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Characterization of acid and alkaline proteases from viscera of farmed giant catfish
Food Bioscience 6 (2014) 9 –16
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Characterization of acid and alkaline proteases from viscera of farmed giant catfish Aten Vannabuna, Sunantha Ketnawaa, Suphat Phongthaia, Soottawat Benjakulb, Saroat Rawdkuena,n a
Food Technology Program, School of Agro-Industry, Mae Fah Luang University, Muang, Chiang Rai 57100, Thailand Department of Food Technology, Faculty of Agro-Industry, Prince of Songkla University, Hat Yai, Songkhla 90112, Thailand
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art i cle i nfo
ab st rac t
Article history:
This work aimed to characterize acid and alkaline proteases extracted from farmed giant
Received 29 July 2013
catfish (Pangasianodon gigas) viscera by using the aqueous two-phase system (ATPS).
Received in revised form
Determinations of optimum pH and pH stability, optimum temperature and thermal
25 December 2013
stability, salt stability and hydrolysis activities against bovine muscle protein and gelatin
Accepted 15 January 2014
were performed. ATPS consisting of 20% polyethylene glycol (PEG1500)–15% MgSO4 or 15%
Keywords:
optimum pH and temperature for acid protease was 3.0 and 40 1C, while alkaline protease
PEG2000–15% NH3C6H5O7 was used for acid and alkaline proteases extraction. The Aqueous two-phase system
was 9.0 and 60 1C. High pH stability of the enzymes was found in the ranges of 1.0–5.0 and
Fish viscera
8.0–12.0 for acid and alkaline proteases, respectively. About 40 and 60% activities reduction
Giant catfish
of acid and alkaline proteases were observed when incubated at 90 1C for 30 min.
Hydrolysate
In addition, 0.5% NaCl addition decreased 450% of total enzyme activities. Hydrolytic
Proteases
activities of the acid and alkaline proteases against bovine muscle protein and gelatin were in the concentration dependent manner as clearly indicated by SDS-PAGE. The results showed that the acid and alkaline proteases obtained from farmed giant catfish viscera could be useful for food protein hydrolysate production. & 2014 Elsevier Ltd. All rights reserved.
1.
Introduction
The value of fishery processing by-products, specifically as a source of enzymes, can be increased by using such starting materials in different disciplines, such as medicine, cosmetics, food, wastewater treatment, and pharmaceutical industries. Digestive enzymes of marine fish have been studied widely, but information on freshwater fish enzymes is limited. The most important proteolytic enzymes in the viscera of fish and aquatic invertebrates are aspartic protease
n
Corresponding author. Tel.: þ66 53 916752; fax: þ66 53 916739. E-mail address: [email protected] (S. Rawdkuen).
http://dx.doi.org/10.1016/j.fbio.2014.01.001 2212-4292 & 2014 Elsevier Ltd. All rights reserved.
(pepsin) and serine proteases (trypsin, chymotrypsin, collagenase and elastase) (Simpson, 2000). Proteases comprise the class of enzymes most used worldwide, accounting for 60% of the world's total enzyme production (Gupta, Beg, & Larenz, 2002). Some proteases have been explored as food processing aids and as reducers of stick-water viscosity in fishmeal processing (Castillo-Yanez, Pavheco-Aguilar, Garcia-Carreno, & Navarrete-Del Toro, 2004). Recently, there has been increasing demand for proteolytic enzymes in the pharmaceutical and food biotechnology industries.
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Food Bioscience 6 (2014) 9 –16
Chiang Rai has one of the highest farmed freshwater fish production capacities in Thailand. Giant catfish is the chief species that is captured locally. Most farm-raised giant catfish are sold raw to restaurants, and in the near future, fish farmers hope to export its meat to other countries, especially in Asia and Europe. With an increase in fish processing, a large amount of internal organs and by-product will be generated. During the filleting of this fish, 50–65% of the body is discarded as processing by-products, which are environmental pollutants. Utilization of these by-products could make more profit to the producer. There have been many reports about the isolation of proteolytic enzymes from fish viscera by various separation techniques, especially various types column chromatography. Most of those operations are time consuming, difficult to scale up, involve expensive reagents, and require technical skill. Partitioning in an aqueous two-phase system (ATPS) or three-phase partitioning (TPP) have shown to be powerful for separating and partial purifying the proteases from farmed giant catfish viscera (Rawdkuen, Vanabun, & Benjakul, 2012; Ketnawa, Benjakul, Ling, Martinez-Alvarez, & Rawdkuen, 2013). ATPS and TPP constitute a favorable method to separate and purify mixtures of proteins; it is fast and economical; and processes are easy to implement (Farruggia, Porfiri, Pico, & Romanini, 2011; Rawdkuen et al., 2012). Biochemical characteristics of the proteases from farmed giant catfish have been studied inadequately, in spite of being the important fishery product of Chiang Rai. Therefore, this study aimed to characterize the isolated acid and alkaline proteases from the viscera of farmed giant catfish.
2.
Materials and methods
2.1.
Materials
L-Cysteine,
sodium dodecyl sulfate (SDS), and bovine serum albumin (BSA) were obtained from Fluka (Buchs, Switzerland). Beta-mercaptoethanol (βME), Coomassie Brilliant Blue G-250, and casien were purchased from Sigma Chemical Co. (St. Louis, MO, USA). N,N,N0 ,N0 -tetramethyl ethylene diamine (TEMED) was purchased from Bio-Rad Laboratories (Hercules, CA, USA). Trichloroacetic acid (TCA), hydrochloric acid, tris(hydroxymethyl)-aminomethane, and other chemicals with analytical grade were obtained from Merck (Darmstadt, Germany). Alive farmed giant catfish (25–30 kg/fish) were catched from the artificial pond of Jarun farm, Phan District, Chiang Rai Province, Thailand. Then they were killed by putting in ice box before subjected to eviscerate by hand. Viscera of farmed giant catfish were collected, put in plastic bag and covered with ice and then transported to the laboratory at Mae Fah Luang University, Chiang Rai within 60 min.
