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Biochemical properties of two isoforms of trypsin purified from the Intestine of skipjack tuna (Katsuwonus pelamis)
Abstract
Two trypsins (A and B) from the intestine of skipjack tuna (Katsuwonus pelamis) were purified by Sephacryl S-200, Sephadex G-50 and DEAE-cellulose with a 177- and 257-fold increase in specific activity and 23% and 21% recovery for trypsin A and B, respectively. Purified trypsins revealed a single band on native-PAGE. The molecular weights of both trypsins were 24 kDa as estimated by size exclusion chromatography and SDS–PAGE. Trypsin A and B exhibited the maximal activity at 55 °C and 60 °C, respectively, and had the same optimal pH at 9.0. Both trypsins were stable up to 50 °C and in the pH range from 6.0 to 11.0. Both trypsin A and B were stabilised by calcium ion. Activity of both trypsins continuously decreased with increasing NaCl concentration (0–30%) and were inhibited by the specific trypsin inhibitors – soybean trypsin inhibitor and N-p-tosyl-l-lysine chloromethyl ketone. Apparent Km and Kcat of trypsin A and B were 0.22–0.31 mM and 69.5–82.5 S−1, respectively. The N-terminal amino acid sequences of the first 20 amino acids of trypsin A and B were IVGGYECQAHSQPPQVSLNA and IVGGYECQAHSQPPQVSLNS, respectively.
... Hence, a better understanding of the properties of trypsin is necessary to generate valuable information for protein degradation in the fish digestive tract. The characterization of trypsin, especially its physicochemical and biochemical properties, has been thoroughly studied from the intestine of various fish including grass carp (Ctenopharyngodon idellus), spotted goatfish (Pseudupeneus maculatus), grey triggerfish (Balistes capriscus), skipjack tuna (Katsuwonus pelamis), smooth hound (Mustelus mustelus), and Brazilian flounder (Paralichthys orbignyanus) [23][24][25][26][27][28]. The activity of trypsin among several sturgeon species has been mostly studied during larval ontogeny in the members of the genus Acipenser such as A. transmontanus [29], A. fulvescens [30], A. oxyrinchus [31,32], A. baerii [33], A. persicus [34], A. nacarii [35,36], A. stellatus [37], and genus Huso such as H. huso [38]. ...
... However, an obvious decrease in the trypsin activity of both ICEs was observed at temperatures above 60 • C, probably due to thermal inactivation of this enzyme caused by protein unfolding [40]. Similar optimum temperature (55 • C) was recorded for trypsins in skipjack tuna [26], gilthead seabream [46], sardinelle [57], and silver mojarra [62]. Optimum temperature of trypsin for both sturgeon species was higher than values reported for cold-water fish such as Atlantic cod (Gadus morhua) [63], grey triggerfish [25], lane snapper (Lutjanus synagris) [64], and Japanese sea bass (L. ...
... However, an obvious decrease in the trypsin activity of both ICEs was observed at temperatures above 60 °C, probably due to thermal inactivation of this enzyme caused by protein unfolding [40]. Similar optimum temperature (55 °C) was recorded for trypsins in skipjack tuna [26], gilthead seabream [46], sardinelle [57], and silver mojarra [62]. Optimum temperature of trypsin for both sturgeon species was higher than values reported for cold-water fish such as Atlantic cod (Gadus morhua) [63], grey triggerfish [25], lane snapper (Lutjanus synagris) [64], and Japanese sea bass (L. ...
This work aimed to determine the physicochemical and biochemical properties of trypsin from beluga Huso huso and sevruga Acipenser stellatus, two highly valuable sturgeon species. According to the results obtained from the methods of casein-zymogram and inhibitory activity staining, the molecular weight of trypsin for sevruga and beluga was 27.5 and 29.5 kDa, respectively. Optimum pH and temperature values for both trypsins were recorded at 8.5 and 55 °C by BAPNA (a specific substrate), respectively. The stability of both trypsins was well-preserved at pH values from 6.0 to 11.0 and temperatures up to 50 °C. TLCK and SBTI, two specific trypsin inhibitors, showed a significant inhibitory effect on the enzymatic activity of both trypsins (p < 0.05). The enzyme activity was significantly increased in the presence of Ca+2 and surfactants and decreased by oxidizing agents, Cu+2, Zn+2, and Co+2 (p < 0.05). However, univalent ions Na+ and K+ did not show any significant effect on the activity of both trypsins (p > 0.05). The results of our study show that the properties of trypsin from beluga and sevruga are in agreement with data reported in bony fish and can contribute to the clear understanding of trypsin activity in these primitive species.
... On the other hand, chymotrypsin activity was more stable in the range pH 5 to 11 in extracts coming from 26°C-adapted fish than in extracts coming from fish reared at 18/22°C. Isoforms for digestive proteases in fish often differ in biochemical features (Klomklao et al., 2009), thus our results were not unexpected in this regard, but the changes in the relative contribution of those isoforms driven by differences among fish rearing temperatures have not been reported before. ...
... Inhibition studies revealed digestive plasticity regarding ambient temperature and the key role of chymotrypsin Our results on the inhibition of caseinolytic activity, as indirect indicative of overall protein digestion capacity, corroborate the key role of serine proteases in digestion, particularly trypsin and chymotrypsin activities, as occur in other fish species (Natalia et al., 2004;Candiotto et al., 2018). Moreover, we found a clear dependence of these enzymes on divalent cations, in agreement with that described for other fish species (Kishimura et al., 2006;Klomklao et al., 2009). ...
... The most plausible explanation for these results is a change in the relative contribution of chymotrypsin isoenzymes, with an increase in the contribution of particular chymotrypsin isoenzymes at lower temperatures that are more sensitive to TPCK. Unfortunately, while trypsin isoenzymes were clearly identified in activity gels, with apparent molecular weights similar to those reported for other fish trypsins (Natalia et al., 2004;Kurtovic et al., 2006;Klomklao et al., 2009), our attempts to identify chymotrypsin isoenzymes in the zymogram gels were unsuccessful using TPCK as done in the tube assays. Other chymotrypsin inhibitor was used, carbobenzoxy-Phe chloromethyl ketone (ZPCK, data not shown), but it did not work either. ...
The study of fish digestive biochemistry is essential to understand factors that affect the net efficiency of food transformation and growth, and therefore aquaculture profitability. The aim of the present study was to assess the activity and functional characteristics of key digestive enzymes in juveniles of greater amberjack (Seriola dumerili), as well as the possible modulation of their relative importance by water temperature. For that, a combination of biochemical assays and substrate-SDS-PAGE were used. Under physiological conditions pepsin activity was negligible. Chymotrypsin was the most active enzyme in the digestive tract of the greater amberjack, while lipase was the enzyme with lower activity, though both enzymes in addition to trypsin were responsive to water temperature as revealed by discriminant analysis. Seriola dumerili showed to have pH-sensitive and, except for chymotrypsin, thermally robust proteases. Inhibition assays showed the major importance of serine proteases and revealed inverse trypsin and chymotrypsin responses to environmental temperature, with higher trypsin contribution in 26°C-fish while higher chymotrypsin contribution in 18°C-fish. Zymograms revealed three isotrypsin and three isochymotrypsin enzymes, with no variation in the presence of particular isoforms among rearing temperatures. However, they confirmed the role of chymotrypsin activity in providing digestive plasticity, with one of the isoforms being more active at lower temperatures. Thus, results indicate that variation in the relative contribution of chymotrypsin isoenzymes to a particular environmental temperature occurs due to different physic-chemical features of isoforms as a source of functional flexibility. This study assessed for the first time the effects of rearing temperature on greater amberjack digestive enzymes, increasing the knowledge on its digestive biochemistry, and aiding in the improvement of management practices for this species industrialization.
... Most enzymes undergo irreversible denaturation in a very acid and alkaline solution, causing the loss of stability. The stability of trypsin at particular pH might be related to the net charge of the enzyme at that pH (Klomklao et al., 2009). Trypsin from grass carp intestine had optimal pH of 8.0 (Liu et al., 2007). ...
... The accumulated heat (30 min at 60°C) with subsequent cooling might induce the conformational changes of enzyme, leading to the loss in activity. The thermal stability of trypsin from common kilka intestine was similar to those from sardine (Bougatef et al., 2007), spotted goatfish (Souza et al., 2007), and skipjack tuna (Klomklao et al., 2009) and was higher than those of grey triggerfish (Jellouli et al., 2009) and zebra blenny (Ktari et al., 2012). However, trypsin thermostability differs among the different species, and experimental conditions might also affect it (Khantaphant and Benjakul, 2010). ...
