Purification and biochemical characterization of a

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Environment  Health  Techniques Protease from P. digitatum and bioactive peptides production

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Research Paper Purification and biochemical characterization of a novel protease from Penicillium digitatum – Use in bioactive peptides production Neyssene Aissaoui, Ferid Abidi, Safa Mahat and M. Nejib Marzouki Laboratory of Protein Engineering and Bioactive Molecules (LIP-MB), National Institute of Applied Sciences and Technology, University of Carthage, Tunis, Cedex, Tunisia

This work reports the production of a novel serine protease enzyme (P. dig-protease) from the fungus Penicillium digitatum. The protease was purified from the culture supernatant to homogeneity using ammonium sulfate precipitation, Sephadex G-150 gel filtration and carboxymethyl-sepharose ion exchange chromatography with a 13-fold increase in specific activity. The apparent molecular weight of P.dig-protease was estimated to be 120 kDa by native high performance liquid chromatography (HPLC), sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) revealed a single polypeptide at about 30 kDa that indicates a tetrameric protein. The proteolytic activity was inhibited by phenylmethylsulfonyl fluoride suggesting a serine-protease enzyme. P.dig-protease stability was investigated over broad range of pH, temperature, salt concentrations, surfactants and metal ions. The purified P.dig-protease was used for the production of bioactive peptides. Red scorpionfish (Scorpaena notata) muscle was hydrolyzed with P.dig-protease in order to obtain peptides with biological activities. Interestingly, the hydrolysate revealed the presence of antioxidant and angiotensin-I converting enzyme inhibitor peptides. Abbreviations: P.dig-protease – protease of Penicillium digitatum; PMSF – phenylmethylsulfonyl f luoride; TCA – trichloroacetic acid; EDTA – ethylenediaminetetraacetic acid; SNH – S. notata hydrolysate; DPPH – 2,2diphenyl-1-picrylhydrazyl; ACE – angiotensin-I converting enzyme Keywords: Penicillium digitatum / Protease purification / Protein hydrolysate / Antioxidant peptides / Angiotensin-I converting enzyme inhibitory activity Received: March 7, 2014; accepted: April 12, 2014 DOI 10.1002/jobm.201400179

Introduction The continual exploration of enzymes and their utilities have expanded their industrial market with the growth of 7.6% per year [1]. Proteases are the most important industrial enzymes accounting for at least 60% of all global industrial enzyme sales [2]. The relevance of this group of enzymes, rich in structural diversity and mechanisms of action is reflected in the importance of their applications in industrial processes. Therefore, the

Correspondence: Dr. Ferid Abidi, National Institute of Applied Sciences and Technology, LIP-MB, North Urban Center, B.P. 676, Tunis Cedex, 1080 Tunisia E-mail: [email protected] Phone: þ216 52 61 57 52 Fax: þ216 71 70 43 29 ß 2014 WILEY-VCH Verlag GmbH & Co. KGaA,Weinheim

industrial demand of proteolytic enzymes, with appropriate specificity and stability to pH, temperature, and chemical agents, continues to motivate the search for new sources [3]. Thermoalkaline proteases are the most commonly used of the alkaline proteases because they can function at a pH range of 7.0–12 and a temperature range of 35–80 °C [4]. Although proteases are widespread in nature, microbes serve as a preferred source of these enzymes because of their rapid growth, the limited space required for their cultivation and the ease with which they can be genetically manipulated to generate new enzymes with altered properties that are desirable for various applications [5]. Enzymes of fungal origin offer a distinct advantage over bacterial proteases in terms of ease of downstream processing [6]. Comparatively, studies on fungal proteases are few and limited mainly

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to species belonging to the genera such as Aspergillus, Conidiobolus, Mucor, Paecilomyces, Penicillium, Rhizopus [6, 7]. These species are widely used for protease production since several species of these genera are regarded as safe (GRAS) strains [8]. Recently, the potential use of enzymes has been catching up fast in a wide range of other biotechnological applications such as bioactive peptide generation [9]. The present work reports the purification and characterization of a serine protease produced by the fungus Penicillium digitatum. Also, we investigated the potential use of the protease on the production of bioactive peptides such as antioxidant and angiotensin-I converting enzyme (ACE) inhibitory peptides.