2.2.
Preparation of crude enzyme extract
Crude enzyme extract was prepared according to the method of Castillo-Yanez et al. (2004) with a slight modification. Viscera from farmed giant catfish were homogenized for
2 min with extraction buffers (10 mM Citrate/HCl pH 3.0 for acid protease or 10 mM Tris–HCl pH 8.0, 10 mM CaCl2 for alkaline protease in the ratio of 1:5 (w/v). The mixture was then centrifuged at 10,000g for 10 min at 4 1C. The pellet was discarded and the supernatant was collected and used as “crude enzyme extract” with the specific activity of 0.60 and 392.91 units/mg proteins for acid and alkaline proteases, respectively.
2.3.
Aqueous two-phase system
The ATPS was prepared in 10-ml centrifuge tubes according to the method of Nalinanoon, Benjakul, Visessanguan, and Kishimura (2009). Acid protease was extracted by using ATPS consisting of 20% polyethylene glycol (PEG1500)–15% MgSO4, while alkaline protease was extracted by using the system consist of 15% PEG2000–15% NH3C6H5O7. Fifty percentage of crude enzyme extract was used in the ATPS. Distilled water was used to adjust the system to obtain the final weight of 8 g. The mixture was mixed thoroughly for 3 min using vortex mixer. Phase separation was achieved by centrifugation at 2000g for 10 min at 4 1C. The top phase was carefully separated using a Pasteur pipette, collected and then dialysed against 100 volumes of distilled water for 12 h before used for further experiments. The specific activity of the top phase of enzyme enriched fraction was 1.29 and 1767.23 units/mg proteins with the purity of 2.16 and 4.35-fold for acid and alkaline proteases, respectively.
2.4.
Proteolytic activity determinations
Acid protease activity against acid-denatured bovine hemoglobin was determined at pH 3.0 and 37 1C, according to the method of Wu et al. (2009). Briefly, 50 ml of appropriately diluted enzyme sample was mixed with 350 ml of 0.25 M HClsodium acetate buffer (pH 3.0), 100 ml of 2.0% acid-denatured bovine hemoglobin was then added to the mixture to initiate the reaction. After incubation at 37 1C for 30 min, the reaction was terminated by addition of 500 ml of 8.0% TCA and then centrifuged at 8000 g for 10 min. The absorbance of the supernatant was measured at 280 nm using a spectrophotometer. One unit of enzymatic activity was defined as the amount of acid protease that catalyzes an increase of absorbance of 1.0 unit per minute at 280 nm under the activity assay conditions. Alkaline protease activity was determined by using casein as a substrate according to the method of Rawdkuen, Chaiwut, Pintathong, and Benjakul (2010) with a slight modification. An alkaline protease sample of 500 ml was mixed with 500 ml of 2% (w/v) casein in 0.1 M Tris–HCl (pH 8.0). The reaction was started by incubation the mixture at 37 1C for 10 min. The reaction was stopped by adding 500 ml of 5% TCA. After centrifugation at 10,000g for 10 min, the absorption of the soluble peptides in supernatant was measured at 280 nm. One of caseinolytic activity units is defined as the amount of enzymes needed to produce an increment of 0.01 absorbance unit per minute at the assayed condition.
Food Bioscience 6 (2014) 9 –16
2.5.
pH profile and stability
The pH profile was determined by assaying the proteolytic activity in different pH (1.0–12.0). The buffer of 100 mM Glycine–HCl (1.0–3.0), Na-acetate (4.0–6.0), Tris–HCl (7.0–9.0) and Glycine–-NaOH (10.0–12.0) were used. The effect of pH on enzyme stability was evaluated by incubating the enzyme enriched fraction at various pH values (1.0–12.0) using different buffers as mentioned above for 30 min. The residual activity after incubation was determined, calculated and reported compared with condition showed the highest value (¼ 100% activity).
2.6.
Thermal profile and stability
The proteolytic activity at different temperatures (30–90 1C) was determined. The assay was performed as mentioned above by using pH 3.0 for acid protease activity determination and 8.0 for alkaline protease activity determination. The thermal stability of proteases was determined by incubating enzyme extract for 1, 3, 5, 10, 15, 20, 30, 40, 50, and 60 min at 90 1C. Aliquots were withdrawn at desired time intervals to test the remaining activity under standard conditions. The non-heated enzyme was considered to be the control (100% activity)
2.7.
11
Effect of NaCl on enzyme activity
The effect of NaCl on the enzyme activity was also determined. NaCl was added into the mixture of reaction assay to obtain the final concentration of 0–2.5% (w/v). The residual activity was determined as mentioned above.
2.8.
Effect of isolated enzymes on proteins hydrolysis
Extracted acid and alkaline proteases were used to hydrolyze bovine muscle protein. The reaction was started by incubating the ground muscle sample (2 g) completely mixed with enzyme at different concentrations (0.17–7.6 units for acid protease at 40 1C for 10 min and 100–5000 units of alkaline protease at 60 1C for 10 min). Pattern of peptides generated was determined by SDS-PAGE using 10% separating gel and 4% stacking gel according to the method of Laemmli (1970). Extracted acid and alkaline proteases were also used to hydrolyze gelatin from farmed giant catfish skin. The reaction was started by incubating the gelatin solution (0.1 g/ml) with enzymes at different concentrations (0.003–0.41 units for acid protease at 40 1C for 10 min and 0.5–30 units for alkaline protease at 60 1C for 10 min) and then terminated by submerging the mixture in boiling water for 3 min. Pattern of peptides generated was determined by SDS-PAGE using 7.5% separating gel and 4% stacking gel.
