. (12) Bolumar, T.; Sanz, Y.; Aristoy, M. C.; Toldrá, F. Purification and characterization of a prolyl aminopeptidase from Debaryomyces hansenii. Appl. Environ. Microbiol. 2003, 69, 227−232. (13) Hatanaka, T.; Yamasato, A.; Arima, J.; Usuki, H.; Yamamoto, Y.; Kumagai, Y. Extracellular production and characterization of Streptomyces X-prolyl dipeptidyl aminopeptidase. Appl. Biochem. Biotechnol. 2011, 164, 475−486. (14) Chen, S. H.; Cao, M. J.; Su, W. J.; Wu, G. P. Purification and characterization of a novel leucine aminopeptidase from the earthworm Eisenia foetida. Process Biochem. 2011, 46, 1641−1648. (15) Zhang, L.; Cai, Q. F.; Wu, G. P.; Shen, J. D.; Liu, G. M.; Su, W. J.; Cao, M. J. Arginine aminopeptidase from white shrimp (Litopenaeus vannamei) muscle: purification and characterization. Eur. Food Res. Technol. 2013, 236, 759−769. (16) Flores, M.; Marina, M.; Toldrá, F. Purification and characterization of a soluble methionyl aminopeptidase from porcine skeletal muscle. Meat Sci. 2000, 56, 247−254. (17) Hatanaka, T.; Arima, J.; Uraji, M.; Uesugi, Y.; Iwabuchi, M. Characterization, cloning, sequencing, and expression of an aminopeptidase N from Streptomyces sp. TH-4. Appl. Microbiol. Biotechnol. 2007, 74, 347−356. (18) Arima, J.; Uesugi, Y.; Iwabuchi, M.; Hatanaka, T. Study on peptide hydrolysis by aminopeptidases from Streptomyces griseus, Streptomyces septatus and Aeromonas proteolytica. Appl. Microbiol. Biotechnol. 2006, 70, 541−547. (19) Cui, C.; Zhao, M.; Yuan, B.; Zhang, Y.; Ren, J. Effect of pH and pepsin limited hydrolysis on the structure and functional properties of soybean protein hydrolysates. J. Food Sci. 2013, 78, 1871−1877. G

DOI: 10.1021/acs.jafc.6b04426 J. Agric. Food Chem. XXXX, XXX, XXX−XXX