... These results provided evidence that the purified enzyme from the intestine of common kilka could most likely be a trypsin. The inhibition pattern of trypsin from common kilka was similar to trypsins from other fish species-such as sardine (Bougatef et al., 2007), grass carp (Liu et al., 2007), grey triggerfish (Jellouli et al., 2009), skipjack tuna (Klomklao et al., 2009), and zebra blenny (Ktari et al., 2012). ...
Trypsin from the intestine of common kilka (Clupeonella cultriventris caspia) was purified using ammonium sulfate precipitation (30–50% saturation), Sephadex G-75, and DEAE-cellulose chromatography with the purity of 30-fold and the yield of 12%. The molecular weight of trypsin was estimated to be 23.2 kDa based on sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). The trypsin had optimal activity at pH 8.0 and 60°C using N-α-benzoyl-DL-arginine-ρ-nitroanilide hydrochloride (BAPNA) as a substrate and showed high stability in the pH range of 7.0–10.0. It was stable up to 50°C. Soybean trypsin inhibitor (SBTI) and N-ρ-tosyl-L-lysine-chloromethylketone (TLCK) significantly inhibited trypsin activity (p < 0.05). Protein hydrolysate from common kilka muscle with different degrees of hydrolysis (DHs; 20, 30, and 40%) was prepared using the purified trypsin, and antioxidative activities were determined. 1,1-Diphenyl-2-picrylhydrazyl (DPPH), 2,2’-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) radical scavenging activities, ferric reducing antioxidant power, and ferrous chelating activity of hydrolysate increased with increasing DH up to 40% (p < 0.05). Therefore, trypsin from intestine of common kilka could be used as a processing aid for production of fish protein hydrolysate with antioxidative activity.
... For instance, an alkaline peptidase (acting on polypeptides) was purified from the viscera of the silver mojarra (Diapterus rhombeus) in a three-step process involving heat treatment, ammonium sulphate fractionation, and molecular exclusion chromatography (Silva et al. 2011). Indeed, many studies have proposed the use of common and simple chromatographic procedures on the purification of gastric and intestinal enzymes from various fish species, such as Nile tilapia (Oreochromis niloticus) (Bezerra et al. 2005) or skipjack tuna (Katsuwonus pelamis) (Klomklao et al. 2009). These protocols proved to be efficient in purifying fish trypsins in a few steps, and were of relative low cost, being easily adapted to industrial scale and affording between 1 and 3 g of purified trypsin per 1 kg of wet waste. ...
... These protocols proved to be efficient in purifying fish trypsins in a few steps, and were of relative low cost, being easily adapted to industrial scale and affording between 1 and 3 g of purified trypsin per 1 kg of wet waste. For instance, two trypsins were attained from the intestine of skipjack tuna (Klomklao et al. 2009). ...
... This extract was purified by chromatography in a Sephacryl S-200 column. Further purification was done by dialysis and other chromatographic operations (Klomklao et al. 2009). Regarding enzymes other than proteases, for instance, hyaluronidase was partially purified by acetone fractionation, ion-exchange column chromatography on a Type-I polystyrene-based anion-exchange resin, Amberlite IRA 420, and subsequently by gel filtration on Sephadex G-200 (Krishnapillai et al. 1999). ...
Fish food is a staple diet product in many countries. Indeed, fish products are important nutritive, and healthy food. Precious nutritive components are found in fish, mainly, vitamins, minerals, proteins with a balanced amino acid profile, and omega 3 polyunsaturated fatty acids (ω3-PUFA). Some of these nutrients are considered functional ingredients, given their positive effect in the regular physiological functioning of the human organism and, also in disease prevention. Accordingly, the growing concern of consumers with food safety and health, has led to a higher demand for fish products. As a result of overexploitation and environmental changes, the world catch have not been able to keep pace, thus leading to an increase in aquaculture production. However, other solutions for the overexploitation problem may be found. There are underexploited resources, low-value fish and fish waste, discards, and by-products. The utilization of these resources is a great challenge for the sector, requiring innovative technological strategies. Many solutions can be obtained from restructuring fish products and extraction, and isolation of relevant components, such as lipids, proteins, enzymes, peptides, chitin, and chitosan. In this way significant amounts of undervalued fish materials are upgraded and find application as foods or nutraceuticals. However, the novelty of these products in the context of a rapidly changing landscape, particularly the nutraceuticals, raises new questions related to the adaptation of the legal regulation that frames production, quality, and marketing of these products.
... Ekstrak kasar enzim tripsin dari usus ikan tuna sirip kuning memiliki aktivitas spesifik (4,908 U/mg) dan ekstrak kasar usus kakap merah (0,076 U/mg). Hasil ini lebih tinggi bila dibandingkan dengan aktivitas spesifik ekstrak kasar dari usus ikan tongkol (0,29 U/ mg), usus ikan cakalang (0,07 U/mg), usus grey trigger fish (0,29 U/mg), pyloric caeca bigeye snapper (0,04 U/mg) (Van Hau dan Benjakul 2006; Klomklao et al. 2009;Klomklao et al. 2009;Jellouli et al. 2009;Lihuana et al. 2019). Namun lebih rendah bila dibandingkan dengan ekstrak kasar tripsin dari pyloric caeca brownstrip red snapper yaitu 21,94 U/mg (Khantaphant dan Benjakul 2010). ...
... Ekstrak kasar enzim tripsin dari usus ikan tuna sirip kuning memiliki aktivitas spesifik (4,908 U/mg) dan ekstrak kasar usus kakap merah (0,076 U/mg). Hasil ini lebih tinggi bila dibandingkan dengan aktivitas spesifik ekstrak kasar dari usus ikan tongkol (0,29 U/ mg), usus ikan cakalang (0,07 U/mg), usus grey trigger fish (0,29 U/mg), pyloric caeca bigeye snapper (0,04 U/mg) (Van Hau dan Benjakul 2006; Klomklao et al. 2009;Klomklao et al. 2009;Jellouli et al. 2009;Lihuana et al. 2019). Namun lebih rendah bila dibandingkan dengan ekstrak kasar tripsin dari pyloric caeca brownstrip red snapper yaitu 21,94 U/mg (Khantaphant dan Benjakul 2010). ...
Jeroan ikan dikenal sebagai salah satu sumber enzim pencernaan yang melimpah, terutama sebagai sumber proteinase pencernaan yang baik, salah satunya adalah tripsin. Jenis dan habitat ikan akan memengaruhi sifat tripsin ikan. Tujuan penelitian ini adalah untuk membandingkan komponen asam amino daging dan aktivitas ekstrak kasar enzim tripsin dan usus ikan tuna sirip kuning (Thunnus albacares) dan ikan kakap merah (Lutjanus campechanus). Kedua jenis ikan diamati morfometrik dan rasio bobot jeroan ikan. Selain itu komposisi kimia, komponen asam amino daging, dan aktivitas ekstrak kasar enzim tripsin dari usus kedua jenis ikan tersebut kemudian dianalisis... Secara umum ikan tuna sirip kuning memiliki komposisi asam amino yang lebih tinggi dibandingkan ikan kakap merah, terutama pada komponen asam amino histidina, leusina, asam aspartat, lisina dan asam glutamat. Komponen asam amino yang paling dominan terdapat pada kedua jenis ikan tersebut adalah asam glutamat. Enzim tripsin ekstrak kasar usus ikan tuna memiliki aktivitas yang lebih tinggi yaitu 4,908 U/mg dibandingkan dengan ekstrak kasar usus ikan kakap merah sebesar 0,076 U/mg.
... The most important proteases in fish viscera are pepsin (an aspartic protease) and serine proteases (trypsin, chymotrypsin, collagenase, and elastase) [17,20,[47][48][49][50][51][52]. Enzymes can be endogenous (pepsin, trypsin, chymotrypsin, collagenase and elastase) or produced by the fish microbiota; they represent a large percentage of bioactive compounds present in fishery by-catch or by-products and are commercially extracted on a large scale [17], but to date their potential application has been only partially disclosed. ...
... Among digestive proteinases detected in the pyloric caeca and intestine of fish, trypsin (EC 3.4.21.4) is a serine proteinase, able to catalyze the hydrolysis of peptide bonds at the carboxyl group of arginine and lysine residues and to activate all pancreatic enzymes [50,51,56]. The use of trypsin extracted from fish viscera has recently increased significantly, due to its high stability and activity at high temperature and pH. ...