Fig. 1 – pH profiles of acid (a) and alkaline protease (c) and stability of acid (b) and alkaline protease (d) at different pHs.
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2.9.
Food Bioscience 6 (2014) 9 –16
Statistical analyses
The experiment was carried out in triplicate using different three lots of samples. Completely randomized design was used throughout the study. The obtained data was statistically analyzed with the SPSS program for Windows (SPSS version 11.5, SPSS Inc., Chicago, IL, USA). Duncan's multiplerange test was used to compare the difference between means. The accepted level of significance for all comparisons was Po0.05. Experiments were conducted in triplicate.
3.
Results and discussion
3.1.
pH profile and stability
The effect of pH on the activity and stability of acid and alkaline proteases from the viscera of farmed giant catfish was determined over a pH range of 1.0–12.0 as shown in Fig. 1. The optimum pH for acid protease was pH 3.0 while pH 9.0 was optimal for alkaline protease. Optimum pH values between 2.0 and 4.0 have been reported for pepsin activities of fish species such as smooth hound (Bougatef et al., 2009), sea beam (Zhou, Fu, Zhang, Su, & Cao, 2007), African coelacanth (Tanji et al., 2007) albacore tuna (Nalinanoon et al., 2009) and Sardinelle (Ben Kahled, Bellaaj, Hmidet, Jellouli, & Nasri, 2011; Bougatef, Souissi, Fakhfakh, & Nasri, 2007). Lakshman, Toyokawa, Toyama, Taira, and Yasuda (2012) reported acid protease from Monascus pilosus optimum pH was 2.5–3.0. Rao, Mizutani, Hirano, Masaki, and Iefuji (2011) reported acid protease from basidiomycetous yeast Cryptococcus optimum pH was 5.0. Many researchers reported that the pH between 8.0 and 10.0 was suitable for enzyme activities of some fish species such as Giant Amazonian fish pirarucu (Freitas-Júnior et al., 2012), hybrid catfish (Klomklao, Benjakul, Kishimura, & Chaijan, 2011), zebra blenny (Ktari et al., 2012) and Tunisian barbel (Sila et al., 2012). The loss of enzyme activity at pH values outside optimum pH is probably due to protein conformational changes caused by repulsion of charges (Klomklao, Kishimura, Nonami, & Benjakul 2009). Under outside optimum pH conditions, their surface charge distributions and even conformations were changed and enzymes could not bind the substrate properly (Benjakul & Morrisey, 1997). High pH stability (495%) of the enzyme was found in the ranges of pH 1.0–5.0 and 8.0–12.0 for acid (b) and alkaline (d) proteases, respectively. Decreased protease activity for about 15–20% was observed at pH value over 6.0 for acid protease and pH value below 7.0 for alkaline protease. The pH stability for acid protease of albacore tuna was pH range 2.0–5.0 (Nalinanoon et al., 2009) and pH stability range 3.0–7.0 for acid protease of basidiomycetous yeast Cryptococcus (Rao et al., 2011). The pH stability for alkaline protease was range 6.0–12.0 such as zebra blenny (Ktari et al., 2012), Tunisian barbel (Sila et al., 2012) and hybrid catfish (Klomklao et al., 2011). The differences in pH stability indicated the different molecular properties including bonding stabilizing the structure as well as enzyme conformation amongst various species and anatomical location (Klomklao, Kishimura, Yabe, & Benjakul, 2007).
3.2.
Thermal profile and stability
The effect of temperature on the acid and alkaline proteases activity and stability was determined by assaying enzyme activity at different temperatures at optimum pH and is shown in Fig. 2. The optimum temperature for acid protease was 40 οC (a) similar to those from other fish species such as pepsins of Sardinelle Ben Kahled et al., 2011, smooth hound (Bougatef et al., 2009), and European eel (Wu et al., 2009). The optimal temperature of alkaline (c) protease was 60 οC. The optimal temperature was similar to that of trypsin from pyloric caeca of Chinook salmon (Klomklao et al., 2011), and Japanese seabass (Cai et al., 2011). Decreasing in activity by 40 and 60% for acid and alkaline proteases was observed when incubated at 90 1C for 30 min. Klomklao et al. (2011) concluded that unfolding of the enzyme molecule during thermal treatment, resulted in inactivation of enzyme activity. Kim, Meyers, Pyeun, and Goldber (1994) also suggested that to increase thermal stability of proteins, the interaction between proteins molecules by any bond such as hydrophobic interaction or disulfide bonds need to be initiated. This finding also similar to the study of Klomklao et al. (2009) who reported that enzyme obtained from fish was less thermal stable when compared to bovine that may caused by lower number of interior interactions or chemical bonding, especially intra-molecular disulfide bond.
3.3.