Materials and methods

Proteolytic activity assay Proteolytic activity was assayed as described by Segers et al. [11] with modifications. The reaction mixture was made up of 50 ml of diluted enzyme, 100 ml of reaction buffer (100 mM Tris–HCl, pH 7.0) and 50 ml of 5% azocasein (w/v) dissolved in 0.1 M Tris-HCl buffer, pH 7.0. The reaction was carried out at 55 °C and stopped after 30 min by adding 600 ml of 10% w/v trichloroacetic acid and left for 15 min on ice, followed by centrifugation at 7000  g for 10 min to remove the precipitated protein. Supernatant (600 ml) was neutralized by adding 700 ml of 1 N NaOH and absorbance at 440 nm was recorded with an UV/Visible spectrophotometer (Shimadzu model 1240, Tokyo, Japan). One unit of enzyme activity was defined as the amount, which yielded an increase A440 of 0.01 in 30 min at 55 °C under the assay conditions, as mentioned above.

Protease production For production of proteases the fungal culture was carried out on complete medium (Potato Dextrose Broth, PDB). Mycelia plugs (4 mm Ø) from a 3 days-old PDA culture were transferred to 15.0 ml of PDB and the culture was grown for 3 days at 25 °C, with shacking at 150 rpm. The medium used for protease production by P. digitatum strain was composed of: KCl, 1.0 g L1; KH2PO4, 6.7 g L1; K2HPO4, 14.3 g L1; MgSO4, 0.5 g L1; NaNO3, 4.3 g L1; (NH4)SO4, 1.4 g L1; yeast extract, 2.0 g L1; 2% Spirulina algae; 1.0 ml of oligo elements. The culture conditions of protease production were 25 °C, agitation at 150 rpm and an initial pH of media of 5.5. The culture was grown in 500 ml Erlenmeyer flasks containing 200 ml medium. The fermented material was filtered and centrifuged at 3000  g and 4 °C for 30 min to remove fungi mycelia. The supernatant was used as a crude enzyme solution.

Enzyme purification The culture supernatant containing the extracellular enzyme was first subjected to ammonium sulfate precipitation at 80% saturation. The supernatant was discarded and the pellet obtained after centrifugation at 5000  g for 30 min was suspended in a minimal volume of 20 mM Tris–HCl buffer, pH 7.0. The partially purified protease was loaded to a Sephadex-G150 (Sigma–Aldrich, USA) column (1.6  40 cm) coupled to fast protein liquid chromatography (FPLC) system and pre-equilibrated with 20 mM Tris–HCl buffer, pH 7.0. Enzyme fractions of 2.0 ml were eluted with the same buffer at a flow rate of 30 mL h1. The eluates were monitored continuously at 280 nm for protein and also assayed individually for protease activity as described above. Fractions with protease activity were pooled. The active fractions were applied to a CM-Sepharose column (1 cm  10 cm) equilibrated with 20 mM acetate buffer pH 5.5. After being washed with the same buffer, bound proteins were eluted with a linear gradient from 0 to 1 M NaCl in the equilibrating buffer. Fractions of 2.0 ml were collected at a flow rate of 40 ml h1. The fractions with high protease activity were collected and stored at 4 °C for further analysis. Fractions showing maximum activity were further analyzed for purity by electrophoresis.

Protein assay Protein was estimated according to the method of Bradford [10] with bovine serum albumin (BSA) as the standard. The protein content was calculated from a standard curve (data not shown). During chromatographic purification steps, protein determination of each fraction was estimated by measuring its absorbance at 280 nm.

High performance liquid chromatography (HPLC) Native molecular weight of purified protease was further analyzed by gel filtration chromatography onto TSKG2000 SWXL column (300  7.8 mm; Tosohaas, Montgomeryville, PA) using a Waters (Milford, MA, USA) HPLC Alliance 2695 system. The column was previously equilibrated with 20 mM Tris–HCl buffer, pH 7.0. Sample (20 mg of proteins) was injected in 20 ml boucle. Elution

Fungal strain P. digitatum employed in this study was provided by the Laboratory of Plant Protection, National Institute of the Agronomic Research of Tunisia. The strain was isolated from infected citrus, identified and maintained on potato dextrose agar (PDA) plates at 20 °C.

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with the same buffer was performed at a flow rate of 0.8 ml min1, and detection was performed with a Waters 996 photodiode array detector at 280 nm. The column was previously calibrated with thyroglobulin (670 kDa), bovine-g-globulin (158 kDa), ovalbumin (43 kDa) and equine myoglobulin (17 kDa). Protease activity was tested in the chromatographic fractions by azocasein assay.

