Characterization of protease activities in a crude extract of germinated cacao

Abstract

Dynamic light scattering (DLS) was applied to the analysis of the hydrodynamic diameter distribution of proteins in extract of germinated cacao. The interactions among divalent cations and their relation with the activity of Xaa-prolyl dipeptidyl aminopeptidase 2 (Xaa-Pro-DAP2) enzyme were evaluated. The peptidases were isolated by 80% saturation of ammonium sulfate from acetone extract of germinated cacao seeds. The results showed a higher activity of Xaa-Pro-DAP2, besides aminopeptidase (APE) and carboxypeptidase (CP) enzymes. Using DLS analysis the size distribution was found to be multi-modal; however with Cu²⁺ and Cd²⁺ at 1 mM, the size distribution was found to be monomodal with a hydrodynamic diameter of 158.5 and 119 nm, respectively. The distribution remained constant from 60 to 80°C, suggesting that thermal stability is related to increase in Xaa-Pro-DAP2 activity an lower protein aggregation. APE activity was slightly activated by Co²⁺ at 1 mM, whereas no significant effect was observed on CP activity.

La dispersión dinámica de luz (DLS) fue aplicada para el análisis de la distribución de diámetros hidrodinámicos de proteínas en un extracto germinado de cacao. Se evaluaron las interacciones con cationes divalentes y su relación con la actividad de la enzima Xaa-prolyl dipeptidyl aminopeptidasa 2 (Xaa-Pro-DAP2). Las peptidasas fueron aisladas de los extractos cetónicos de semillas de cacao germinadas, por precipitación con sulfato de amonio al 80% de saturación. Los resultados mostraron una alta actividad de Xaa-Pro-DAP2, además de las enzimas de aminopeptidasa (APE) y carboxipeptidasa (CP). De acuerdo con el análisis DLS, la distribución de tamaños es multi-modal con diámetros hidrodinámicos de 158.5 y 119 nm, respectivamente. La distribución permaneció constante desde 60°C hasta 80°C, sugiriendo estabilidad térmica relacionada con el incremento de la actividad de Xaa-Pro-DAP2 y la baja agregación de proteínas. La actividad de APE fue ligeramente activada por Co²⁺ a 1 mM, mientras que no se observó efecto importante sobre la actividad de CP.

Keywords:

• cacao
• crude extract
• proteases
• dynamic light scattering

Palabras clave:

• cacao
• extracto crudo
• proteasas
• dispersión dinámica de luz

1. Introduction

Theobroma cacao L. has been described as a plant whose seed contains vicilin-type globulin, but not legumin, as a vacuolar storage protein (Voigt, Biehl & Wazir, 1993). Proteolysis is initiated by an endopeptidase with high specificity for seed reserve proteins, which are degraded partially as a consequence of the rupture of specific peptide bonds. The resulting polypeptide chains would are then susceptible to the action of previously inactive endopeptidases and, eventually, to that of carboxypeptidases (CP) (Abecia-Soria, Pezoa-García & Amaya-Farfan, 2005; Dunaevsky, Sarbakanova & Belozersky, 1989). Among cacao proteins, proline (0.72–1.97 g/100 g of cacao) predominates, and because of its specific conformation it poses many restrictions on the structural aspects of peptides and proteins, and grants particular biological properties to a large range of physiologically important biomolecules (Kalvatchev, Garzaro & Guerra, 1998; Kratzer et al., 2009). In contrast to the plethora of reports regarding Xaa-prolyl dipeptidyl aminopeptidase (Xaa-Pro-DAP2, EC 3.4.14.5) in lactic acid bacteria, there are few reports on this enzyme in plants (Besanova, Kovacs, Psenak & Barth, 1987; Davy et al., 2000; . Stano et al., 1997, Stano et al., 1994a; Stano, Kovács, Nemec & Neubert, 1994b), and given the above-mentioned proline content in cacao seeds, the study of this enzyme is relevant.

Many enzymes incorporate divalent cations (Mg²⁺, Ca²⁺, Zn²⁺, Ba²⁺, Cd²⁺) within their structure to stabilize the folded conformation of the protein or to facilitate possible direct participation in chemical reactions catalysed by these enzymes. According to Atanasov et al. (2013), the presence of metal ions leads to conformational changes in enzymes and the release of hydrolysis products, using natural or artificial substrates. Chan, Ho, Law and Yuen (2002) found that higher enzyme activity in the presence of metal ions in low-substrate concentrations led to a more stable enzyme–substrate complex and higher release rate of phosphate from the phosphor-enzyme complex. Metals commonly bind with the protein portion of the enzyme by the formation of coordinate bonds with certain amino acid chains. Histidine residues are always found in association with transition metal-binding sites on proteins and are normally associated also with divalent metal ion binding (Copeland, 2000).

Dynamic light scattering (DLS) can be used to study various applications in the protein field (Kaszuba, Connah, McNeil-Watson & Nobbmann, 2007). As such, DLS is very sensitive to the onset of protein aggregation arising from subtle changes in solution conditions. When a protein denatures, the hydrophobic residues buried within the interior of the folded structure are exposed to the solvent. This entropically unfavourable state is soon replaced, however, by one wherein the hydrophobic residues on one protein associate with those on another protein chain. This non-specific aggregation of denatured proteins is easily monitored with the Zetasizer Nano because of the molar mass dependence of the scattering intensity. Because of its high sensitivity to particles of high molecular weight, DLS is also a useful tool for monitoring the effects of salts on protein aggregation.