Effect of NaCl on enzyme activity
Effect of NaCl on acid and alkaline proteases activity is depicted in Fig. 3. Relative enzyme activity was decreased more than 50% for both acid and alkaline proteases when 0.5% (w/v) NaCl was added. The slightly decreased protease activity was observed as NaCl concentration increased. Ben Kahled et al. (2011) reported the relative activity of acid protease from Sardinelle at 10% NaCl was approximately 20%. Gildberg, Olsen, and Bjarnason (1990) also reported that pepsin activity from Atlantic cod was significant decreased when added NaCl concentration at the level of 5% and 10%. Trypsin from Giant Amazonian fish pirarucu activity decreased with increasing NaCl concentration, showing 65%, 51% and 42% of residual activity at concentrations of 5%, 10% and 15% NaCl (w/v), respectively (Freitas-Júnior et al., 2012). Trypsin activity from hybrid catfish continuously decreased gradually with increasing NaCl (Klomklao et al., 2011). The decrease in activity might be due to the denaturation of enzymes (Ben Kahled et al., 2011; Klomklao et al., 2011). It could be described by the salting out phenomenon. An increase in ionic strength causes a reduction in enzyme activity by an enhanced hydrophobic–hydrophobic interaction between protein chains and the competition for water of ionic salts, leading to the induced enzyme precipitation (Klomklao et al., 2009). Proteases from giant catfish may have not potential in accelerating the hydrolysis of high-salt products such as fish sauce.
3.4.
Effect of isolated enzymes on proteins hydrolysis
The protein patterns of bovine muscle and gelatin treated with acid and alkaline proteases at different concentrations is
Food Bioscience 6 (2014) 9 –16
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Fig. 2 – Thermal profiles (30–90 1C) of acid (a) and alkaline (c) proteases and thermal stability of acid (b) and alkaline (d) proteases at 90 1C for 0–60 min.
shown in Fig. 4. The myosin heavy chain (MHC) and actin (AC) are the major proteins in muscle foods as shown in lane C (muscle protein without enzyme addition). Degradation of MHC and AC were observed when the unit of enzyme increased. A disappearance of MHC of bovine muscle was observed when addition levels were 3.5 units and 3000 units for acid and alkaline proteases, respectively. Cao et al. (2011) reported that most muscular proteins of fish and shrimp could be degraded by pepsins from Japanese seabass and they play important roles in food digestion. They also reported that trypsins from the hepatopancreas of Japanese seabass are active in hydrolyzing native protein substrates to some degrees while complete decomposition of food proteins for nutrition absorbance should be expected by coupling trypsins and chymotrypsins together with other proteinases such as pepsin (Cao et al., 2011) and amino peptidases (Wu et al., 2009). Nalinanon, Benjakul, Kishimura, and Shahidi (2011) reported that protein hydrolysate from the muscle of ornate threadfin bream produced by skipjack tuna pepsin can be used as a promising source of functional peptides with antioxidant properties. Walnut proteins were hydrolyzed separately using three different proteases to obtain antioxidant peptides (Chen, Yanga, Sun, Niua, & Liu, 2012). Klomklao, Kishimura, and Benjakul (2013) reported that protein hydrolysate from toothed pony fish muscle produced
by hybrid catfish protease contained a high amount of essential amino acids (48.22%) and had arginine and lysine as the dominant amino acids. The smooth hound muscle protein hydrolysate produced by the low molecular weight alkaline proteases generally showed a greater antioxidative activity (Bougatef et al., 2009). Pattern of hydrolyzed gelatin were clearly observed with increasing units of enzymes. Without enzyme addition to the farmed giant catfish skin gelatin (Lane C, Fig. 4C & D), high molecular weight protein components were observed at the top of separating gel. These bands intensity were decreased in concurrent with the increasing of enzyme concentrations. It can be concluded that the reduction of high molecular weight proteins was hydrolyzed by the added enzymes. Complete hydrolysis of major protein components in gelatin was observed with the addition of acid protease at 0.15 units and alkaline protease at 15 units. From the hydrolytic patterns, the acid and alkaline proteases from farmed giant catfish viscera can further be used for gelatin hydrolysate production. Lin, Li and Li (2012) reported that hydrolysate of squid skin gelatin obtained by treatment with pepsin was a good source of peptides with ACE-inhibitory activity and had an antihypertensive effect by oral administration. Gelatin protein from Nile tilapia was hydrolyzed using alcalase, pronase E, trypsin and pepsin acts as a candidate against
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Food Bioscience 6 (2014) 9 –16
Fig. 3 – Effect of NaCl addition on the activity of acid (a) and alkaline (b) proteases.
Fig. 4 – Effect of acid (a, c) and alkaline proteases (b, d) on proteins degradation at different enzyme concentrations. MHC: myosin heavy chain, AC: actin, M: Molecular weight markers, C: Control protein samples, and T: isolated enzymes. Numbers represent the unit of enzyme application.
oxidative stress and could be used as a potential functional food ingredient (Kim, Ngo, Qian, Ryu, & Park, 2010). Alemán et al. (2011) reported that gelatin obtained from giant squid inner and outer tunics was hydrolyzed by seven commercial proteases to produce bioactive hydrolysates. Hydrolysates derived from gelatin using alcalase combined with pyloric caeca extract from bigeye snapper showed the high 2,2azinobis (3-ethyl-benzothiazoline-6-sulfonic acid) radical
scavenging activity (Phanturat, Benjakul, Visessanguan, & Roytrakul, 2010).
4.
Conclusion
Acid and alkaline proteases isolated from viscera of farmed giant catfish showed high activity in the acid condition and
Food Bioscience 6 (2014) 9 –16
alkaline condition for acid and alkaline proteases, respectively. In addition, they exhibited the maximal activity at 40 1C for acid protease and 60 1C for alkaline protease. However, the enzymes were not stable at high temperature and high NaCl concentration. The isolated enzymes could be used as a biotechnological alternative for gelatin hydrolysate production and other applications.