In order to determine optimal temperature, the enzymatic assay was carried out at different temperatures (40–75 °C), at pH 7.0. Thermal inactivation was examined by pre-incubating the enzyme at 35, 45, and 55 °C for 2 h 30 at intervals of 30 min and the residual activity was measured at 55 °C, pH 7.0 and expressed as percentage of initial activity taken as 100% (non-heated enzyme).

Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) analysis SDS–PAGE was carried out according to the method suggested by Laemmli [12] using 5% stacking gel and 10% separating gel. Samples were mixed with sample buffer containing SDS and b-mercaptoethanol and heated at 100 °C before electrophoresis. Electrophoresis was performed at 50 V and protein bands were visualized after staining the gel with 0.25% Coomassie Brilliant Blue R250 in 30% ethanol–10% acetic acid and destained with 30% ethanol–10% acetic acid. The molecular weight of protein bands was determined by comparing with the bands of standard molecular mass marker.

Storage stability Stability of the enzyme at 4 °C and at room temperature (25 °C) was investigated by incubating the protease at these two temperatures during 1 month storage. The residual activity was measured at 55 °C, pH 7.0 and expressed as percentage of initial activity taken as 100%.

Zymography Zymography was performed on SDS–PAGE according to the method of Schmidt et al. [13] with slight modification. The resolving gel was copolymerized with 0.05% gelatin. After migration at 100 V, the gel was submerged in 100 mM Tris–HCl buffer (pH 7.0) containing 2.5% Triton X-100, with shaking for 30 min to remove SDS. Triton X100 was removed by washing the gel three times with 100 mM Tris–HCl buffer (pH 7.0). The gel was then incubated in 100 mM Tris–HCl buffer (pH 7.0) for 40 min at 55 °C. Finally, the gel was stained with 0.25% Coomassie Brilliant Blue R250 in 30% ethanol–10% acetic acid and destained with 30% ethanol–10% acetic acid. The development of a clear zone on the blue background of the gel indicated the presence of protease activity. Effect of pH and temperature on protease activity and stability Optimum pH was determined by performing standard activity assays in a pH range from 4.0 to 11.0 at 55 °C using suitable buffers (Acetate buffer, pH 4.0–5.0–6.0; Tris–HCl buffer, pH 7.0–7.5–8.0–8.5–9.0; glycine–NaOH buffer, pH 10.0–11.0). For the measurement of pH stability, the enzyme was kept at 4 °C for 12 h in different buffers at 100 mM, at pH values ranging from 4.0 to 11.0. Residual proteolytic activity was estimated as described earlier and expressed as percentage of the initial activity taken as 100%. ß 2014 WILEY-VCH Verlag GmbH & Co. KGaA,Weinheim

Effect of salt on enzyme activity and stability To determine the effect of NaCl on enzyme activity, NaCl solution with concentrations ranging from 0 to 30% w/v were added to the reaction mixture and the enzymatic assay was carried out at optimum conditions (55 °C, pH 7.0). For stability, the purified enzyme was pre-incubated for 1 h at 4 °C with an equal volume of NaCl solution with concentrations ranging from 0 to 30% w/v. The enzyme activity assayed in absence of salt was taken as 100%. Effect of enzyme inhibitors and surfactants The effect of enzyme inhibitors (2.0 and 5.0 mM) on the purified protease was studied using phenylmethylsulfonyl fluoride (PMSF), b-mercaptoethanol, and ethylenediaminetetraacetic acid (EDTA). The purified enzyme was pre-incubated with each inhibitor for 30 min at 4 °C, and then the remaining enzyme activity was estimated using azocasein as substrate. The activity of the enzyme assayed in the absence of inhibitors was taken as 100%. The effect of some surfactants (Tween 20, Triton X-100, and SDS) on enzyme stability was studied by preincubating the purified protease for 30 min at 4 °C with each additive. The residual activity was measured at pH 7.0 and 55 °C. The activity of the enzyme without any surfactant was taken as 100%. Effect of metal ions on enzyme activity The effect of various metal ions (5.0 and 10.0 mM) on protease activity was determined by adding the monovalent (Naþ and Kþ) and divalent (Ca2þ, Mn2þ, Zn2þ, Cu2þ, Mg2þ) metal ions to the reaction mixture. After incubation for 30 min at 4 °C, substrate was added and the residual activity of enzyme was measured. Protease activity without metal was taken as 100%.