The aim of this study was to evaluate the effect of different metal ions on the distribution of hydrodynamic diameters of peptidases under different temperatures in a crude extract of cacao using the technique of DLS, and their relationship with exopeptidase activity, thereby contributing to characterization of the proteolytic system of T. cacao L.

2. Materials and methods

2.1. Seed material and growth conditions

We used cacao (Theobroma cacao L.) seeds of the white almond ‘criollo’ genotype, cultivated in the municipality of Cunduacan, Tabasco State, Mexico. Seeds were germinated for 10 days, during which the highest levels of Xaa-Pro-DAP activity were obtained (Sánchez-Mundo, Bautista-Muñoz & Jaramillo-Flores, 2010) before enzymatic extraction. Mucilage was removed from seeds and these were placed to germinate in wet agrolite at 30°C. For the analysis and preparation, only cotyledons were used.

2.2. Acetone dry powder

Dry cacao powder (DCP) was obtained according to the method described by Hansen, Del Olmo and Burri (1998). Fat was removed from the powder by the addition of 10 ml/g 100% hexane at 4°C and stirring constantly for 2 h. Finally, the solvent was eliminated by vacuum rotary evaporation from the sample and was dried at 30°C. To remove polyphenols, the defatted seed powder was extracted five times with 80% (v/v) aqueous acetone and then three times with 100% acetone. The suspensions were centrifuged at 4°C for 5 min at 5000 g and 20,000 g for the final three extractions. Finally, the solvent was evaporated from the resulting paste at room temperature and ground until a homogeneous appearance was achieved. The dry powder, which was yellowish-white, was stored at −20°C and served as an enzyme source for the determination of enzyme activities.

2.3. Enzyme extract

The enzyme extract was obtained from 15 mg of DCP and 30 mg of polyvinylpolypyrrolidone (PVPP) in 900 µl of 0.1 M phosphate buffer (pH 7.0) containing 1% Triton X-100. The mixture was stirred for 30 min at 37°C and then centrifuged at 20,000 g for 10 min at 4°C.

2.4. Ammonium sulfate fractionation

The supernatant obtained (in 2.3) was precipitated at 40% saturation of ammonium sulfate. The precipitate was separated by centrifugation at 17,000 g for 20 min at 4°C, the supernatant obtained was subjected again to precipitation at 80% saturation of ammonium and the pellet obtained was dissolved in 20 mM sodium phosphate buffer (pH 7.0) and dialysed for 24 h at 4°C with the same buffer, and then concentrated by ultrafiltration with a 10 kDa molecular weight cut-off membrane (Amicon® Millipore, Inc., Beverly, MA). The protein concentration and peptidase activity were then determined.

2.5. Protein determination

The protein concentration was determined by the method of Folin–Lowry as modified by Markwell, Haas, Bieber and Tolbert (1978), using bovine serum albumin as standard.

2.6. Peptidase activity measurement

Xaa-Pro-DAP2, aminopeptidase (APE) and carboxypeptidase (CP) activity was assayed by measuring the hydrolysis of p-nitroanilide substrates (Bachem, Bubendorf, Switzerland and Sigma-Aldrich, St Louis, MO). Thus, arginine-proline-p-nitroanilide (Arg-Pro-pNA), glycine-proline-p-nitroanilide (Gly-Pro-pNA), alanine-alanine-proline-p-nitroanilide (Ala-Ala-Pro-pNA) and alanine-p-nitroanilide (Ala-pNA) at concentrations of 1 and 10 mM were used to analyse Xaa-Pro-DAP2 activity. APE activity was tested with Ala-pNA, lysine-p-nitroanilide (Lys-pNA) and leucine-p-nitroanilide (Leu-pNA), CP-type activity was tested with benzoyl-tirosine-p-nitroanilide (Bz-Tyr-pNA). The incubation mixture consisted of 30 µl substrate (1 mM and 10 mM), 250 µl of 0.1 M sodium phosphate (pH 7.0) containing 1% Triton X-100, 120 µl distilled water and 100 µl of enzyme extract. After incubation at 37°C for 30 min, the reaction was stopped by the addition of 400 µl ZnSO₄ (5%) and 100 µl Ba (OH)₂ (7.5%). The mixture was centrifuged (15,000 g for 10 min), and absorbance of the released p-nitroaniline in the clear supernatant was determined at 405 nm. One unit of enzyme (U) was defined as the amount of enzyme producing 1 µmol of p-nitroaniline per minute at 37°C under assay conditions.

2.7. Effect of inhibitors and metal ions on protease activity

The effects of enzyme inhibitors on protease activity were studied using bestatine, pefabloc, leupeptin, pepstatin, diprotine, 4-(2-aminoethyl) benzenesulfonyl fluoride hydrochloride (AEBSF), phenylmethylsulfonyl fluoride (PMSF), ethylenediaminetetraacetic acid (EDTA), p-chloromercuribenzoate (PCMB), dithiothreitol (DTT), β-mercaptoethanol and cysteine. The enzyme solution was incubated with inhibitors for 30 min at 37°C, and then the enzyme assay was applied using Ala-Pro-pNA as substrate. The reaction was stopped by the addition of 400 µl ZnSO₄ (5%) and 100 µl Ba (OH)₂ (7.5%). The mixture was centrifuged (15,000 g for 10 min), and absorbance of the released p-nitroaniline in the clear supernatant was determined at 405 nm. Activity was expressed as a percentage of the activity obtained in the absence of the added inhibitor.