The search for new biological sources of commercial value is a major goal for the sustainable management of natural resources. The huge amount of fishery by-catch or processing by-products continuously produced needs to be managed to avoid environmental problems and keep resource sustainability. Fishery by-products can represent an interesting source of high added value bioactive compounds, such as proteins, carbohydrates, collagen, polyunsaturated fatty acids, chitin, polyphenolic constituents, carotenoids, vitamins, alkaloids, tocopherols, tocotrienols, toxins; nevertheless, their biotechnological potential is still largely underutilized. Depending on their structural and functional characteristics, marine-derived biomolecules can find several applications in food industry, agriculture, biotechnological (chemical, industrial or environmental) fields. Fish internal organs are a rich and underexplored source of bioactive compounds; the fish gut microbiota biosynthesizes essential or short-chain fatty acids, vitamins, minerals or enzymes and is also a source of probiotic candidates, in turn producing bioactive compounds with antibiotic and biosurfactant/bioemulsifier activities. Chemical, enzymatic and/or microbial processing of fishery by-catch or processing by-products allows the production of different valuable bioactive compounds; to date, however, the lack of cost-effective extraction strategies so far has prevented their exploitation on a large scale. Standardization and optimization of extraction procedures are urgently required, as processing conditions can affect the qualitative and quantitative properties of these biomolecules. Valorization routes for such raw materials can provide a great additional value for companies involved in the field of bioprospecting. The present review aims at collecting current knowledge on fishery by-catch or by-products, exploring the valorization of their active biomolecules, in application of the circular economy paradigm applied to the fishery field. It will address specific issues from a biorefinery perspective: (i) fish tissues and organs as potential sources of metabolites, antibiotics and probiotics; (ii) screening for bioactive compounds; (iii) extraction processes and innovative technologies for purification and chemical characterization; (iv) energy production technologies for the exhausted biomass. We provide a general perspective on the techno-economic feasibility and the environmental footprint of the production process, as well as on the definition of legal constraints for the new products production and commercial use.
... Recently, interest has grown to search high-value functional biomolecules from the fishery wastes, notably enzymes. Nevertheless, several researchers investigated proteases from the visceral wastes from marine fish (Klomklao et al., 2009;Nasri et al., 2011). But characterization of fish proteases especially from the visceral wastes of freshwater fish is seldom reported. ...
... The ionic strength is increased with the increasing salt concentration. In the high ionic strength, the enzyme activity is reduced due to superior hydrophobic-hydrophobic interaction between proteins of the enzyme and also enhanced affinity of ionic salts for water thereby resulting precipitation of enzyme (Klomklao et al., 2009). ...
Acidic and alkaline proteases from visceral waste of Labeo rohita (Hamilton,
1822) were isolated, partially purified by ammonium sulphate precipitation
followed by dialysis, their kinetics and characteristics studied. The purification
fold increased from 1.24 to 2.49 and 1.19 to 1.55 in acidic and alkaline protease
respectively along the purification steps. The molecular weight was found in the
range of 15-35 kDa and 25-63 kDa respectively in acidic and alkaline proteases.
The pH and temperature optima for acidic and alkaline proteases were 3 and 10, at
40°C and 60°C respectively. The Protease activity was decreased by 40% and
60%, when incubated at 90°C for 30 min. Both the proteases showed a decreased
activity of more than 50% after incubation with NaCl concentration of 0.5%.
Degree of hydrolysis (DH) of the proteases on muscle protein increased with
increase of enzyme concentrations. Both soybean trypsin inhibitor and EDTA
exhibited high percentage of inhibition, when proteases were incubated with 50
mM of both the inhibitors. The study showed that proteases from Rohu visceral
waste of could find use in applications where maximum activity at moderate
temperature and low NaCl concentration is desired.
... Many proteases from fish, such as Oreochromis niloticus [7,11], Stolephorus indicus [1] and Raja clavata [12], showed a high activity within pH ranges of 7.0-9.5. The decline in the proteolytic activity at a pH outside of the optimal pH might be involved in the change of charge distribution and conformation of both the enzyme and substrate [8,13]. ...
... Thereafter, the enzyme activity was constant with a further increment of NaCl concentration at 20%. In accordance with several reports on fish, such as Pangasianodon gigas [8], Katsuwonus pelamis [13] and S. aurita [17], the reduction of protease activity was observed with an increasing NaCl concentration. The decline in activity might be due to an increase in ionic strength, which is described by the salting out effect [18,19]. ...
Proteolytic enzymes play an important role in determining the quality of blue crab during postmortem storage. Activity of endogenous proteases is involved in the texture softening and autolysis of blue crab, which limits the customer acceptance and marketing price. This research aimed to characterize the protease activity of crude enzyme extract from the hepatopancreas of blue crab. The optimum activity of crude protease extract was found at pH 7.0 and 50°C. The crude protease enzyme was highly stable over a wide pH range of 4.0-11.0 and showed high stability at temperatures below 40°C. In addition, the protease activity continuously decreased with an increasing concentration of NaCl (0-15% w/v). Therefore, an understanding of the endogenous proteases in the blue crab could be used to develop appropriate storage methods during its distribution process.
... Fish oils are isolated from muscles and perivisceral organs, such as liver and intestine, and proteins can be obtained from fish byproducts in general 1 3 BAPNA was used, the catalytic activity of the enzyme was not significantly reduced with the increase in pH value [22]. Casein [21,25], and Nα-p-Tosyl-l-arginine methyl ester hydrochloride (TAME) [24,37,38] are used in less extent. A fluorimetric assay to measure trypsin activity was reported by Marcushi et al. [22] using carbobenzoxy-Phe-Arg-7-amido-4-methylcoumarin (z-FRMCA). ...
... Enzyme kinetics studies include the determination of optimum pH, pH stability, optimum temperature, thermostability, as well as kinetic parameters (Micahelis constant, K M ; maximal velocity, V max ) [21,32,35,[39][40][41]. The effect of inhibitors (phenylmethylsulfonyl fluoride, PMSF; ethylenediaminetetraacetic acid, EDTA; pepstatin; soybean trypsin inhibitor (SBTI) and iodoacetic acid), as well as activators (CaCl 2 and NaCl) should be addressed [20,22,23,30,38]. Some authors [19][20][21]25] also studied the effect of metal ions (e.g. ...
Fish processing industry has experienced significant growth, playing an important role in the world economy. The increased exploration of marine resources contributes to the generation of considerable amounts of biowaste, which ends up as discards. In the face of the resultant disposal and environmental problems, many efforts have been made to deal with the fishery waste in more efficient ways. Nowadays, these by-products are regarded as important sources of high added value compounds, such as hydroxyapatite, collagen, gelatin, lipids, enzymes, hydrolysates and bioactive peptides, with great potential for human health applications. The present paper aims to review the current methods of extraction and characterization of added value products from fish by-products, as well as their actual and potential applications.
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... The use of Ca 2+ improved protease production [27]. The presence of 10 mM Ca 2+ activates trypsinogen to trypsin and increases the thermal stability of the enzyme incubated for 8 h at a temperature of 30°C [28]. Stabilization was achieved by changing the conformation of trypsin molecules into a more compact structure [29]. ...
Trypsin production from skipjack tuna (Katsuwonus pelamis) viscera is one significant way to increase the value of fish’s industrial waste. The present work reports the biochemical properties of trypsin from skipjack tuna viscera. The trypsin was fractionated using 0–60% ammonium sulfate and dialyzed. The enzyme was characterized to find the optimum temperature and pH for the substrate N-α-benzoyl-dl-arginine-p-nitroanilide. The 40–50% ammonium sulfate fractionation showed the highest activity at a specific activity of 1.66 U/mg and yield of 69.91%. Specific activity increased after dialysis to 2.17 U/mg with 4.49 times purity and yield of 39.20%. The molecular weights of the enzymes were estimated as 25, 29, and 35 kDa based on the enzyme activity separated by electrophoresis. The enzyme worked optimally at a temperature and pH of 50–60°C and 8.0, respectively. Metal ions (Ca²⁺, K⁺, Na⁺, Mg²⁺) at a concentration of 20 mM showed no influence on the activity. Enzyme activity was inhibited by Zn²⁺ at 20 mM, phenyl methyl sulfonyl fluoride (PMSF), benzamidine, and soybean trypsin inhibitor (SBTI), which confirmed the characteristics of a serine protease.