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Determination of Km and Vmax Kinetic parameters of the enzyme were determined by measuring its activity with various concentrations of the substrate azocasein (1–20 mg ml1). Kinetic constants, including the apparent Michaelis–Menten constant (Km) and the maximal velocity (Vmax), were calculated from Lineweaver–Burk plot. Preparation of protein extract Fish sample (red scorpionfish: Scorpaena notata) was purchased from the fish market of Tunis. The fish were placed in ice with a fish/ice ratio of 1:2 w/w and transported to the laboratory. Upon arrival, fish were washed and the meat was separated manually. The meat (250 g) was minced, using a grinder, and suspended in 250 ml distilled water. Quantification of the protein concentration was performed by Bradford [10] method using BSA as a standard. Preparation of S. notata hydrolysate (SNH) Prior to hydrolysis, sample was first cooked at 90 °C for 20 min to inactivate endogenous enzymes. Hydrolysis reaction was started by the addition of the enzyme (P.digprotease) at an enzyme-substrate ratio of 5:1 (U mg1). The reaction was conducted at pH 7.0, 55 °C using the pHstat method, as described by Adler-Nissen [14]. The pH of the mixture was maintained constant during hydrolysis using 1 M NaOH. The degree of hydrolysis (DH), defined as the percent ratio of the number of peptide bonds broken (h) to the total number of peptide bonds in the substrate studied (htot) was calculated from the amount of base (NaOH) added to keep the pH constant during the hydrolysis as given below: DHð%Þ ¼

h B  Nb 1 1  100 ¼    100 htot Mp a htot

where B is the amount of alkali consumed (ml) to keep the pH constant during hydrolysis, Nb is the normality of the base, Mp is the mass of the substrate (g) (N  6.25), htot is the content of peptide bonds, and a is the average degree of dissociation of the a-NH2 groups released during hydrolysis expressed as: a¼

10pHpK 1 þ 10pHpK

where pH and pK are the values at which the proteolysis was conducted. The enzymatic reaction was stopped by heating the solution at 80 °C for 20 min followed by centrifugation at 7000  g for 30 min. Supernatant was then ultrafiltrated ß 2014 WILEY-VCH Verlag GmbH & Co. KGaA,Weinheim

using 10 000 MWCO membranes (Millipore) in order to remove the enzyme and the non-hydrolyzed proteins. The ultrafiltration was conducted using Amicon ultra-15 centrifugal filter devices (Millipore Corporation, USA). The protein hydrolysate obtained was stored at 20 °C and used for further analyses as described below. Antioxidant activity The 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical-scavenging activity of the hydrolysates was determined according to the method of Bersuder et al. [15]. Five hundered microliters of each sample was mixed with 500 ml of 99.5% ethanol and 125 ml of DPPH radical solution (0.02% in 99.5% ethanol). The mixture was then incubated at room temperature for 1 h in the dark and the reduction of DPPH radicals was measured at 517 nm using a UV spectrophotometer (Shimadzu model 1240, Tokyo, Japan). The DPPH-radical scavenging activity was expressed as: Inhibitionð%Þ ¼

Absorbance control  Absorbance sample Absorbance control  100

The control was conducted in the same manner, except that distilled water was used instead of sample. Trolox was used as positive standard. Lower absorbance of the reaction mixture indicated higher free radical scavenging activity. Angiotensin-I converting enzyme (ACE) inhibitory assay The ACE inhibitory activity was measured by the spectrophotometric assay of Nakamura et al. [16]. A volume of 80 ml of sample solution was pre-incubated for 3 min at 37 °C with 200 ml of borate buffer (pH 8.3) containing 300 mM NaCl and 5 mM Hip–His–Leu (HHL) as a substrate. The reactions were then initiated by adding 20 ml of 0.1 U ml1 ACE from rabbit lung, prepared in the same buffer. After incubation at 37 °C for 60 min, the enzymatic reactions were terminated by adding 250 ml of 1 N HCl. The hippuric acid (HA) formed was extracted with ethyl acetate (1.7 ml). After centrifugation (4000  g for 5 min), 1.0 ml of supernatant was transferred into a test tube. After removal of ethyl acetate by heat evaporation at 95 °C for 10 min, hippuric acid was redissolved in 1 ml of distilled water and the absorbance of the extract at 228 nm was determined using a UV–visible spectrophotometer (Shimadzu model 1240, Tokyo, Japan). The inhibition activity was calculated using the following equation: Ac  As Inhibition activityð%Þ ¼ 100 Ac  Ab

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where, Ac is the absorbance of the buffer (control), As is the absorbance of the reaction mixture (sample), Ab is the absorbance when the stop solution was added before the reaction occurred (blank). The IC50 value was defined as the concentration of hydrolysate (mg ml1) required to reduce the hippuric acid liberation by 50%.