The effect of various metal ions (0.1 mM and 1.0 mM) on Xaa-Pro-2 activity was investigated by adding the monovalent (Na⁺) or divalent metal ions (Ba²⁺, Zn²⁺, Cu²⁺, Co²⁺, Cd²⁺, Mg²⁺ and Ca²) to the reaction mixture. The activity of the enzyme without metallic ions was considered as 100% (García-Alvarez, Bordallo, Gascón & Suárez-Rendueles, 1985; Haddar, Bougatef, Agrebi, Sellami-Kamoun & Nasri, 2009).

2.8. DLS

The DLS technique was applied for analysis of the hydrodynamic diameter distribution of proteins in the enzymatic extract and proteins in the enzymatic extract + ions (Na⁺, Ba²⁺, Zn²⁺, Cu²⁺, Co²⁺, Cd²⁺, Mg²⁺ and Ca²⁺) at 25, 30, 40, 50, 60, 70 and 80°C in a Malvern Zetasizer Nano Series S-90 (Malvern Instruments, Malvern, UK). The protein concentration utilized was 0.017 mg/ml according to ISO 13321 (International Standard ISO 13321; Jachimska, Wasilewska & Adamczyk, 2008; Kaszuba et al., 2007).

2.9. Effect of temperature and Cd²⁺ and Co²⁺ ions on peptidases activity

Peptidase activity in the presence of Cd²⁺ and Co²⁺ions was determined by incubating of enzymatic extract and ions at 30–80°C for 60 min, after which it was assayed according to Sánchez-Mundo et al. (2010).

2.10. Statistical analyses

Statistical tests and analysis of variance were performed for all substrates, inhibitors and metal ions to determine whether there was a significant difference among chemical substances. Data obtained were analysed using SPSS package software v. 18.0 (IBM Corp, Armonk, NY). Statistical significance was assessed by analyses of variance and significant differences were detected using Duncan´s multiple range test using a 5% significance level.

3. Results and discussion

3.1. Enzymatic activity in crude extract

The results of enzymatic activity on the assayed substrates are shown in Table 1. The degree of hydrolysis at a substrate concentration of 10 mM was: Gly-Pro-pNA > Ala-Pro-pNA and Leu-pNA > Lys-pNA and Ala-pNA > Arg-Pro-pNA, Ala-Ala-Pro-pNA and Bz-Tyr-pNA. The highest enzyme activity was found at 10 mM concentration for Gly-Pro-pNA substrate, with a value 0.796 ± 0.006 U/mg protein, and the lowest enzyme activity was detected at 10 mM concentration using Bz-Tyr-pNA as substrate, with a value 0.254 ± 0.003 U/mg protein. The difference was significant between substrates. However, no significant differences were found between Bz-Tyr-pNA and Ala-Ala-Pro-pNA and Arg-Pro-pNA.

Table 1. Exopeptidase activity of Theobroma cacao towards various chromogenic substrates¹.

Tabla 1. Actividad exopeptidasa de Theobroma cacao frente a varios sustratos cromogénicos¹.

Substrate^1	Relative activity^1 (%)
	(mean value ± SD (n = 3))
	1 mM	10 mM
Gly-Pro-pNA	0.092 ± 0.001^c,d	0.796 ± 0.006^h
Ala-Pro-pNA	0.315 ± 0.001^b,c,d	0.542 ± 0.007^g
Leu-pNA	0.221 ± 0.008^b,c,d	0.493 ± 0.006^g
Lys-pNA	0.275 ± 0.005^c,d	0.366 ± 0.001^f
Arg-Pro-pNA	0.249 ± 0.007^b,c,d	0.293 ± 0.001^e
Ala-pNA	0.278 ± 0.009^b,c	0.285 ± 0.009^e,f
Ala-Ala-Pro-pNA	0.249 ± 0.001^b	0.277 ± 0.008^e
Bz-Tyr-pNA	0.194 ± 0.001^a	0.254 ± 0.003^e

Notes: Means with same letter in the same column are not significantly different according to Duncan’s multiple range test at α = 0.05.

¹An enzyme unit is defined as the amount of enzyme needed to liberate 1 µmol pNA/min under assay conditions.

¹Medias con la misma letra en la misma columna no son estadísticamente diferentes de acuerdo con la Prueba de Rangos Múltiples de Duncan con un α = 0,05.

²Una unidad de enzima es definida como la cantidad de enzima necesaria para liberar un µmol de pNA por minuto bajo las condiciones de ensayo.

Of the substrates with proline at the N-penultimate position, the highest degree of hydrolysis was obtained with Gly-Pro-pNA at 10 mM, followed by Ala-Pro-pNA and Arg-Pro-pNA, which shows the preference of Xaa-Pro-DAP2 for the Gly amino acid adjacent to proline, followed by alanine and, finally, by arginine.

With increase in substrate concentration activity increased, so that with Gly-Pro-pNA 8.7-fold higher activity was obtained with 10 mM than with 1 mM, whereas with Leu-pNA and Ala-Pro-pNA the increase was 2.2- and 1.7-fold, respectively; for the remaining substrates, the variations were very small or absent. The highest Xaa-Pro-DAP2 activity in lactic bacteria reported was for substrates with a non-charged N-terminal (Ala or Gly) or basic residues (Arg) (Sanz & Toldrá, 2001). Given the presence of these enzymes during germination of the cacao seed and its specificity for proline (an amino acid representing 0.72–1.97% of cacao proteins), their role, in conjunction with other peptidases, is to participate in the hydrolysis of reserve proteins.