... Where gastric samples are of interest for cell studies, increasing pH from 3.0 (INFOGEST method) to pH 7.5 with sodium hydroxide or sodium bicarbonate will halt pepsin activity. However, trypsin exhibits activity at pH 6 to 9 and stability from pH 4 to 11 (Klomklao et al. 2009) so pH adjustment alone is unlikely to inhibit trypsin in intestinal samples. Where polyphenols were tracked, Cilla et al. (2009) added 1.5% formic acid to decrease the pH of samples post intestinal digestion to pH 2. The samples were then diluted in media not only to reach a pH of 7.0-7.5 but also the addition of media would inhibit any reversible trypsin activity (Cilla et al. 2009). ...
As food transits the gastrointestinal tract, food structures are disrupted and nutrients are absorbed across the gut barrier. In the past decade, great efforts have focused on the creation of a consensus gastrointestinal digestion protocol (i.e., INFOGEST method) to mimic digestion in the upper gut. However, to better determine the fate of food components, it is also critical to mimic food absorption in vitro. This is usually performed by treating polarized epithelial cells (i.e., differentiated Caco-2 monolayers) with food digesta. This food digesta contains digestive enzymes and bile salts, and if following the INFOGEST protocol, at concentrations that although physiologically relevant are harmful to cells. The lack of a harmonized protocol on how to prepare the food digesta samples for downstream Caco-2 studies creates challenges in comparing inter laboratory results. This article aims to critically review the current detoxification practices, highlight potential routes and their limitations, and recommend common approaches to ensure food digesta is biocompatible with Caco-2 monolayers. Our ultimate aim is to agree a harmonized consensus protocol or framework for in vitro studies focused on the absorption of food components across the intestinal barrier.
... Combined works are mainly evident with Fereidoon Shahidi, who have often worked together (Benjakul et al., 2012). Soottawat Benjakul is also found to be contributing with Chantachum et al. (2000), Klomklao et al. (2009), Thiansilakul et al. (2007, Phanturat et al. (2010) and Nalinanon et al. (2011). A similar collaboration is also witnessed among Asbjörn Gildberg, Rasa Šližytė and Hördur G. Kristinsson (Thorkelsson et al., 2009). ...
Food loss and waste have become an issue of global significance, considering their concurrent effects on the socioeconomic and environmental facet of society. Despite this domain gaining prolific attention recently, issues hampering the effective utilization of residues from fish processing usually go unidentified in developing economies such as India. This occurs mainly owing to fragmented supply chains, inappropriate handling, discontinuous cold chains, inadequate temperature monitoring and so on, affecting quality and causing underuse. Any researcher trying to understand the prospects of utilizing these fish processing co-streams in a developing economy with the vision of improving consumption, economic sustainability, reducing discards and promoting circularity faces a lacuna. The authors address this demand in research by identifying the validity of this domain both in the global and native research community by conducting a detailed review using bibliometric analysis and content analysis. Data from Scopus with 717 documents, comprising 612 research articles from 78 countries, 1597 organizations and 2587 authors, are analysed. Results signify (i) developing a focus on hydroxyapatite production, bio-methane generation, transesterification processes, biomass and the rest raw material generated from fish processing, and (ii) reduced research on supply chain-related aspects despite their considerable importance. To comprehend this deficiency, especially in the Indian stance, barriers hindering the utilization of generated by-products are identified, and recommendations for improvements are proposed. The results will provide the struts for a circular and sustainable supply chain for processed seafood in developing economies.
... During the processing of canned tuna, about 50% of skipjack tuna materials are generated as by-products (19)(20)(21). On the contrary, several functional molecules, such as collagen/gelatin, unsaturated fatty acid, protease, and polysaccharides, are generated from skipjack tuna by-products and exhibited huge application prospects and various biological activities (22,23). Moreover, some bioactive peptides have been prepared from protein hydrolysates of tuna by-products, such as bone/frame (24), scale (25), roe (4), milt (21), and head and viscera (26,27). ...
To effectively utilize skipjack tuna (Katsuwonus pelamis) processing by-products to prepare peptides with high angiotensin-I-converting enzyme (ACE) inhibitory (ACEi) activity, Neutrase was selected from five kinds of protease for hydrolyzing skipjack tuna dark muscle, and its best hydrolysis conditions were optimized as enzyme dose of 1.6%, pH 6.7, and temperature of 50°C using single factor and response surface experiments. Subsequently, 14 novel ACEi peptides were prepared from the high ACEi protein hydrolysate and identified as TE, AG, MWN, MEKS, VK, MQR, MKKS, VKRT, IPK, YNY, LPRS, FEK, IRR, and WERGE. MWN, MEKS, MKKS, and LPRS displayed significantly ACEi activity with IC50 values of 0.328 ± 0.035, 0.527 ± 0.030, 0.269 ± 0.006, and 0.495 ± 0.024 mg/mL, respectively. Furthermore, LPRS showed the highest increasing ability on nitric oxide (NO) production among four ACEi peptides combining the direct increase and reversing the negative influence of norepinephrine (NE), and MKKS showed the highest ability on directly decreasing and reversing the side effects of NE on the secretion level of endothelin-1 (ET-1) among four ACEi peptides. These findings demonstrate that seafood by-product proteins are potential ACEi peptide sources and prepared ACEi peptides from skipjack tuna dark muscle, which are beneficial components for functional food against hypertension and cardiovascular diseases.
... Recently, interest has grown to search high-value functional bio-molecules from the fishery wastes, notably enzymes. Nevertheless, several researchers investigated proteases from the visceral wastes from marine fish (Klomklao et al, 2009;Nasri et al, 2011). But characterization of fish proteases especially from the visceral wastes of freshwater fish is seldom reported. of the family Cyprinidae native to eastern Asia. ...
Acidic and alkaline proteases from visceral waste of Labeo fimbriatus (Bloch, 1795) were isolated, partially
purified by ammonium sulphate precipitation followed by dialysis, their kinetics and characteristics studied. The crude enzyme
was partially purified and its molecular weight was studied. The enzyme showed highest activity and purification-fold when
precipitated at 40–60% ammonium sulfate. The purification fold increased from 1.23 to 2.49 and 1.17 to 1.51 in acidic and
alkaline protease respectively along the purification steps. Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDSPAGE)
showed a molecular weight of 15-35 kDa and 25-63 kDa respectively in acidic and alkaline proteases. The pH and
temperature optima for acidic and alkaline proteases were 3 and 10, at 40°C and 60°C, respectively. The acidic and alkaline
protease activity was decreased by 40% and 60%, when incubated at 90°C for 30 min. Degree of hydrolysis (DH) of the
proteases on muscle protein increased with increase of enzyme concentrations. The study showed that proteases from Fringed
lipped carp visceral waste of could find use in applications, where maximum activity at moderate temperature is desired.
... Fish viscera are really a good source of digestive enzymes, such as pepsin and the serine proteases, trypsin and chymotrypsin and serine proteases such as elastase and collagenase (Rossano et al., 2011) [55] . One of the main proteases detected in fish viscera, especially in the pyloric ceaca and intestine is trypsin, a member of a large family of serine proteases, isolated and characterized thoroughly based on their physiochemical and enzymatic properties from several species of fish, example, carp (Cyprinus carpio), tambaqui (Colossoma macropomum), true sardine (Sardinops melanostictus), arabesque greenling (Pleuroprammus azonus), Alaska pollock (Theragra chalcogramma) and Skipjack tuna (Katsuwonus pelamis) (Cao et al., 2000;Bezerra et al., 2001;Kishimura et al., 2006;Kishimura et al., 2008& Klomklao et al., 2009 . Nasri et al. (2011) studied the alkaline protease activities in the viscera of goby (Zosterisessor ophiocephalus), thornback ray (Raja clavata), and scorpionfish (Scorpaena scrofa). ...
... Inactivation of enzymatic activity during thermal treatment is due to unfolding of the enzyme molecule (Klomklao et al., 2011). Klomklao et al. (2009) . Inhibition of Rohu alkaline proteases at 250 µM concentration of SBTI was reported to be 78.1% (Kumar et al., 2007). ...