Acknowledgments The authors would like to thank Mae Fah Luang University, Office of the Higher Education Commission and the Thailand Research Fund under the TRF Senior Research Scholar program for financial support.
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Characterization of acid and alkaline proteases from viscera of farmed giant catfish Aten Vannabuna, Sunantha Ketnawaa, Suphat Phongthaia, Soottawat Benjakulb, Saroat Rawdkuena,n a
Food Technology Program, School of Agro-Industry, Mae Fah Luang University, Muang, Chiang Rai 57100, Thailand Department of Food Technology, Faculty of Agro-Industry, Prince of Songkla University, Hat Yai, Songkhla 90112, Thailand
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art i cle i nfo
ab st rac t
Article history:
This work aimed to characterize acid and alkaline proteases extracted from farmed giant
Received 29 July 2013
catfish (Pangasianodon gigas) viscera by using the aqueous two-phase system (ATPS).
Received in revised form
Determinations of optimum pH and pH stability, optimum temperature and thermal
25 December 2013
stability, salt stability and hydrolysis activities against bovine muscle protein and gelatin
Accepted 15 January 2014
were performed. ATPS consisting of 20% polyethylene glycol (PEG1500)–15% MgSO4 or 15%
Keywords:
optimum pH and temperature for acid protease was 3.0 and 40 1C, while alkaline protease
PEG2000–15% NH3C6H5O7 was used for acid and alkaline proteases extraction. The Aqueous two-phase system
was 9.0 and 60 1C. High pH stability of the enzymes was found in the ranges of 1.0–5.0 and
Fish viscera
8.0–12.0 for acid and alkaline proteases, respectively. About 40 and 60% activities reduction
Giant catfish
of acid and alkaline proteases were observed when incubated at 90 1C for 30 min.
Hydrolysate
In addition, 0.5% NaCl addition decreased 450% of total enzyme activities. Hydrolytic
Proteases
activities of the acid and alkaline proteases against bovine muscle protein and gelatin were in the concentration dependent manner as clearly indicated by SDS-PAGE. The results showed that the acid and alkaline proteases obtained from farmed giant catfish viscera could be useful for food protein hydrolysate production. & 2014 Elsevier Ltd. All rights reserved.
1.
Introduction
The value of fishery processing by-products, specifically as a source of enzymes, can be increased by using such starting materials in different disciplines, such as medicine, cosmetics, food, wastewater treatment, and pharmaceutical industries. Digestive enzymes of marine fish have been studied widely, but information on freshwater fish enzymes is limited. The most important proteolytic enzymes in the viscera of fish and aquatic invertebrates are aspartic protease
n
Corresponding author. Tel.: þ66 53 916752; fax: þ66 53 916739. E-mail address: [email protected] (S. Rawdkuen).
http://dx.doi.org/10.1016/j.fbio.2014.01.001 2212-4292 & 2014 Elsevier Ltd. All rights reserved.
(pepsin) and serine proteases (trypsin, chymotrypsin, collagenase and elastase) (Simpson, 2000). Proteases comprise the class of enzymes most used worldwide, accounting for 60% of the world's total enzyme production (Gupta, Beg, & Larenz, 2002). Some proteases have been explored as food processing aids and as reducers of stick-water viscosity in fishmeal processing (Castillo-Yanez, Pavheco-Aguilar, Garcia-Carreno, & Navarrete-Del Toro, 2004). Recently, there has been increasing demand for proteolytic enzymes in the pharmaceutical and food biotechnology industries.
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Chiang Rai has one of the highest farmed freshwater fish production capacities in Thailand. Giant catfish is the chief species that is captured locally. Most farm-raised giant catfish are sold raw to restaurants, and in the near future, fish farmers hope to export its meat to other countries, especially in Asia and Europe. With an increase in fish processing, a large amount of internal organs and by-product will be generated. During the filleting of this fish, 50–65% of the body is discarded as processing by-products, which are environmental pollutants. Utilization of these by-products could make more profit to the producer. There have been many reports about the isolation of proteolytic enzymes from fish viscera by various separation techniques, especially various types column chromatography. Most of those operations are time consuming, difficult to scale up, involve expensive reagents, and require technical skill. Partitioning in an aqueous two-phase system (ATPS) or three-phase partitioning (TPP) have shown to be powerful for separating and partial purifying the proteases from farmed giant catfish viscera (Rawdkuen, Vanabun, & Benjakul, 2012; Ketnawa, Benjakul, Ling, Martinez-Alvarez, & Rawdkuen, 2013). ATPS and TPP constitute a favorable method to separate and purify mixtures of proteins; it is fast and economical; and processes are easy to implement (Farruggia, Porfiri, Pico, & Romanini, 2011; Rawdkuen et al., 2012). Biochemical characteristics of the proteases from farmed giant catfish have been studied inadequately, in spite of being the important fishery product of Chiang Rai. Therefore, this study aimed to characterize the isolated acid and alkaline proteases from the viscera of farmed giant catfish.
2.
Materials and methods
2.1.
Materials
L-Cysteine,
sodium dodecyl sulfate (SDS), and bovine serum albumin (BSA) were obtained from Fluka (Buchs, Switzerland). Beta-mercaptoethanol (βME), Coomassie Brilliant Blue G-250, and casien were purchased from Sigma Chemical Co. (St. Louis, MO, USA). N,N,N0 ,N0 -tetramethyl ethylene diamine (TEMED) was purchased from Bio-Rad Laboratories (Hercules, CA, USA). Trichloroacetic acid (TCA), hydrochloric acid, tris(hydroxymethyl)-aminomethane, and other chemicals with analytical grade were obtained from Merck (Darmstadt, Germany). Alive farmed giant catfish (25–30 kg/fish) were catched from the artificial pond of Jarun farm, Phan District, Chiang Rai Province, Thailand. Then they were killed by putting in ice box before subjected to eviscerate by hand. Viscera of farmed giant catfish were collected, put in plastic bag and covered with ice and then transported to the laboratory at Mae Fah Luang University, Chiang Rai within 60 min.