According to the activity towards Leu-pNA, a substrate for aminopeptidases, and by the inhibitory action shown by bestatine, as identified APE activity, the highest degree of hydrolysis was with Leu-pNA followed by Lys-pNA at 10 mM (Table 1). Likewise, we assessed the Ala-pNA substrate, finding a lower degree of hydrolysis as compared with Leu-pNA and Lys-pNA at 10 mM. The non-specific Type N APE identified in a large number of lactic bacteria demonstrated Lys-pNA, Leu-pNA (Gómez De La Cruz, 1996) and Ala-pNA (Magboul & McSweeney, 2000) activities. In fermented cacao, the aminopeptidase activity is considered as the second most important enzyme after aspartyl peptidase (AP) (Hansen et al., 1998). Because of hydrolysis of the substrate Bz-Tyr-pNA, reported as specific for carboxypeptidase-type enzymes with preference for hydrophobic amino acids in the carboxyl terminal, CP activity was confirmed.

3.2. Effect of peptidase inhibitors and reducing agents

Table 2 shows the effect of different inhibitors on the activity of the Xaa-Pro-DAP2 enzyme, revealing that at 0.1 mM the optimal inhibitors were bestatine, PCMB, cysteine, diprotine A and pepstatin A, with 52, 48, 47, 47 and 45% inhibition, respectively; whereas at 1 mM, the best inhibitors were pepstatin, diprotine A, AEBSF and pefabloc. Diprotine A (Ile-Pro-Ile) has been identified as a competitive inhibitor of peptidases with dipeptidyl peptidase IV (DPP IV) activity (Rigolet, Xi., Rety & Chich, 2005). In barley, a relative activity of 3% was reported with diprotine A at 0.1 mM (Davy et al., 2000) and, in Lactobacillus delbrueckii ssp. bulgaricus LBU-147, 15% relative activity was reported with the Ile-Pro-Ile sequence of diprotine A (Miyakawa, Kobayashi, Shimamura & Tomita, 1991). A reduction of 48% in Xaa-Pro-DAP2 activity in the presence of PCMB at 0.1 mM, as well as the recovery of enzymatic activity by reducing agents (β-mercapto-ethanol and cysteine), suggests the presence of a cysteine peptidase. Circular dichroism (CD) studies (data not shown) demonstrated a low secondary structure or ‘random coil’ type, which suggests the presence of cysteine peptidases in cacao extract. In Serratia marcescens the activity of endonuclease, containing SH-groups, was affected by PCMB, showing secondary structure changes in the CD spectrum (Filimonova et al., 2001).

Table 2. Effect of inhibitors on the exopeptidase activity of Theobroma cacao L¹.

Tabla 2. Efecto de inhibidores sobre la actividad exopeptidase de Theobroma cacao L¹.

Inhibitor	Relative activity^1 (%)
	(mean value ± SD (n = 3))
	0.1 mM	1.0 mM
Leupeptin	76 ^b,c	94 ^i,j
Pepstatin A	55 ^a	53 ^f
Bestatine	48 ^a	79 ^g
Diprotine A	53 ^a	44 ^f
AEBSF	89 ^d	48 ^f
Pefabloc	84 ^c,d	44 ^f
PMSF	76 ^b,c	81 ^g
EDTA	71 ^b	83 ^g,h
PCMB	52 ^e	78 ^g
β-mercaptoethanol	69 ^b	84 ^g,h
Cysteine	53 ^a	90 ^h,i
Dithiothreitol	70 ^b	77 ^g

Notes: Means with same letter in the same column are not significantly different according to Duncan´s multiple range test at α = 0.05.

¹Expressed as a percentage of hydrolysis of Ala-Pro-pNA in the absence of any added chemical agent, which was given a value of 100%.

¹Medias con la misma letra en la misma columna no son estadísticamente diferentes de acuerdo con la Prueba de Rangos Múltiples de Duncan con un α = 0,05.

²Expresada como un porcentaje de hidrólisis de Ala-Pro-pNA en ausencia de algún agente químico, al cual le fue dado un valor de 100%.

The metallo-peptidase inhibitors, bestatine and EDTA, reduced Xaa-Pro-DAP2 activity at 0.1 mM by 52 and 29%, respectively. These inhibition levels indicate the presence of a metallo-protein. Inhibitors such as PMSF, bestatine and EDTA at 1.0 mM had a lower inhibitory effect on Xaa-Pro-DAP2 activity. This is due to the presence of different peptidases in the enzymatic extract, which are also inhibited by various compounds, reflecting lower proteolytic action, increasing indirectly the Xaa-Pro-DAP2 activity under study; this is the case for the aminopeptidase enzyme reported as serine peptidase (Haddar et al., 2009).

The serine peptidase blockers, AEBSF and pefabloc, at higher concentrations, inhibited 52 and 56% of enzymatic activity, respectively, suggesting that serine residues may participate in enzyme catalysis, as occurs with other microbial Xaa-Pro-DAPs (Magboul & McSweeney, 2000). However, another serine peptidase inhibitor, PMSF (at 0.1 mM) reduced Xaa-Pro-DAP2 activity by only 24%; leupeptin, which also inhibits cysteine peptidases, had the same effect on enzymatic activity. In contrast to Xaa-Pro-DAP1 obtained at 40% saturation (Sánchez-Mundo et al., 2010), in that obtained at 80% saturation the differences shown by the diverse serine peptidase inhibitors revealed that the inhibition mechanism of acylation of the active site of the enzyme is more effective than sulfonation. Results revealed the presence of at least three structural characteristics of Xaa-Pro-DAP peptidases in the enzymatic extract of T. cacao: (1) serine peptidase type; (2) cysteine peptidase type; and (3) a metallic character, apart from a CP and an APE. APE and CP enzymes have been reported as metallopeptidases (Biehl et al., 1991; Pérez-Guzmán, Cruz y Victoria, Cruz-Camarillo & Hernández-Sánchez, 2006, 2004b, Ramírez-Zavala, Mercado-Flores, Hernández-Rodríguez & Vila-Tanaca, 2004b).