Technical characteristics and detergent compatibility of visceral alkaline proteases of three freshwater fish, namely Labeo rohita, Pangasianodon hypophthalmus and Cyprinus carpio of different feeding habits, were studied. Crude enzyme extract was partially purified by (NH4)2SO4 precipitation and dialysis. The molecular weight was in the range of 20–63 kDa. The enzyme purification folds post‐dialysis were found to be 1.55, 1.81 and 2.17 in case of Rohu, Pangas and Common carp respectively. The alkaline protease from Rohu, Pangas and Common carp exhibited maximum activity at pH 10.0, 9.0 and 11.0 respectively. The enzyme temperature optima observed were 60°C (Rohu and Pangas) and 70°C (Common carp). SBTI and EDTA inhibited more than 90% of the activity at conc. of 50 mM. Exposure of the proteases to non‐ionic surfactants like Tween 20–80 retained about 92%–100% and 76%–100% of their activity at conc. (v/v) of 1% and 5% respectively. Proteases were found less stable in the presence of SDS. There was moderate to lesser influence of H2O2 and sodium perborate on the proteolytic activity. The alkaline protease from omnivorous fish was found superior compared to the herbivore and carnivore in respect of pH and temperature optima and stability with detergents and oxidizing agents.
... Thus, the potential of fish and shellfish viscera for recovery of proteases is large [114]. Some examples of commonly extracted gastric, intestinal and hepatopancreatic proteases are pepsin trypsin, chymotrypsin, collagenase, and elastase [115], followed by non-proteolytic enzymes, such as transglutaminase, lipases, and chitinolytic enzymes obtained from various fish species, such as the Nile tilapia (Oreochromis niloticus) [116] or skipjack tuna (Katsuwonus pelamis) [117]. Therefore, visceral by-products have been evaluated as a favourable source of gastric, intestinal, and hepatopancreatic enzymes [118][119][120]. ...
The global demand for dietary proteins and protein-derived products are projected to dramatically increase which cannot be met using traditional protein sources. Seafood processing by-products (SPBs) and microalgae are promising resources that can fill the demand gap for proteins and protein derivatives. Globally, 32 million tonnes of SPBs are estimated to be produced annually which represents an inexpensive resource for protein recovery while technical advantages in microalgal biomass production would yield secure protein supplies with minimal competition for arable land and freshwater resources. Moreover, these biomaterials are a rich source of proteins with high nutritional quality while protein hydrolysates and biopeptides derived from these marine proteins possess several useful bioactivities for commercial applications in multiple industries. Efficient utilisation of these marine biomaterials for protein recovery would not only supplement global demand and save natural bioresources but would also successfully address the financial and environmental burdens of biowaste, paving the way for greener production and a circular economy. This comprehensive review analyses the potential of using SPBs and microalgae for protein recovery and production critically assessing the feasibility of current and emerging technologies used for the process development. Nutritional quality, functionalities, and bioactivities of the extracted proteins and derived products together with their potential applications for commercial product development are also systematically summarised and discussed.
... The optimum temperature for trypsin activity in C. othonopterus was 60°C, but not significantly different from that at 55 or 65°C, coinciding with the values for two trypsin isoforms of Katsuwonus pelamis, 55 and 60°C [57] and the trypsins of C. macrocephalus × C. gariepinus [51], O. tshawytscha [15], Sardina pilchardus [58] and Mugil cephalus [59], with an optimum temperature at 60°C. In C. parvipinnis and C. xanthulus, the optimum temperature was 65°C, the same as for Centropomus undecimalis [60], Atractosteus tropicus [61], Brycon orbignyanus [62] and T. thynnus [63]. ...
Trypsin and pancreatic lipase promote the digestion of proteins and lipids, respectively, when they are secreted into the anterior intestine; however, since the pancreas is a diffuse tissue in fish, the characterization and quantification of pancreatic enzymes is uncommon. The objective of this study was to partially characterize and compare the enzymatic activities of lipase and trypsin within the gastrointestinal tract of Cynoscion parvipinnis, Cynoscion othonopterus and Cynoscion xanthulus, to contribute to the knowledge of the digestive physiology of these important commercial sciaenids and to reveal whether they have potential for biotechnological applications. The presence of lipase and trypsin was confirmed by zymography and the molecular weights of both enzymes were determined by electrophoresis. For lipase, molecular weights of 65.8 and 69.5 kDa were determined for C. othonopterus and C. xanthulus, respectively. For C. parvipinnis, two lipases of 61.5 and 36.0 kDa were determined. In all three species the largest lipase activity was observed in the anterior intestine, followed by pyloric caeca, with optimum activity observed at pH 8.0 and at temperatures ranging between 40 and 45°C. Molecular weights of trypsin were 24.4, 23.6 and 23.7 kDa in C. othonopterus, C. parvipinnis, and C. xanthulus, respectively. The optimum pH of activity ranged between 7.0 and 9.0 and optimum temperature between 55 and 65°C for all species. These enzymes meet certain criteria that make them potential candidates for some industrial applications, such as the food industry and the production of detergents.
... Trypsins from spleen of skipjack, yellowfin, and tongol were purified. They had MW of 24 kDa and showed the optimal pH around 8.5-9.0 using Nα-ρ-tosyl-L-arginine methyl ester hydrochloride (TAME) as a substrate (Klomklao et al. 2006a,b;Klomklao et al. 2009b). Purified trypsin from Luphiosilurus alexandri pyloric cecum with a molecular weight of 24 kDa showed an optimum at pH 9.0 and 50 C when Nα-benzoyl-DL-arginine ρ-nitroanilide (BAPNA) was used as a substrate (dos Santos et al. 2016). ...
The current fish processing practically generates large amounts of byproducts, accounting for up to 75% of the total fish weight. Fish processing byproducts contain a wide range of nutritional components, especially lipid and protein fractions as well as functional compounds or nutraceuticals. Collagen, gelatin, as well as hydrolyzed collagen can be produced from collagenous materials such as bone, scale, or skin, etc. In addition to fish proteins and oil, other valuable components, including enzymes, nucleic acids, minerals, and other bioactive compounds such as chondroitin sulfate, etc. can be recovered from those leftovers. To ensure better utilization of fish processing byproducts for applications in food, nutraceutical, cosmetic, or medical products, it is necessary to use high quality byproducts; increase the yield of recovered products; develop the standardized and controlled processes accounting for variation in raw material, providing stable, healthy, and high‐quality products; and measure and enhance the selected properties, especially bioactivities.
... The process of carotenoproteins extraction assisted by proteolytic enzymes facilitated the release of proteins and lipids from shells of blue crab, while minerals and chitin were still associated with shells. These findings were in agreement with previous researches (Senphan et al., 2014;Klomklao et al., 2009). ...
Carotenoids, natural pigments, are a group of chemically heterogeneous molecules, present in numerous taxonomical clusters. Because of their various bioactivities, carotenoids are day-by-day applied in numerous fields. The present work aimed to investigate an efficient extraction process of carotenoids from blue crab shells and their identification by HR-ESI-MS technique. In this context, different methods (enzymatic, maceration, Soxhlet, etc.) and solvents (variable polarity index) were tested. Maceration using the binary system hexane/isopropanol (50/50) was found to be the most efficient process, producing high carotenoids content and low total phenolic and soluble protein amounts (p < 0.05). When combined with an enzymatic pretreatment, this procedure was found to be remarkably (p < 0.05) more efficient and selective especially towards astaxanthin (p < 0.05). The HR-ESI-MS identified 23 compounds, depending on the adopted extraction approach. The compounds identified may have potential for applications in food or pharmaceutical industries.
... The treatment at 0 MPa, when the solution passes through the valve into the equipment without pressure application, can promote small changes in enzyme configuration due to shear, friction and others stress caused by the process. According to Benjakul [27]. This indicated that the pH could have changed the enzyme native configuration and, consequently, the enzyme susceptibility to HPH treatment. ...
Alkaline proteases have great importance in the pharmaceutical, textile, food industries and research studies. High pressure is an emerging and relatively new technology that is capable of modifying various molecules. The use of High Pressure Homogenization (HPH) has been widely evaluated in the modulation of enzyme activity, either to inactivate, improve or stabilize its activity. The effect of HPH treatment on Bacillus licheniformis LBA 46 semi-purified protease activity was investigated at different pH values and temperatures of activity. The enzyme was treated up to 200 MPa at pH 4, 7 and 9 and the residual activity was evaluated on the day of the treatment and after 24 h of refrigerated storage. The protease activity and stability measured at 40, 60 and 90°C between the control and the treated samples showed similar residual activity values. This suggests that the enzyme was resistant to the HPH treatment (50-200 MPa). Though HPH is a promising method to change enzymes characteristics, it was not able to change the protease from Bacillus licheniformis LBA 46.
Effect of high pressure homogenization on the activity and stability of protease from Bacillus licheniformis LBA 46 in different pH values.