2.2.
Preparation of crude enzyme extract
Crude enzyme extract was prepared according to the method of Castillo-Yanez et al. (2004) with a slight modification. Viscera from farmed giant catfish were homogenized for
2 min with extraction buffers (10 mM Citrate/HCl pH 3.0 for acid protease or 10 mM Tris–HCl pH 8.0, 10 mM CaCl2 for alkaline protease in the ratio of 1:5 (w/v). The mixture was then centrifuged at 10,000g for 10 min at 4 1C. The pellet was discarded and the supernatant was collected and used as “crude enzyme extract” with the specific activity of 0.60 and 392.91 units/mg proteins for acid and alkaline proteases, respectively.
2.3.
Aqueous two-phase system
The ATPS was prepared in 10-ml centrifuge tubes according to the method of Nalinanoon, Benjakul, Visessanguan, and Kishimura (2009). Acid protease was extracted by using ATPS consisting of 20% polyethylene glycol (PEG1500)–15% MgSO4, while alkaline protease was extracted by using the system consist of 15% PEG2000–15% NH3C6H5O7. Fifty percentage of crude enzyme extract was used in the ATPS. Distilled water was used to adjust the system to obtain the final weight of 8 g. The mixture was mixed thoroughly for 3 min using vortex mixer. Phase separation was achieved by centrifugation at 2000g for 10 min at 4 1C. The top phase was carefully separated using a Pasteur pipette, collected and then dialysed against 100 volumes of distilled water for 12 h before used for further experiments. The specific activity of the top phase of enzyme enriched fraction was 1.29 and 1767.23 units/mg proteins with the purity of 2.16 and 4.35-fold for acid and alkaline proteases, respectively.
2.4.
Proteolytic activity determinations
Acid protease activity against acid-denatured bovine hemoglobin was determined at pH 3.0 and 37 1C, according to the method of Wu et al. (2009). Briefly, 50 ml of appropriately diluted enzyme sample was mixed with 350 ml of 0.25 M HClsodium acetate buffer (pH 3.0), 100 ml of 2.0% acid-denatured bovine hemoglobin was then added to the mixture to initiate the reaction. After incubation at 37 1C for 30 min, the reaction was terminated by addition of 500 ml of 8.0% TCA and then centrifuged at 8000 g for 10 min. The absorbance of the supernatant was measured at 280 nm using a spectrophotometer. One unit of enzymatic activity was defined as the amount of acid protease that catalyzes an increase of absorbance of 1.0 unit per minute at 280 nm under the activity assay conditions. Alkaline protease activity was determined by using casein as a substrate according to the method of Rawdkuen, Chaiwut, Pintathong, and Benjakul (2010) with a slight modification. An alkaline protease sample of 500 ml was mixed with 500 ml of 2% (w/v) casein in 0.1 M Tris–HCl (pH 8.0). The reaction was started by incubation the mixture at 37 1C for 10 min. The reaction was stopped by adding 500 ml of 5% TCA. After centrifugation at 10,000g for 10 min, the absorption of the soluble peptides in supernatant was measured at 280 nm. One of caseinolytic activity units is defined as the amount of enzymes needed to produce an increment of 0.01 absorbance unit per minute at the assayed condition.
Food Bioscience 6 (2014) 9 –16
2.5.
pH profile and stability
The pH profile was determined by assaying the proteolytic activity in different pH (1.0–12.0). The buffer of 100 mM Glycine–HCl (1.0–3.0), Na-acetate (4.0–6.0), Tris–HCl (7.0–9.0) and Glycine–-NaOH (10.0–12.0) were used. The effect of pH on enzyme stability was evaluated by incubating the enzyme enriched fraction at various pH values (1.0–12.0) using different buffers as mentioned above for 30 min. The residual activity after incubation was determined, calculated and reported compared with condition showed the highest value (¼ 100% activity).
2.6.
Thermal profile and stability
The proteolytic activity at different temperatures (30–90 1C) was determined. The assay was performed as mentioned above by using pH 3.0 for acid protease activity determination and 8.0 for alkaline protease activity determination. The thermal stability of proteases was determined by incubating enzyme extract for 1, 3, 5, 10, 15, 20, 30, 40, 50, and 60 min at 90 1C. Aliquots were withdrawn at desired time intervals to test the remaining activity under standard conditions. The non-heated enzyme was considered to be the control (100% activity)
2.7.
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Effect of NaCl on enzyme activity
The effect of NaCl on the enzyme activity was also determined. NaCl was added into the mixture of reaction assay to obtain the final concentration of 0–2.5% (w/v). The residual activity was determined as mentioned above.
2.8.
Effect of isolated enzymes on proteins hydrolysis
Extracted acid and alkaline proteases were used to hydrolyze bovine muscle protein. The reaction was started by incubating the ground muscle sample (2 g) completely mixed with enzyme at different concentrations (0.17–7.6 units for acid protease at 40 1C for 10 min and 100–5000 units of alkaline protease at 60 1C for 10 min). Pattern of peptides generated was determined by SDS-PAGE using 10% separating gel and 4% stacking gel according to the method of Laemmli (1970). Extracted acid and alkaline proteases were also used to hydrolyze gelatin from farmed giant catfish skin. The reaction was started by incubating the gelatin solution (0.1 g/ml) with enzymes at different concentrations (0.003–0.41 units for acid protease at 40 1C for 10 min and 0.5–30 units for alkaline protease at 60 1C for 10 min) and then terminated by submerging the mixture in boiling water for 3 min. Pattern of peptides generated was determined by SDS-PAGE using 7.5% separating gel and 4% stacking gel.