Table 3 shows the specificity of each of the tested inhibitors, as well as the inhibitory effect on the enzymes identified in this study, including the carboxypeptidase- and aminopeptidase-type metallopeptidases of different plant and microorganism sources. In general, the Xaa-Pro-DAPs of different species of Lactobacillus and Lactococcus have been identified as serine and cysteine peptidases due to their inhibition by PMSF and PMCB, respectively (Magboul & McSweeney, 2000; Pérez-Guzmán et al., 2006), while Xaa-Pro-DAP1 has been identified in cacao (Sánchez-Mundo et al., 2010). However, in this study Xaa-Pro-DAP2 revealed a different profile of inhibition. In Hordem vulgare, one of the few reports in this field on plants (Davy et al., 2000), DPP IV showed inhibition profiles similar to those found for cacao Xaa-Pro-DAP2 identified in this study, sensitive to AEBSF and diprotine, inhibitors of serine peptidases specific for proline. On the other hand, bestatine, EDTA and leupeptin also showed inhibitory effects on APE and CP from different sources (Biehl et al., 1991; Ramírez-Zavala, Mercado-Flores, Hernández-Rodríguez & Vila-Tanaca, et al., 2004a), as detected in this study.

Table 3. Effect of various inhibitors on peptidase activity of different sources.

Tabla 3. Efecto de diversos inhibidores sobre la actividad peptidasa de diferentes orígenes.

Protease inhibitor	Specificity of inhibitor	Enzymes/origin	References
AEBSF	Irreversible inhibitor of serine proteases. Inhibits by acylation of the active site of the enzyme. Serine proteases	Xaa-Pro-DAP2^e/T. cacao	Current study
		DPP IV: H. vulgare	Davy et al. (2000)
Pefabloc	Water-soluble and relatively non-toxic irreversible inhibitor of thrombin and other serine proteases. Inhibits by acylation of the active site of the enzyme	Xaa-Pro-DAP2/ T. cacao	Current study
		Xaa-Pro-DAP/ Lb. curvatus DPC2024	Magboul and McSweeney (2000)
PMSF	Irreversibly inhibits serine proteases by sulfonylation of the serine residue in the active site of the protease. Does not inhibit metallo-, aspartic or most cysteine proteases.	Xaa-Pro-DAP/Lc. lactis ssp. cremoris NRRL634	Pérez-Guzmán et al. (2006)
		CP: K. marxianus	Ramírez-Zavala et al. (2004b)
		Xaa-Pro-DAP1/ T. cacao	Sánchez-Mundo et al. (2010)
Bestatine	Metalloprotease inhibitor. Inhibits cell surface aminopeptidases (notably B) and leucine aminopeptidase	APE: U. maydis	Biehl et al. (1991)
Pepstatin	Pentapeptide derivative. Reversible inhibitor of aspartyl proteases (AP)	AP: T. cacao	Guilloteau, Laloi, Michaux, Bucheli, and McCarthy (2005)
Leupeptin	Tripeptide aldehyde. Reversible competitive inhibitor of serine and cysteine proteases	CP: K. marxianus	Ramirez-Zavala et al. (2004b)
			Hook and Peng Loh (1984)
PCMB	A highly specific sulfhydryl reagent	Xaa-Pro-DAP/Lb. sanfranciscensis CB1	Gallo et al. (2005)
		Xaa-Pro-DAP/Lb. curvatus DPC 2024	Magboul and McSweeney (2000)
		Xaa-Pro-DAP1/ T. cacao	Sánchez-Mundo et al. (2010)
EDTA	Reversible inhibitor of metalloproteases	APE: U. maydis	Mercado-Flores et al. (2004)
		Xaa-Pro-DAP1/T. cacao	Sánchez-Mundo et al. (2010)
Diprotine	Reversible inhibitor of the metallo-protease dipeptidylaminopeptidase IV	DPP IV: H. vulgare	Davy et al. (2000)
		Xaa-Pro-DAP2/T. cacao	Current study

Data obtained in this work represent one of the few reports on plant Xaa-Pro-DAP, aside from one study on barley, in contrast to studies on acid-lactic bacteria, which use proline-rich substrates for their growth. These stud results share characteristics with the cacao Xaa-Pro-DAP2 of this study: sensitivity to serine peptidase inhibitors (PMSF, AEBSF), inhibition by diprotine A and PCMB and, in some cases, reduction in activity by chelating agents.

