Characterization of protease activities in a crude extract of germinated cacao

Figures & data

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.

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%.

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

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%.

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).

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.

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).

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).

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).

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