... However, some fish trypsins are stable over a wider pH range, such is the case of the Tunisian barbel (Barbus callensis) and striped seabream (Lithognathus mormyrus) trypsins that preserve above 90% of the enzymatic activity in a pH range of 5-12 Elhadj-Ali et al. 2009); the zebra blenny trypsin (Salaria basilisca) that conserves 100% of the enzymatic activity in a pH range of 7.0-12.0; and the trypsins of pectoral rattail (Coryphaenoides pectoralis) and Bogue (Boops boops) that conserve above 70% of the enzymatic activity in a pH range of 6-11 Klomklao et al. 2009c; Barkia et al. 2010). In all of them, the stability test was carried out at 40 °C for 1 h. ...
In fishes, trypsins are adapted to different environmental conditions, and the biochemical and kinetic properties of a broad variety of native isoforms have been studied. Proteolytic enzymes remain in high demand in the detergent, food, and feed industries; however, our analysis of the literature showed that, in the last decade, some fish trypsins have been studied for the synthesis of industrial peptides and for specific biomedical uses as antipathogenic agents against viruses and bacteria, which have been recently patented. In addition, innovative strategies of trypsin administration have been studied to ensure that trypsins retain their properties until they exert their action. Biomedical uses require the production of high-quality enzymes. In this context, the production of recombinant trypsins is an alternative. For this purpose, E. coli-based systems have been tested for the production of fish trypsins; however, P. pastoris-based systems also seem to show great potential in the production of fish trypsins with higher production quality. On the other hand, there is a lack of information regarding the specific structures, biochemical and kinetic properties, and characteristics of trypsins produced using heterologous systems. This review describes the potential uses of fish trypsins in biomedicine and the enzymatic and structural properties of native and recombinant fish trypsins obtained to date, outlining some prospects for their study.
... The molecular weight of S. notprotease was similar to digestive proteases purified from other fish species such as tongol tuna (Thunnus tonggol), skipjack tuna (Katsuwonus pelamis), and Tunisian barbel (Barbus callensis). [14,22,23] Effect of temperature and pH on protease activity and stability Temperature profile of S. not-protease is shown in Fig. 3A. The low molecular weight protease was active in the temperature range of 30 and 50°C with an optimum at 40°C. ...
ABSTRACT
A new protease was isolated from the intestine of small red scorpionfish.
After ammonium sulfate precipitation, the enzyme was purified to homogeneity
by a two-step chromatography with 6.69% recovery and fivefold
increase in specific activity. The molecular weight of the protease was about
24 kDa as estimated by size exclusion chromatography and sodium dodecyl
sulfate–polyacrylamide gel electrophoresis. The enzyme optimum pH and
temperature were pH 10.0 and 40°C, respectively. The protease was stable
at temperatures below 40°C and over a broad pH range (8.0–11.0). The Km
and Kcat values were 0.36 mmol L−1 and 1.61 s−1, respectively. Interestingly,
the enzyme presented specificity for casein, egg albumin, bovine serum
albumin, and gelatin with obtained degrees of hydrolysis ranging from
3.14% to 6.58%, after 2 h of hydrolysis. Moreover, gluten hydrolysate
analysis confirmed protein hydrolysis and solubilization. These results suggested
the potential use of the protease in the preparation of food protein
hydrolysates with interesting properties.
... Tuna is one of the most economically impo rtant groups of fish species. The main forms for international tuna trade are the raw material for canning, tuna for direct consumption (sashimi), smoked tuna and dried tuna (Klo mklao, Kishimura, Nonami & Benjakul, 2009). In Thailand, the tuna processing industry has been economically important as an essential income generator. ...
Protein hydrolysates from skipjack tuna viscera prepared using Alcalase 2.4 L (VPH) with different degree of hydrolysis (DH: 10%, 20% and 30%) were prepared and determined for their antioxidative activity. VPH with 20% DH had the highest DPPH, ABTS radical scavenging activities and ferrous chelating activity (P<0.05). However, ferric reducing antioxidant activity of hydrolysates increased as DH increased (P<0.05). When VPH with 20% DH was determined for its pH and thermal stability, ABTS radical scavenging activity remained constant or slightly decreased in a wide pH range (2-11) and during heating (100o C) for 180 min. Functional properties of VPH (20% DH) at different concentrations were also investigated. The emulsifying and foaming properties were governed by its concentrations used. Hydrolysis by Alcalase at 20% DH increased protein solubility to above 91% over a wide pH range (3-9). Therefore, VPH could be used as a food additive possessing both antioxidant activity and functional properties. © Published by Central Fisheries Research Institute (CFRI) Trabzon, Turkey.
... The molecular weight of S. notprotease was similar to digestive proteases purified from other fish species such as tongol tuna (Thunnus tonggol), skipjack tuna (Katsuwonus pelamis), and Tunisian barbel (Barbus callensis). [14,22,23] Effect of temperature and pH on protease activity and stability Temperature profile of S. not-protease is shown in Fig. 3A. The low molecular weight protease was active in the temperature range of 30 and 50°C with an optimum at 40°C. ...
A new protease was isolated from the intestine of small red scorpionfish. After ammonium sulfate precipitation, the enzyme was purified to homogeneity by a two-step chromatography with 6.69% recovery and fivefold increase in specific activity. The molecular weight of the protease was about 24 kDa as estimated by size exclusion chromatography and sodium dodecyl sulfate–polyacrylamide gel electrophoresis. The enzyme optimum pH and temperature were pH 10.0 and 40°C, respectively. The protease was stable at temperatures below 40°C and over a broad pH range (8.0–11.0). The Km and Kcat values were 0.36 mmol L⁻¹ and 1.61 s⁻¹, respectively. Interestingly, the enzyme presented specificity for casein, egg albumin, bovine serum albumin, and gelatin with obtained degrees of hydrolysis ranging from 3.14% to 6.58%, after 2 h of hydrolysis. Moreover, gluten hydrolysate analysis confirmed protein hydrolysis and solubilization. These results suggested the potential use of the protease in the preparation of food protein hydrolysates with interesting properties.
... The optimal temperature of P. segnis proteases was similar to crude enzymes extracted from Macrobrachium rosenbergii [6], golden gray mullet [14], farmed giant catfish [16], Scomber japonicus [30], zebra blenny [31] and skipjack tuna [45] viscera. However, it was higher than optimal temperatures of goby and scorpionfish digestive alkaline proteases, which showed optimal temperatures at 50 and 55 • C, respectively [27], as well as silver mojarra (Diapterus rhombeus) purified protease (55 • C) [42]. ...
Since chitin is closely associated with proteins, deproteinization is a crucial step in the process of extracting chitin. Thus, this research aimed to extract chitin from Portunus segnis and Penaeus kerathurus shells by means of crude digestive alkaline proteases from the viscera of P. segnis, regarding deproteinization step, as an alternative to chemical treatment. Casein zymography revealed that five caseinolytic proteases bands exist, suggesting the presence of at least five different major proteases. The optimum pH and temperature for protease activity was pH 8.0 and 60 °C, respectively, using casein as a substrate. The crude enzymes extract was highly stable at low temperatures and over a wide range of pH from 6.0 to 12.0. The crude alkaline protease extract was found to be effective in the deproteinization of blue crab and shrimp shells, to produce chitin. The best efficiency in deproteinization (84.69 ± 0.65% for blue crabs shells and 91.06 ± 1.40% for shrimp shells) were achieved with an E/S ratio of 5 U/mg of proteins after 3 h incubation at 50 °C. These results suggest that enzymatic deproteinization of crab and shrimp wastes by fish endogenous alkaline proteases could be a potential alternative in the chitin production process.
... In the low pH range, the activity of cobia trypsin was negligible. This is commonly observed with other fish trypsin, primarily due to conformational changes under acidic conditions, or enzyme denaturation [9,37,38]. ...