3.3. Effect of divalent cations and sodium on Xaa-Pro-DAP2 activity

Table 4 depicts the effect of divalent cations and sodium on the enzymatic activity. The presence of Cu²⁺ at 0.1 mM decreased activity by 46%, cations Ba²⁺, Mg²⁺ and Ca²⁺ at 0.1 mM decreased it by 38, 34 and 30%, respectively, while it was decreased by 27 and 20%, respectively, with Cd²⁺ and Co²⁺. Ca²⁺ and Mg²⁺ caused 46% inhibition at 1 mM and Zn²⁺ 30%, whereas only a slight effect (10–14%) was observed in the presence of Ba²⁺ and Na⁺. Increasing the concentration of Ba^2+, Cd^2+, Cu²⁺, Na⁺ and Co²⁺ to 1 mM, the inhibitory effect was lower, with Co²⁺ resulting in a full recovery of the enzymatic activity of Xaa-Pro-DAP2. These findings are probably due to interactions among peptidases with lower specificity for substrates such as AP, APE, and CP, in such a way that the activity of Xaa-Pro-DAP2 is indirectly favoured. For the APE of Ustilago maydis, the presence of Cu²⁺, Hg²⁺ and Zn²⁺ at 1 mM induced complete inhibition of the purified enzyme. Co²⁺, Ni²⁺ and Mn²⁺ had a strong inhibitory effect on APE (Mercado-Flores, Noriega-Reyes, Ramírez-Zavala, Hernández-Rodríguez & Villa-Tanaca, 2004). Cu²⁺, Hg²⁺ and Zn²⁺ are reported as strong inhibitors of the Xaa-Pro-DAP2 activity of Lactobacillus helveticus ITG LH1 and L. delbreckii subsp. bulgaricus. In Lactobacillus sakei, Cu²⁺and Hg²⁺ resulted in 53 and 78% inhibition, respectively, whereas other cations had no significant inhibitory effect on activity (Sanz & Toldra, 2001). The effect of the ionic strength of sodium chloride revealed that activity increases when the former increases in the medium. Monovalent cation coordination plays an influential role in many enzyme-catalysed reactions (Di Cera, 2006; Page & Di Cera, 2006). Due to limited electrostatic properties, Na⁺ and K⁺ are optimal reagents for stabilization of the active conformational state of an enzyme or for facilitating electrostatic interactions between enzyme and substrate (Page & Di Cera, 2008).

Table 4. Effect of divalent cations and sodium ion on exopeptidase activity of Theobroma cacao L¹.

Tabla 4. Efecto de cationes divalentes e ion sodio sobre la actividad exopeptidasa de Theobroma cacao L¹.

Metal salt	Relative activity^1 (%)
	(mean value ± SD (n = 3))
	0.1 Mm	1.0 mM
BaCl2	62^a,b,c	90^h,i,j
CoCl2	80^d	102^j
CdCl2	73^c,d	76^i,j
ZnCl2	81^d	70^g
CuCl2	54^a,b	74^g,h
CaCl2	70^e	54^f
MgCl2	66^a	54^f
NaCl	51^a	86^h,i

Notes: Means with same letter in the same column are not significantly different according to Duncan’s multiple range test at α = 0.05.

¹Expressed as a percentage of hydrolysis of Ala-Pro-pNA in the absence of any added metal salt: given a value of 100%.

¹Medias con la misma letra en la misma columna no son estadísticamente diferentes de acuerdo con la Prueba de Rangos Múltiples de Duncan con un α = 0,05.

²Expresada como un porcentaje de hidrólisis de Ala-Pro-pNA en ausencia de alguna sal metálica añadida, al cual le fue dado un valor de 100%.

3.4. Analysis of protein–cation interaction by DLS

The effect of salts on the aggregation of the protein was monitored by DLS due to its high sensitivity, which makes it a useful tool for this type of study. The intensity-averaged diameter (in nanometres) and polydispersity index (PdI) values (an estimate of distribution width) were determinate as defined in ISO 13321 Standard (1996). Regarding the enzymatic extract, the size distribution intensity at 25°C revealed three protein groups of around 8.281, 151.4 and 4987 nm in diameter, showing a major association at the interval of 151.4 nm with an intensity of 73.1%. Interactions (protein–cation) of the enzymatic extract with the tested cations at both 0.1 and 1.0 mM at 25°C, according to the distribution of hydrodynamic diameters, showed three aggregation groups (≤10 nm, 10 ≤ 100 nm and 100 ≤ 4000 nm). The observation of a higher intensity in the signal demonstrates that the most important protein–cation interactions were for CdCl₂ and CuCl₂ at 0.1 mM; CdCl_{2 has} three groups of hydrodynamic diameter: 12.7 nm (20%), 141 nm (76%) and 5021 nm (4%), while CuCl₂ has 9.9 nm (8%), 104.9 nm (90%) and 4801 nm (2%). Noteworthy are the interactions with CaCl₂ at 0.1 mM and 1 mM, which reached their highest intensity among the cations tested with a hydrodynamic diameter of 153.1–131.6 nm. This size distribution is multi-modal (i.e. it reveals the presence of different protein groups, indicating protein aggregation that is reflected in loss of enzymatic activity of Xaa-Pro-DAP2 (Table 4)). Incrementing the ionic strength in the medium by adding salts at 1 mM (Figure 1a) induces a lower variation in size distribution, but with the same multi-modal behaviour, indicating that the aggregation induced by cations at 1 mM could be due to binding of a metallic ion at a specific site of the protein. There is growing evidence that metal ions can accelerate the aggregation process of several proteins, as occurs in the denaturation and aggregation process of β-lactoglobulin A (BLG-A) in the presence of copper and zinc ions (Stirpe et al., 2008). However, interactions involving CuCl₂ and CdCl₂ are prominent, in which the size distribution is monomodal, with hydrodynamic diameters of 158.5 and 119 nm with PdI = 0.242 and 0.211, respectively, indicating a tendency towards homogeneous molecular size or polydispersed medium; this is related to increase in the enzymatic activity of Xaa-Pro-DAP2 resulting from lower protein aggregation and leading to denaturation induced by the ions. CoCl₂ favours the aggregation of several groups of varying molecular size, in contrast to its effect on activity, which is re-established at 1 mM.