Results Protease production Protease was produced by growing P. digitatum in a liquid basal medium supplemented with 2% algae (Spirulina) at 25 °C, 150 rpm for 10 days. Results indicate that the enzyme titers increased with time to a maximal protease activity (9210 UP) after 9 days. Thereafter, the enzyme production decreases gradually. The decline in enzyme activity may be due to protease self-hydrolysis and nutrient limitations. In fact, culture at 9 days was used for further enzyme purification. Protease purification P.dig-protease was purified by a two-step procedure. After ammonium sulfate precipitation, a Sephadex G-150 column was used to purify the enzyme, obtaining several peaks (Fig. 1). However, only the second peak (fractions 22–33) presented proteolytic activity. Fractions were pooled and submitted to cation exchange column CMsepharose. Protease was eluted on a single peak at a NaCl concentration of 0.25 M. The elution profiles of proteins and protease activity are shown in Fig. 2. Table 1 summarizes the results of the purification process. Approximately, 13-fold purification of the crude enzyme was achieved with a recovery of 17.9%. Specific activity of the protease increased from 68619.2 U mg1

Figure 1. Purification profile of protease from P. digitatum by gel filtration on Sephadex G-150 column. The concentrated enzyme preparation was applied to a 1.6 cm  40 cm column, equilibrated and eluted with 20 mM Tris–HCl buffer (pH 7.0) at a flow rate of 30 ml h1. ß 2014 WILEY-VCH Verlag GmbH & Co. KGaA,Weinheim

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for the crude protease to 886 940 U mg1 after the final purification step. Molecular weight and structure analysis of P.digprotease Zymogram activity staining showed a single band of clear zone of proteolytic activity against the blue background indicating the presence of only one isoform. The apparent molecular mass of the purified P.dig-protease was estimated to be 120 kDa after HPLC gel filtration analysis on TSK 2000 column (Fig. 3A). The enzyme was eluted at a retention time of 12 min. The oligomeric state of the pure protease was investigated using SDS polyacrylamide gel under reduced conditions (Fig. 3B). The enzyme migrated as a single band of at about 30 kDa after treatment with b-mercaptoethanol in denaturating PAGE indicating its homogeneity. This result suggested that the protease is probably formed of four subunits with the same molecular weight. Determination of Km and Vmax The kinetic of the purified P.dig-protease were studied by varying azocasein concentrations from 1 to 20 mg ml1 at the optimal pH. Protease activity was enhanced with increasing substrate concentrations. The Km and Vmax values determined through a Lineweaver–Burk plot were 7.31 mg ml1 and 322.5 U ml1, respectively. Effect of pH and temperature on P.dig-protease activity and stability P.dig-protease was found to be highly active in the pH range of 5.0–8.0 with an optimum at pH 7.0 suggesting that it is a neutral protease. The relative activities at pH 6.0 and 8.0 were about 90 and 70%, respectively (data not shown). The pH stability profile showed that the purified protease was highly stable in a pH range of 7.0–11.0 maintaining approximately 80% of the initial activity at pH 10.0–11.0 and 70% of its activity at pH 6.0 with maximum stability at pH 7.0 (data not shown). Analysis of temperature dependent activity of protease was determined from 40 to 75 °C. P.dig-protease was active in the temperature range of 50 and 60 °C with an optimum at 55 °C (data not shown). The thermostability of the protease was carried out at temperatures 35, 45, and 55 °C for 2 h 30 min at pH 7.0. P.dig-protease was stable at lower temperatures as the enzyme retains 100% of its initial activity and around 50% of its original activity after 2 h 30 min of pre-incubation at 35 and 45 °C, respectively (Fig. 4A). Whereas, pre-incubation at higher temperature (55 °C) revealed that enzyme activity was affected and not more than 18% of relative activity was noticed after 2 h 30 min of pre-incubation. The study of enzyme stability after conservation at room temperature

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Figure 2. Elution profile of protease from P. digitatum from a CM-Sepharose column. The enzyme was eluted with a linear gradient of NaCl (0– 1 M) in 20 mM acetate buffer (pH 5.5) at a flow rate of 40 ml h1 and zymogram activity staining of the eluted protease (the clear zone is indicated by an arrow).