Fig. 1 – pH profiles of acid (a) and alkaline protease (c) and stability of acid (b) and alkaline protease (d) at different pHs.
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2.9.
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Statistical analyses
The experiment was carried out in triplicate using different three lots of samples. Completely randomized design was used throughout the study. The obtained data was statistically analyzed with the SPSS program for Windows (SPSS version 11.5, SPSS Inc., Chicago, IL, USA). Duncan's multiplerange test was used to compare the difference between means. The accepted level of significance for all comparisons was Po0.05. Experiments were conducted in triplicate.
3.
Results and discussion
3.1.
pH profile and stability
The effect of pH on the activity and stability of acid and alkaline proteases from the viscera of farmed giant catfish was determined over a pH range of 1.0–12.0 as shown in Fig. 1. The optimum pH for acid protease was pH 3.0 while pH 9.0 was optimal for alkaline protease. Optimum pH values between 2.0 and 4.0 have been reported for pepsin activities of fish species such as smooth hound (Bougatef et al., 2009), sea beam (Zhou, Fu, Zhang, Su, & Cao, 2007), African coelacanth (Tanji et al., 2007) albacore tuna (Nalinanoon et al., 2009) and Sardinelle (Ben Kahled, Bellaaj, Hmidet, Jellouli, & Nasri, 2011; Bougatef, Souissi, Fakhfakh, & Nasri, 2007). Lakshman, Toyokawa, Toyama, Taira, and Yasuda (2012) reported acid protease from Monascus pilosus optimum pH was 2.5–3.0. Rao, Mizutani, Hirano, Masaki, and Iefuji (2011) reported acid protease from basidiomycetous yeast Cryptococcus optimum pH was 5.0. Many researchers reported that the pH between 8.0 and 10.0 was suitable for enzyme activities of some fish species such as Giant Amazonian fish pirarucu (Freitas-Júnior et al., 2012), hybrid catfish (Klomklao, Benjakul, Kishimura, & Chaijan, 2011), zebra blenny (Ktari et al., 2012) and Tunisian barbel (Sila et al., 2012). The loss of enzyme activity at pH values outside optimum pH is probably due to protein conformational changes caused by repulsion of charges (Klomklao, Kishimura, Nonami, & Benjakul 2009). Under outside optimum pH conditions, their surface charge distributions and even conformations were changed and enzymes could not bind the substrate properly (Benjakul & Morrisey, 1997). High pH stability (495%) of the enzyme was found in the ranges of pH 1.0–5.0 and 8.0–12.0 for acid (b) and alkaline (d) proteases, respectively. Decreased protease activity for about 15–20% was observed at pH value over 6.0 for acid protease and pH value below 7.0 for alkaline protease. The pH stability for acid protease of albacore tuna was pH range 2.0–5.0 (Nalinanoon et al., 2009) and pH stability range 3.0–7.0 for acid protease of basidiomycetous yeast Cryptococcus (Rao et al., 2011). The pH stability for alkaline protease was range 6.0–12.0 such as zebra blenny (Ktari et al., 2012), Tunisian barbel (Sila et al., 2012) and hybrid catfish (Klomklao et al., 2011). The differences in pH stability indicated the different molecular properties including bonding stabilizing the structure as well as enzyme conformation amongst various species and anatomical location (Klomklao, Kishimura, Yabe, & Benjakul, 2007).
3.2.
Thermal profile and stability
The effect of temperature on the acid and alkaline proteases activity and stability was determined by assaying enzyme activity at different temperatures at optimum pH and is shown in Fig. 2. The optimum temperature for acid protease was 40 οC (a) similar to those from other fish species such as pepsins of Sardinelle Ben Kahled et al., 2011, smooth hound (Bougatef et al., 2009), and European eel (Wu et al., 2009). The optimal temperature of alkaline (c) protease was 60 οC. The optimal temperature was similar to that of trypsin from pyloric caeca of Chinook salmon (Klomklao et al., 2011), and Japanese seabass (Cai et al., 2011). Decreasing in activity by 40 and 60% for acid and alkaline proteases was observed when incubated at 90 1C for 30 min. Klomklao et al. (2011) concluded that unfolding of the enzyme molecule during thermal treatment, resulted in inactivation of enzyme activity. Kim, Meyers, Pyeun, and Goldber (1994) also suggested that to increase thermal stability of proteins, the interaction between proteins molecules by any bond such as hydrophobic interaction or disulfide bonds need to be initiated. This finding also similar to the study of Klomklao et al. (2009) who reported that enzyme obtained from fish was less thermal stable when compared to bovine that may caused by lower number of interior interactions or chemical bonding, especially intra-molecular disulfide bond.
3.3.