Figure 1. Effect of temperature on size distribution of interactions (protein–cation) by DLS in the enzymatic extract with Ba²⁺, Zn²⁺, Cu²⁺, Co²⁺, Cd²⁺, Mg²⁺, Ca²⁺ and Na¹⁺ at 1 mM: (a) 25°C, (b) 40°C, (c) 60°C + (insert: CuCl₂ 1 mM), (d) 80°C + (insert: CdCl₂ 1 mM).

Figura 1. Análisis mediante DLS del efecto de la temperatura sobre la distribución de tamaño de interacciones (proteína-catión) en el extracto enzimático con Ba²⁺, Zn²⁺, Cu²⁺, Co²⁺, Cd²⁺, Mg²⁺, Ca²⁺ y Na¹⁺ at 1 mM (a) 25°C,(b) 40°C, (c) 60°C + (inserto: CuCl₂ 1 mM), (d) 80°C + (inserto: CdCl₂ 1 mM).

Increments of temperature revealed a higher variation in the distribution of hydrodynamic diameter generated in the interaction with the salts studied; however, the formation of molecular aggregates (protein–cation interaction) was observed between 10 and 70 nm (Figure 1b), this being very evident at 0.1 mM at both 40 and 50°C.

At 40°C, with 0.1 and 1.0 mM, aggregates larger than 5000 nm were observed (Figure 1b) that became denatured at temperatures higher than 50°C (Figure 1c), generating large aggregates, except those formed with MgCl₂, indicating that this probably results in polymer stability. For CuCl₂ at 1 mM, the monomodal behaviour was maintained up to 60°C, with an average size of 154.9 nm and PdI = 0.224 (insert, Figures 1c, 2a). A similar distribution was obtained with CdCl₂ at 1 mM (insert, Figures 1d, 2b), but was maintained up to 80°C (i.e. these cations favour protein stabilization, since there was no evident increase in protein sizes after denaturation.

Figure 2. Effect of temperature on size distribution of interactions (protein–cation) by DLS in the enzymatic extract: (a) Cu²⁺ at 25, 30, 40, 50 and 60°C; (b) Cd²⁺ at 25, 30, 40, 50, 60, 70 and 80°C.

Figura 2. Análisis mediante DLS del efecto de la temperatura sobre la distribución de tamaño de interacciones (proteína-catión) en el extracto enzimático (a) Cu²⁺at 25, 30, 40, 50 y 60°C (b) Cd²⁺ at 25, 30, 40, 50, 60, 70 y 80°C.

3.5. Effect of temperature and Cd²⁺ and Co²⁺ ions on peptidase activity

The effect of temperature and the ions Cd²⁺ and Co²⁺ on peptidase activity was examined at various temperatures. Xaa-Pro-DAP2 showed 1.6 U/mg protein at 25°C, which increased to 2.9 and 19.7 U/mg at 50 and 80°C, respectively, indicating activation by increasing temperature (Figure 3a). In the presence of CoCl₂ at 0.1 mM, activity showed the same behaviour, while at 1 mM a maximal activity of 25.7 U/mg protein was reached at 70°C, decreasing to 19.74 U/mg at the end of the assay (80°C) (Figure 3b). However, the addition of CdCl₂ at 1.0 mM achieved higher thermal stability of the Xaa-Pro-DAP2 enzyme at 80°C, reaching 28 U/mg protein (Figure 3c). At 0.1 mM, the CdCl₂ effect was very similar. Accordingly, Xaa-Pro-DAP-type enzymes can be ascribed to the only protein groups detected with DLS in the presence of CdCl₂, corresponding to 34.87 nm at 50°C until 80°C with 53.48 nm.

Figure 3. Effect of temperature on enzymatic activity of Xaa-Prolyl dipeptidyl aminopeptidase (Xaa-Pro-DAP2). (a) Control: Xaa-Pro-DAP2 activity without cations in the enzyme extract. (b) Xaa-Pro-DAP2 activity in the enzyme extract in the presence of CoCl₂ at a concentration of 1 mM. (c) Xaa-Pro-DAP2 activity in the enzyme extract in the presence of CdCl₂ at concentration of 1.0 mM. Mean values are connected by a line, bars represent standard deviations (n = 3).

Figura 3. Efecto de la temperatura sobre la actividad enzimática de Xaa-Prolyl dipeptidyl aminopeptidase (Xaa-Pro-DAP2). (a) Control: Actividad de Xaa-Pro-DAP2 en el extracto enzimático en ausencia de cationes (b) Actividad de Xaa-Pro-DAP2 en el extracto enzimático en presencia de CoCl₂ a 1 mM y (c) Actividad de Xaa-Pro-DAP2 en el extracto enzimático en presencia de CdCl₂ a 1.0 mM. Los valores de las medias están unidos por una línea, las barras representan la desviación standard (n = 3).