A thermostable alkaline peptidase was purified from the processing waste of cobia (Rachycentron canadum) using bovine pancreatic trypsin inhibitor (BPTI) immobilized onto Sepharose. The purified enzyme had an apparent molecular mass of 24 kDa by both sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and mass spectrometry. Its optimal temperature and pH were 50 °C and 8.5, respectively. The enzyme was thermostable until 55 °C and its activity was strongly inhibited by the classic trypsin inhibitors N-ρ-tosyl-l-lysine chloromethyl ketone (TLCK) and benzamidine. BPTI column allowed at least 15 assays without loss of efficacy. The purified enzyme was identified as a trypsin and the N-terminal amino acid sequence of this trypsin was IVGGYECTPHSQAHQVSLNSGYHFC, which was highly homologous to trypsin from cold water fish species. Using Nα-benzoyl-dl-arginine ρ-nitroanilide hydrochloride (BApNA) as substrate, the apparent km value of the purified trypsin was 0.38 mM, kcat value was 3.14 s⁻¹, and kcat/km was 8.26 s⁻¹ mM⁻¹. The catalytic proficiency of the purified enzyme was 2.75 × 10¹² M⁻¹ showing higher affinity for the substrate at the transition state than other fish trypsin. The activation energy (AE) of the BApNA hydrolysis catalyzed by this enzyme was estimated to be 11.93 kcal mol⁻¹ while the resulting rate enhancement of this reaction was found to be approximately in a range from 10⁹ to 10¹⁰-fold evidencing its efficiency in comparison to other trypsin. This new purification strategy showed to be appropriate to obtain an alkaline peptidase from cobia processing waste with high purification degree. According with N-terminal homology and kinetic parameters, R. canadum trypsin may gathers desirable properties of psychrophilic and thermostable enzymes.
... Sutanbawa and Aknes (2006) reported that processing waste from the tuna canning industry amounts to 450,000 million tons per year. Due to biological resource limitations and environmental concerns, interest in recycling and utilization of tuna waste has been increasing (Klomklao et al. 2009;Ferraro et al. 2010;Seo et al. 2012). In the fish processing industry, the yield is generally based on the gutted fish with the head. ...
Skipjack tuna, Katsuwonus pelamis, are distributed throughout the Pacific Ocean in the tropical and subtropical areas, including South Korea, Japan and Indonesia. The antidiabetic and antioxidant potential of 70% ethanol (EtOH) extract of skipjack tuna heart were investigated via protein tyrosine phosphatase 1B (PTP1B), α-glucosidase, human recombinant aldose reductase (HRAR), 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical, peroxynitrite (ONOO-), 2,2′-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS) radical, and total reactive oxygen species (ROS). The 70% EtOH tuna heart extract exhibited potent inhibitory activity against PTP1B, α-glucosidase and HRAR with inhibition percentages of 85.42, 82.70 and 51.1%, respectively, at a concentration range of 1-2 mg/mL. In addition, it was a potent inhibitor against DPPH, ABTS, ONOO-, and ROS with inhibition percentages of 69.45, 58.31, 96.20 and 34.02%, respectively, at a concentration of 1 mg/mL. The total phenolic content present in tuna extract was 15.80 mg/g GAE. The results demonstrate the potential antidiabetic and antioxidant activities of tuna heart extract.
... At extreme pH, strong intramolecular electrostatic repulsion caused by high net charge results in swelling and unfolding of the protein molecules (Benjakul et al., 2000;Klomklao et al., 2007). Inactivation of enzyme activity at acidic pH was also reported for the anionic trypsins from capelin (Hjelmeland and Raa, 1982), true sardine , Atlantic white croaker (Pavlisko et al., 1997), yellowfin tuna (Klomklao et al., 2006), and skipjack tuna (Klomklao et al., 2009b). For thermal stability, crude proteinases from oxeye scad muscle was stable at temperature below 40°C, but the activity sharply decreased above 50°C (Fig. 4B). ...
The autolytic profile of oxeye scad mince was characterized. Mince showed higher proteolytic activity than washed mince. The highest autolysis was observed at 65 and 60°C for mince and washed mince, respectively. Both mince and washed mince showed the optimum pH for autolysis at pH 9.0, and their activities decreased with increasing NaCl concentration (0–3.5%). Autolysis of washed mince was markedly inhibited by soybean trypsin inhibitor (SBTI), suggesting that myofibril-associated proteinase was serine proteinase. Sarcoplasmic proteinase was characterized to be heat-activated alkaline proteinase having the optimal pH and temperature of 9.0 and 60°C, respectively. The activities were stable at pH range of 8.0–11.0 at 20–40°C. The crude proteinase was inhibited by N-p-tosyl-L-lysine chloromethyl ketone, SBTI, and phenylmethanesulfonyl fluoride, suggesting the predominance of serine proteinases, especially trypsin. NaCl suppressed the activity while β-mercaptoethanol, dithiothreitol, and CaCl2 activated the activity. Therefore, trypsin-like proteinase is a major endogenous proteinase responsible for autolysis in oxeye scad muscle. The present results can be used as scientific guidelines to predict the gel strength of surimi made from oxeye scad muscle.
... Sutanbawa and Aknes (2006) reported that processing waste from the tuna canning industry amounts to 450,000 million tons per year. Due to biological resource limitations and environmental concerns, interest in recycling and utilization of tuna waste has been increasing ( Klomklao et al. 2009;Ferraro et al. 2010;Seo et al. 2012). In the fish processing industry, the yield is generally based on the gutted fish with the head. ...
... Sutanbawa and Aknes (2006) reported that processing waste from the tuna canning industry amounts to 450,000 million tons per year. Due to biological resource limitations and environmental concerns, interest in recycling and utilization of tuna waste has been increasing (Klomklao et al. 2009; Ferraro et al. 2010; Seo et al. 2012 ). In the fish processing industry, the yield is generally based on the gutted fish with the head. ...
... The role of trypsin in the T. orientalis was in accordance with a previous report by Matus de la Parra et al. (2007). SBTI has been shown to inhibit serine proteases, mainly trypsins from T. thynnus (Essed et al. 2002), Katsuwonus pelamis (Klomklao et al. 2007(Klomklao et al. , 2009, and Thunnus albacores (Klomklao et al. 2006). The SBTI inhibited more than 88% and 95% of the enzyme activity of purified trypsin from K. pelamis and T. albacores, respectively. ...
The digestive process of the Pacific bluefin tuna (PBT), Thunnus orientalis, was simulated through two phases of in vitro digestion: acidic digestion with porcine pepsin, followed by alkaline digestion with pancreatic crude extract (PCE) obtained from the PBT to hydrolyze fish meal (FM) and soybean meal (SBM) as protein substrates. The crude protein from FM resulted in a lower degree of hydrolysis (73.3%) compared with SBM (79.2%). However, the resulting digested products showed that FM contained 35% more small peptides, with sizes 150 kDa). The SBM had an increase of only 1.3% in the similar peptide cut‐offs found after hydrolysis. These results suggested that FM appeared to be a better source of protein according to the amount of low‐molecular weight peptides. In addition, the proteolytic activity of PCE showed that 88.9% of its alkaline proteolytic activity corresponded to trypsin and 2.9% corresponded to chymotrypsin activity. The results shown here demonstrate that peptide sizes are important in identifying suitable protein sources for aquafeed production to reinforce the primary results obtained from the in vitro digestibility using the pH‐Stat system. These results also contribute to a better understanding of the digestibility process in aquatic organisms.
... However, the enzyme was very active even at high salt concentrations, showing 90, 82, 76, 72, 66 and 59% residual activity at 5, 10, 15, 20, 25 and 30% of NaCl, respectively (P < 0.05). Klomklao et al. (2009b) also observed the same effect in two trypsin isoforms from the fish skipjack tuna, where trypsin A and B retained about 40 and 50% of their activity in 25% NaCl, respectively. The same results were reported by Klomklao et al. (2011), who found that trypsin activity from the hybrid catfish decreased with increasing NaCl concentrations. ...
Peptidases were extracted from the viscera of farmed giant catfish (Pangasianodon gigas) in an aqueous two-phase system (ATPS) of 15% (w/w) polyethylene glycol (PEG-2000) and 15% (w/w) sodium citrate. The recovery of the enzymes was 273% with 12-fold purification. Protein pattern, activity and inhibitory staining confirmed that the proteins with molecular weights of 12 and 31 kDa were a mixture of proteolytic enzyme. The optimum pH and temperature of the enzyme were 8.0 and 70C, respectively. Besides, it retained more than 50% of activity at the highest salt concentration (30% w/v). The enzyme was strongly inhibited by serine protease inhibitors (>80% inhibition), while low inhibition with cysteine-, aspartic- and metallo-protease inhibitors (<20% inhibition). The enzyme activity was extremely inhibited by the metal ions Ag+, Cu2+ and Fe2+. The peptidases from the giant catfish viscera have potential applications in food processing where high temperatures (60–70C) and/or high salt content are used, or even in detergent formulation.Practical ApplicationsExtraction and characterization of a proteolytic enzyme from viscera, fishery processing by-product, could add value to it. According to its properties, the isolated peptidases could be potentially applied to food industry, especially in fish sauce production, protein hydrolysate production, or in neutraceutical and in detergent industry.