(25 °C) and 4 °C was assayed. The enzyme retained full activity at 4 °C while there was marginal loss of 15% at room temperature after 1 month of incubation (Fig. 4B). So, purified protease showed good stability in conservation conditions. This property is of great interest for enzyme utilization in main industrial applications. Effect of salt on P.dig-protease activity and stability P.dig-protease showed optimum proteolytic activity and stability at 1% w/v salt concentration and could retain about 78% of its activity at salt concentration as high as 10% w/v. Moreover, enzyme retained 55% of its optimum activity even at a very high salt concentration of 30% w/v. Further, the enzyme was stable even at higher salt concentration as it retained 48% of its activity after a preincubation of 1 h in 30% w/v salt concentration (Fig. 5). Effect of enzyme inhibitors and surfactants In order to identify the type of P.dig-protease, different enzyme inhibitors such as specific group reagents and a chelating agent were pre-incubated with the purified enzyme and their effect on enzyme activity was then assessed (Table 2).

P.dig-protease was strongly inhibited by PMSF. It retained only 2.52% of its initial activity after 30 min incubation in the presence of 5.0 mM PMSF. Partial inhibitory effect on the protease activity was observed with the chelating agent EDTA, which inhibited the enzyme activity by 27.17 and 39.68% at a final concentration of 2.0 and 5.0 mM, respectively. b-mercaptoethanol was practically without influence on the activity of the purified enzyme, indicating its stability in the presence of reducing agents. The effect of surfactants on P.dig-protease activity is shown in Table 3. Enzyme presented a moderate loss of activity in the presence of the non-ionic surfactants Tween 20 and Triton X-100 at the two tested concentrations 1 and 5%. It retained 62.54 and 65.37% of its initial activity in the presence of 5% Tween 20 and Triton X-100, respectively. The strong anionic surfactant SDS caused a moderate inhibition of 38.28 and 58.83% at a concentration of 1 and 5%, respectively. Effect of metal ions on P.dig-protease activity The effect of various metal ions (at two different concentrations, 5.0 and 10.0 mM) on the activity of P.

Table 1. Purification of the protease produced by P. digitatum. Step Crude extract Ammonium sulfate precipitation Gel filtration Ion exchange

Total protein (mg)

Total activity (103 U)

Specific activity (103 U mg1)

81.45 10.64

5589 3514.5

68.62 330.32

1 4.81

100 68.88

5.45 1.13

2750.8 1002.3

504.73 886.94

7.35 12.9

49.2 17.9

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Purification fold

Recovery (%)

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Figure 3. (A) HPLC profile of purified protease (retention time Tr ¼ 12 min) and standard curve and (B) SDS–PAGE of the purified P.dig-protease after treatment with b-mercaptoethanol. Lane 1: molecular weight markers; lane 2: purified protease treated with b-mercaptoethanol.

dig-protease is presented in Table 4. The addition of Kþ, Naþ, Mg2þ, and Mn2þ at 5.0 mM and 10.0 mM showed little influence on enzyme activity. Ca2þ increased enzyme activity by about 20% compared to the control at a concentration of 10.0 mM. Zn2þ greatly affected the enzyme activity, with more than 94% inhibition at a concentration of 5.0 mM, whereas the protease activity

was completely inhibited by Cu2þ at the two different concentrations. Hydrolysis of fish protein extract Extracted proteins from S. notata were hydrolyzed with P. dig-protease. The extent of protein degradation by P.digprotease was measured by assessing the degree of

Figure 4. (A) Effect of temperature on the stability of P.dig-protease. The enzyme was incubated at different temperatures and samples were withdrawn at 30 min intervals up to 180 min. Percent residual activity was calculated. Standard deviations were based on three replicates and (B) Stability of P.dig-protease during conservation at 4 °C and room temperature (25 °C). Standard deviations were based on three replicates. ß 2014 WILEY-VCH Verlag GmbH & Co. KGaA,Weinheim

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Table 3. Effect of surfactants on enzyme activity of the purified P.dig-protease. Residual activity (%) Surfactant

1%

5%

None (control) Tween 20 Triton X-100 SDS

100 64.25 70.35 61.72

100 62.54 65.37 41.17

Purified enzyme was pre-incubated with various surfactants for 30 min at 4 °C and the remaining activity was determined with azocasein substrate at pH 7.0 and 55 °C. Enzyme activity measured in the absence of any surfactant was taken as 100%. Figure 5. Effect of salt on enzyme activity and stability. Standard deviations were based on three replicates.

hydrolysis. The DH increased almost linearly (p < 0.05) with increasing hydrolysis time in the first hour, and reached a plateau (DH ¼ 15.12%) after 4 h of hydrolysis (Fig. 6). In fact, available substrate decreases as time of reaction increases.

inhibitory activity of SNH increased with increasing hydrolysate concentrations. The results so obtained revealed that S. notata hydrolysate exhibited an IC50 of 1.35 mg ml1.