APE activity showed a decreasing trend following thermal denaturation, from 0.7 U/mg protein at 40°C to 0.5 U/mg protein at 80°C (Figure 4a). The activity levels of APE in the enzymatic extract were low as compared with Xaa-Pro-DAP2. However, in the presence of CoCl₂ at 1 mM, APE activity became stabilized, with 1.0 and 1.2 U/mg protein at 60 and 80°C, respectively (Figure 4b). Increases of 1.7- to 50-fold the original activity of CP (Cheng, Ramakrishnan & Chan, 1999; Kishimura, Hayashi & Ando, 2006) and of APE enzymes (Bolumar, Sanz, Aristoy, & Toldrá, 2003; Dong et al., 2005), activated by cobalt, were attained using 4 µM to 1 mM CoCl₂. Nevertheless, inhibition of APE by Co²⁺ at 10 mM has also been detected (Mercado-Flores et al., 2004; Mohamed et al., 2009). The effect of CdCl₂ on APE activity was not significant, although the lowest enzymatic activity was detected at 60°C (0.483 U/mg protein), a value below that obtained in the absence of the cation (Figure 4c).

Figure 4. Effect of temperature on enzymatic activity of aminopeptidase (APE) in the enzymatic extract. (a) Control: APE activity without cations. (b) APE activity in the presence of CoCl₂ at a concentration of 1 mM. (c) APE activity in the presence of CdCl₂ at a concentration of 1.0 mM. Mean values are connected by a line, bars represent standard deviations (n = 3).

Figura 4. Efecto de la temperatura sobre la actividad enzimática de aminopeptidasa (APE) en el extracto enzimático. (a) Control: Actividad APE en ausencia de cationes; (b) Actividad APE activity en presencia de CoCl₂ a 1 mM y (c) Actividad APE en presencia de CdCl₂ a 1.0 mM. Los valores de las medias están unidos por una línea, las barras representan la desviación standard (n = 3).

Finally, CP-type activity depicted a random behaviour, recording 0.6 U/mg protein at 40°C to 0.4 U/mg protein at 80°C (Figure 5a). The presence of CoCl₂ at 0.1 mM and 1 mM did not exert any evident effect on CP activity (Figure 5b), whereas the addition of CdCl₂ at both concentrations was able to slightly recover slightly CP activity, from 0.34–0.30 U/mg protein at 60°C to 0.64–0.63 U/mg protein at the end of the assay (Figure 5c).

Figure 5. Effect of temperature on enzymatic activity of carboxypeptidase (CP). (a) Control: CP activity without cations in the enzyme extract. (b) CP activity in the enzyme extract in the presence of CoCl₂ at a concentration of 1 mM. (c) CP activity in the enzyme extract in the presence of CdCl₂ at a concentration of 1.0 mM. Mean values are connected by a line, bars represent standard deviations (n = 3).

Figura 5. Efecto de la temperatura sobre la actividad enzimática de carboxipeptidasa (CP) en el extracto enzimático. (a) Control: Actividad de CP en ausencia de cationes; (b) actividad de CP en presencia de CoCl₂ a 1 mM and (c) actividad CP en presencia de CdCl₂ a 1.0 mM. Los valores de las medias están unidos por una línea, las barras representan la desviación standard (n = 3).

4. Conclusions

In the enzymatic extract of germinated T. cacao, Xaa-Pro-DAP2 activity with a preference for Gly-Pro-pNA and Ala-Pro-pNA was identified. Also detected was lysine and leucine aminopeptidase activity, as well as carboxypeptidase-type activity. The Xaa-Pro-DAP2 enzyme was inhibited by the divalent ions Cu²⁺ (0.1 mM) and Ca²⁺ and Mg²⁺ (1 mM); in the absence of divalent ions, activity was increased by the effect of temperature with values of 2.9 U/mg protein at 50°C and 19.7 U/mg protein at 80°C. The addition of Cd²⁺ ions favoured enzymatic stability, with 28 U/mg protein detected at the end of the assay (80°C). According to DLS analysis, in the presence of Cd²⁺ (1 mM) only one protein group of 53.48 nm diameter at 80°C was detected, which can be attributed to a Xaa-Pro-DAP-type enzyme, indicating thermostability. APE activity was stabilized by 1 mM Co^2+, whereas Co²⁺ had no significant effect on CP activity. Despite APE and CP activity levels being lower than those detected for Xaa-Pro-DAP2, the three enzymes were able to maintain residual activity up to 80°C, indicating low thermal denaturation.

In the present study, the effect of some metal ions on the protease activities was assayed. The metallic ions Cd²⁺ and Co²⁺ at 1 mM may have been responsible for the thermal stability observed during the reaction catalysed for proteases in the cacao extract. Also, the DLS study showed that Cd²⁺ and Cu²⁺ at 1 mM induced monomodal distribution, possibly due to conformational changes. Prepurified Xaa-Pro-DAP1 showed increased activity in substrates with Ala-Pro-pNA residues (Sánchez-Mundo et al., 2010), whereas the present study showed higher specificity on substrates with Arg-Pro-pNA residues. Moreover, the effect of cations on activity and stability varied since Xaa-Pro-DAP1 had no effect on stability. The results contribute to the study of Xaa-Pro-DAP2, APE and CP enzymes, which are important because of their participation in degrading reserve proteins during the germination of cacao, especially given the scarce information available on these types of vegetable-derived enzymes. Following our findings here, for further research is possible especially in regard to determining which enzyme components link with metal ions.

Disclosure statement

No potential conflict of interest was reported by the authors.

Additional information

Funding

This work was partially supported by funds from CONACyT Scholarship 217805 and PIFI. The authors M.E.J.F. and C.B.M. would like to acknowledge SNI/COFAA-IPN; EDI/IPN SIP/IPN Grants SIP-20150143 and SECITI-ICYTDF/213/2012 for financial support.