Effect of NaCl on enzyme activity
Effect of NaCl on acid and alkaline proteases activity is depicted in Fig. 3. Relative enzyme activity was decreased more than 50% for both acid and alkaline proteases when 0.5% (w/v) NaCl was added. The slightly decreased protease activity was observed as NaCl concentration increased. Ben Kahled et al. (2011) reported the relative activity of acid protease from Sardinelle at 10% NaCl was approximately 20%. Gildberg, Olsen, and Bjarnason (1990) also reported that pepsin activity from Atlantic cod was significant decreased when added NaCl concentration at the level of 5% and 10%. Trypsin from Giant Amazonian fish pirarucu activity decreased with increasing NaCl concentration, showing 65%, 51% and 42% of residual activity at concentrations of 5%, 10% and 15% NaCl (w/v), respectively (Freitas-Júnior et al., 2012). Trypsin activity from hybrid catfish continuously decreased gradually with increasing NaCl (Klomklao et al., 2011). The decrease in activity might be due to the denaturation of enzymes (Ben Kahled et al., 2011; Klomklao et al., 2011). It could be described by the salting out phenomenon. An increase in ionic strength causes a reduction in enzyme activity by an enhanced hydrophobic–hydrophobic interaction between protein chains and the competition for water of ionic salts, leading to the induced enzyme precipitation (Klomklao et al., 2009). Proteases from giant catfish may have not potential in accelerating the hydrolysis of high-salt products such as fish sauce.
3.4.
Effect of isolated enzymes on proteins hydrolysis
The protein patterns of bovine muscle and gelatin treated with acid and alkaline proteases at different concentrations is
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Fig. 2 – Thermal profiles (30–90 1C) of acid (a) and alkaline (c) proteases and thermal stability of acid (b) and alkaline (d) proteases at 90 1C for 0–60 min.
shown in Fig. 4. The myosin heavy chain (MHC) and actin (AC) are the major proteins in muscle foods as shown in lane C (muscle protein without enzyme addition). Degradation of MHC and AC were observed when the unit of enzyme increased. A disappearance of MHC of bovine muscle was observed when addition levels were 3.5 units and 3000 units for acid and alkaline proteases, respectively. Cao et al. (2011) reported that most muscular proteins of fish and shrimp could be degraded by pepsins from Japanese seabass and they play important roles in food digestion. They also reported that trypsins from the hepatopancreas of Japanese seabass are active in hydrolyzing native protein substrates to some degrees while complete decomposition of food proteins for nutrition absorbance should be expected by coupling trypsins and chymotrypsins together with other proteinases such as pepsin (Cao et al., 2011) and amino peptidases (Wu et al., 2009). Nalinanon, Benjakul, Kishimura, and Shahidi (2011) reported that protein hydrolysate from the muscle of ornate threadfin bream produced by skipjack tuna pepsin can be used as a promising source of functional peptides with antioxidant properties. Walnut proteins were hydrolyzed separately using three different proteases to obtain antioxidant peptides (Chen, Yanga, Sun, Niua, & Liu, 2012). Klomklao, Kishimura, and Benjakul (2013) reported that protein hydrolysate from toothed pony fish muscle produced
by hybrid catfish protease contained a high amount of essential amino acids (48.22%) and had arginine and lysine as the dominant amino acids. The smooth hound muscle protein hydrolysate produced by the low molecular weight alkaline proteases generally showed a greater antioxidative activity (Bougatef et al., 2009). Pattern of hydrolyzed gelatin were clearly observed with increasing units of enzymes. Without enzyme addition to the farmed giant catfish skin gelatin (Lane C, Fig. 4C & D), high molecular weight protein components were observed at the top of separating gel. These bands intensity were decreased in concurrent with the increasing of enzyme concentrations. It can be concluded that the reduction of high molecular weight proteins was hydrolyzed by the added enzymes. Complete hydrolysis of major protein components in gelatin was observed with the addition of acid protease at 0.15 units and alkaline protease at 15 units. From the hydrolytic patterns, the acid and alkaline proteases from farmed giant catfish viscera can further be used for gelatin hydrolysate production. Lin, Li and Li (2012) reported that hydrolysate of squid skin gelatin obtained by treatment with pepsin was a good source of peptides with ACE-inhibitory activity and had an antihypertensive effect by oral administration. Gelatin protein from Nile tilapia was hydrolyzed using alcalase, pronase E, trypsin and pepsin acts as a candidate against
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Fig. 3 – Effect of NaCl addition on the activity of acid (a) and alkaline (b) proteases.
Fig. 4 – Effect of acid (a, c) and alkaline proteases (b, d) on proteins degradation at different enzyme concentrations. MHC: myosin heavy chain, AC: actin, M: Molecular weight markers, C: Control protein samples, and T: isolated enzymes. Numbers represent the unit of enzyme application.
oxidative stress and could be used as a potential functional food ingredient (Kim, Ngo, Qian, Ryu, & Park, 2010). Alemán et al. (2011) reported that gelatin obtained from giant squid inner and outer tunics was hydrolyzed by seven commercial proteases to produce bioactive hydrolysates. Hydrolysates derived from gelatin using alcalase combined with pyloric caeca extract from bigeye snapper showed the high 2,2azinobis (3-ethyl-benzothiazoline-6-sulfonic acid) radical
scavenging activity (Phanturat, Benjakul, Visessanguan, & Roytrakul, 2010).
4.
Conclusion
Acid and alkaline proteases isolated from viscera of farmed giant catfish showed high activity in the acid condition and
Food Bioscience 6 (2014) 9 –16
alkaline condition for acid and alkaline proteases, respectively. In addition, they exhibited the maximal activity at 40 1C for acid protease and 60 1C for alkaline protease. However, the enzymes were not stable at high temperature and high NaCl concentration. The isolated enzymes could be used as a biotechnological alternative for gelatin hydrolysate production and other applications.
Acknowledgments The authors would like to thank Mae Fah Luang University, Office of the Higher Education Commission and the Thailand Research Fund under the TRF Senior Research Scholar program for financial support.
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