Discussion

Antioxidant and angiotensin-I converting enzyme inhibitor activities of SNH DPPH scavenging activity revealed that SNH can reduce the signal intensity of DPPH. Figure 7A shows the DPPH radical-scavenging activity of fish hydrolysate at different concentrations. SNH exhibited 15% of inhibition at 100 mg ml1, which is lower than the radical scavenging activity of Trolox (100%) at the same concentration. However, at a higher concentration (500 mg ml1), the hydrolysate exhibited a total inhibition (100%) of DPPH. The S. notata hydrolysate obtained by treatment with P.dig-protease was also assayed for ACE inhibitory activity. SNH at a concentration of 2.33 mg ml1 decreased the liberation of hippuric acid from the substrate and exhibited 68.2  1.4% of inhibition ACE activity, while no activity was detected with the undigested fish proteins (t ¼ 0) (data not shown). As shown in Fig. 7B, ACE

In the present study, we have explored the production of an extracellular protease by the fungus P. digitatum. The time (216 h) to reach maximal protease production is longer than that reported by other authors. Sandhya et al. [17] reported 72 for Aspergillus oryzae. Kunert and Kopecek [18] reported 96 h for Aspergillus fumigatus and Gandolfi Boer and Peralta [19] reported 144 h for Aspergillus tamarii. No data on protease production by P. digitatum has been previously reported. The protease (P.dig-protease) was then purified and characterized. After the final purification step, the enzyme was purified 13-fold with a specific activity of 886 940 U mg1 and 17.9% recovery. The purified protease protein has a native molecular weight of at about 120 kDa determined by HPLC and was homogeneous on SDS–PAGE presenting a single polypeptide with a molecular mass estimated to be 30 kDa suggesting a

Table 2. Effect of various enzyme inhibitors on the activity of the purified P.dig-protease.

Table 4. Effect of metal ions on enzyme activity. The protease activity was determined by pre-incubating the enzyme in the presence of various metal ions for 30 min at 4 °C.

Residual activity (%) Inhibitors

2 mM

5 mM

None (control) PMSF EDTA b-mercaptoethanol

100 20.37 72.83 100

100 2.52 60.32 97.32

Residual activity (%)

Purified enzyme was pre-incubated with various enzyme inhibitors for 30 min at 4 °C and the remaining activity was determined with azocasein substrate at pH 7.0 and 55 °C. Enzyme activity measured in the absence of any inhibitor was taken as 100%. ß 2014 WILEY-VCH Verlag GmbH & Co. KGaA,Weinheim

Metal ions

5 mM

10 mM

None (control) Ca2þ Cu2þ Mn2þ Mg2þ Zn2þ Naþ Kþ

100 105.82 1.61 90.52 110.32 5.82 103.65 98.96

100 119.31 1.41 81.37 96.55 7.37 105.17 94.82

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Figure 6. Degree of hydrolysis (DH) of fish (S. notata) protein extract during hydrolysis with protease from P. digitatum at 5:1 (U mg1) enzyme/protein substrates ratio.

tetrameric form of the P.dig-protease protein. The native apparent molecular weight is higher than that reported for most of serine proteases whose molecular weight range generally between 18 and 35 kDa [4] with a few exceptions. The highest molecular weight of 124 kDa for protease from Aspergillus fumigates is reported by Wang et al. [20]. The Km and Vmax values determined through a Lineweaver–Burk plot were 7.31 mg ml1 and 322.5 U ml1, respectively, which is in the range, as reported in literature [21]. The results suggested requiring more substrate for saturation, though hydrolysis is more active (high Vmax). The observed high Vmax suggested that P.dig-protease will be good to be used in controlled proteolysis producing bioactive peptides. The enzyme showed good activity over a wide temperature range, with an optimum temperature at 55 °C and optimum pH of 7.0, which may be beneficial for potential application. These findings are in accordance

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with the results reported by Ogundero and Osunlaja [22] concerning an Aspergillus clavatus protease, which had a maximum activity at pH 7.8. However, our results differ from other fungi proteases such as Aspergillus nidulans [23], Aspergillus parasiticus [24] and A. clavatus CCT2759 [25] showing pH optima between 8.0 and 9.5. Proteases from fungi show similar results for optimum temperature such as 45 °C for A. tamarii [19] and 50 °C for Fusarium culmorum [26]. It is well known that the presence of high salt concentrations destabilizes ion pairs and salt bridges and alters electrostatic interactions between charged amino acids, leading to enzyme denaturation. The effect of salt on P.dig-protease activity and stability confirmed the potential application of this enzyme in laundry detergents at neutral pH as P.dig-protease is stable at high salt concentrations. P.dig-protease was stable in the presence of non-ionic surfactants (Tween 20 and Triton X-100) but inhibited at a concentration of 5% w/v SDS. This is a desirable characteristic because salt is used as a core component in granulation of the protease prior to its addition to detergents. Complete inhibition of protease activity by PMSF indicates that the enzyme is a serine protease with a serine residue in its active site. PMSF is known to sulfonate the essential serine residue on the active site of the enzyme resulting in the complete loss of enzyme activity [27]. The decrease in activity by EDTA reveals the requirement of metal ion(s) for the enzymatic activity because EDTA removes metal ion(s) through chelation. The inhibition of the protease by both PMSF and EDTA was also reported for a serine alkaline protease from Bacillus licheniformis RSP-09-37 [28]. P.dig-protease activity was greatly affected in the presence of metal ions Zn2þ and Cu2þ. These findings

Figure 7. (A) 1,1-diphenyl-2-picrylhydrazyl free radical-scavenging activity of SNH obtained by treatment with P.dig-protease. (B) ACE inhibitory activity of SNH at different concentrations. ß 2014 WILEY-VCH Verlag GmbH & Co. KGaA,Weinheim

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are similar to those of Hajji et al. [29] who reported that the protease from A. clavatus ES1 was inhibited in the presence of 5.0 mM Zn2þ and Cu2þ. Other proteases were also inhibited in the presence of Cu2þ [3, 30]. This metal can react with cysteines promoting their oxidation and formation of cystine (Cys–Cys) [31], probably affecting enzyme structure and/or the access to the active site. P.dig-protease was then used for controlled hydrolysis of S. notata protein extract. Hydrolysis curve of S. notata is similar to other hydrolytic curves that were reported for the enzymatic hydrolysis of pacific whiting solid wastes [32], yellow stripe trevally [33], and salmon byproducts [34]. Results obtained after hydrolysis of fish proteins indicate that the enzyme can be effective in the production of antioxidant and ACE inhibitor peptides. Recently, a number of studies have demonstrated that peptides derived from different marine protein hydrolysates act as potential antioxidants and have been isolated from marine organisms such as sardinelle [35], mackerel [36], yellow stripe trevally [33], and yellow fin sole [37]. During hydrolysis, a wide variety of larger, medium, and smaller peptides are generated, depending on enzyme specificity. The amino acid composition and sequence, the size and the configuration are reported to be the main factors influencing on the antioxidant properties of proteins and their hydrolysates [38]. Several studies have also reported that peptides from various marine sources exhibited ACE inhibitory activity [39–41]. The obtained results of ACE inhibitory activity indicate that hydrolysis is necessary to breakdown fish muscle proteins to release active peptides since no activity was detected with the undigested fish proteins (t ¼ 0). The IC50 value of SNH (1.35 mg ml1) is lower than those of hydrolysates from oyster, scallop, codfish skin and herring skin presenting an IC50 >10 mg ml1 [42], whereas it is higher than those of smooth hound and goby (0.13 and 0.73 mg ml1, respectively) [41, 43]. Inhibition of ACE activity is considered to be a useful therapeutic approach in the treatment of high blood pressure, since it reduces the activity of angiotensin-II and increases the level of bradykinin. The separation of the most active peptide/peptides from the hydrolysate and elucidation of their chemical structure are in progress.

Acknowledgments This work was supported by the financial project of LIPMB Laboratory, INSAT, Carthage University, Ministry of Higher Education and Scientific Research of Tunisia. The ß 2014 WILEY-VCH Verlag GmbH & Co. KGaA,Weinheim

authors acknowledge the support of Professor Mohamed Rabeh Hajlaoui, Laboratory of Plant Protection, National Institute for Agricultural Research, INRA, Tunisia (Rue Hedi Karray, 2049 Ariana, Tunisia).

Conflict of interest statement The authors declare that they have no competing interests.

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