... Trypsins from other fish species such wileyonlinelibrary.com/jsfa as grey triggerfish, 13 zebra blenny, 14 Pacific saury 39 and skipjack tuna 42 were also effectively inhibited by TLCK as well as SBTI. ...
Fish proteases, especially trypsin could be used to prepare fish protein hydrolysate with antioxidative activities. In this study, trypsin from the pyloric caeca of unicorn leatherjacket was purified using ammonium sulfate precipitation and SBTI-Sepharose 4B column. Hydrolysate from Indian mackerel protein isolate with different degrees of hydrolysis (DHs) (20, 30 and 40%) was prepared using the purified trypsin and antioxidative activities of hydrolysate including DPPH, ABTS radical scavenging activities, ferric reducing antioxidant power and ferrous chelating activity were determined.
Trypsin was purified with the purity of 26.43 fold and the yield of 13.43%. It had the molecular weight (MW) of 23.5 kDa with the optimal activity at pH 8.0 and 55 °C. It displayed high stability in the pH range of 6.0-11.0 and was stable up to 50 °C. SBTI (0.05 mmol L(-1) ) and TLCK (5 mmol L(-1) ) completely inhibited trypsin activity. Antioxidative activities of hydrolysate from Indian mackerel protein isolate increased with increasing degree of hydrolysis (DH) up to 40% (P< 0.05). Based on SDS-PAGE, hydrolysate with 40% DH showed MW lower than 6.5 kDa.
The purified protease from unicorn leatherjacket pyloric caeca was identified as trypsin based on its ability to hydrolyse specific synthetic substrate and the response to specific trypsin inhibitor. The purified trypsin could hydrolyze Indian mackerel protein isolate and the resulting hydrolysate exhibited antioxidative activity, depending upon DHs.
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Abalones reveal unique taste after processing, mainly because of their abundant free amino acids (FAAs) and nucleotides. FAAs are nutrition components that can contribute to the unique taste. However, which factor(s) is responsible for the accumulation of FAAs still need further studies. To analyze the production of FAAs, we studied the variation of FAAs during 7 days of storage at 4°C. The content of taste-active amino acids, including Asp, Glu, Ser, and Gly increased by 1.7-fold, 2.0-fold, 3.0-fold, and 8.4-fold, respectively. The relative activity of cathepsin L and aminopeptidase (AP) increased significantly during the cold storage period. To identify AP in abalone and its function in mediating the production of FAAs, an aminopeptidase with wide substrate specificity was then extracted and purified from abalone muscle to homogeneity. Purified AP with a molecular mass of 100 kDa exhibited its maximum activity at 30°C, pH 7.5, and was further confirmed by LC-MS. Bestatin specifically inhibited the activity of AP, and metalloproteinase inhibitors EDTA, EGTA and 1, 10-phenanthroline also suppressed its activity to different degrees. Based on its highest activity to substrate Leu-MCA and its peptide sequences, the purified enzyme was identified as leucine aminopeptidase (LAP). Our present study indicated the essential role of AP for FAAs accumulation during cold storage of abalone.
This work aimed to determine the physicochemical and biochemical characterization of the intestinal trypsin from beluga ( Huso huso ) and sevruga ( Acipenser stellatus ), two highly valuable sturgeon species, by a series of assays. According to the results obtained from casein-zymogram and inhibitory activity staining method, indicating the existence of trypsin in the intestinal crude extract of both species, molecular weight of the enzyme was estimated to be 27.5 and 29.5 kDa in sevruga and beluga, respectively. Optimum pH and temperature of both trypsins were recorded at 8.5 and 55°C by BAPNA (a specific substrate), respectively. The stability of both trypsins was well preserved at pH from 6.0 to11.0 and temperatures of up to 50°C. TLCK and SBTI, two specific trypsin inhibitors, showed a significant inhibitory effect on the enzymatic activity of both trypsins ( P < 0.05). The enzyme activity was significantly increased in the presence of Ca + 2 and surfactants and decreased by oxidizing agents, Cu + 2 , Zn + 2 and Co + 2 ( P < 0.05). However, univalent ions Na ⁺ and K ⁺ did not show any significant effect on the activity of both trypsins ( P > 0.05). Therefore, the results of our study can contribute to the clear understanding of the intestinal trypsin activity in beluga and sevruga under evaluated experimental conditions.
Specificity studies can contribute to the understanding of how substrate binding and catalysis change among similar enzymes. This study evaluated through the substrate specificity, physicochemical and kinetic properties of a trypsin from the pyloric caeca of pirarucu (Arapaima gigas). For such purposes, two series of FRET-peptides (Abz-RXFK-Eddnp and Abz-XRFK-Eddnp) were used. The mature trypsin molecular mass was 23.5 kDa and N-terminal sequence was IVGGYECPRNSVPYQVSLNVGYH. Moreover, higher trypsin catalytic efficiency was observed for the substrates containing Arg, Tyr and Ser at P1’ and Lys and Val at P2. However, catalytic deficiency was found in the presence of Pro, Trp, Asp, Gly, Glu, Ala at P1’ and Gly, Glu, Trp, Asn, Gln and Tyr at P2. Using z-FR-MCA as a substrate, the optimum pH (9.25) and temperature (45 °C) were determined by pseudo-first-order kinetics. The enzyme retained all of its initial activity after 180 min incubation at temperatures up to 45 °C. Kinetic assays in the presence of Ca²⁺ showed an increase of approximately 7-fold in enzyme catalytic efficiency (kcat/Km). On the other hand, Na⁺, K⁺ and Mg²⁺ were able to inhibit pirarucu trypsin. Therefore, the knowledge of kinetic and biochemical properties, as well as enzyme specificity, are important for the evaluation of biotechnological use of this enzyme and may contribute to the development of more efficient substrates for fish trypsin.
Previously we purified and characterized a sarcoplasmic serine proteinase (SSP) from the belly muscle of the threadfin bream as a modori-inducing proteinase. In our attempt to clarify the structure and physiological functions of SSP, we successfully cloned the full-length cDNA of SSP (ORF 726 bp). The deduced amino acid sequence of SSP (241 residues) was highly homologous to fish trypsinogen. The distribution of SSP mRNA and the proteinase activity in the tissue indicated that SSP was mainly synthesized and existed in the digestive system under physiological conditions. After ice storage of the threadfin bream without gutting, a high SSP activity was detected only in the belly muscle because of SSP leaked from the viscera. Therefore, it is desirable to use edible proteinase inhibitor to inactivate the leaked SSP during production of surimi-based products or to take effective measures to prevent the proteinase leakage during post-harvest storage.
Molecular weights of trypsin, chymotrypsin, and lipase from anterior intestine and pyloric caeca of Totoaba macdonaldi were evaluated, as well as optimum temperature and pH for activity of the proteases. Trypsin was 24.1 kDa and effectively hydrolyzed Nα-benzoyl-DL-arginine 4-nitroanilide hydrochloride at optimum pH and temperature of 8 and 65°C, respectively. Chymotrypsin was 25.9 kDa and showed higher hydrolytic activity for N-benzoyl-L-tyrosine ethyl ester at pH 8 and 45°C, with a wider range of statistically similar activity values. Two pancreatic lipases of 70.2 and 47.5 kDa were detected, which could be the uncleaved and the final form of a colipase-dependent pancreatic lipase, since enzyme activity was detected without supplementation of bile salts and supplementing them inhibited activity.
Anionic trypsin from albacore tuna spleen was purified by chromatographic separations on Q-Sepharose, Superdex 75 and Arginine Sepharose 4B. The trypsin migrated as single bands in both SDS-PAGE and native-PAGE. The molecular weight of purified trypsin was estimated to be 30 kDa using SDS-PAGE. The enzyme exhibited maximal activity at pH 9.0 and 55 °C for hydrolysis of Boc-Val-Pro-Arg-MCA. pH and temperature stabilities of the trypsin were well maintained in the pH range of 6–11 and over a temperature range from 20 up to 50 °C. The enzyme was effectively inhibited by soybean trypsin inhibitor, N‑tosyl‑L‑phenyl‑alanine chloromethyl ketone (TLCK) and Pefabloc SC. The N-terminal amino acid sequence of 20 residues of the purified enzyme was IVGGYECQAHSQPHQVSLNA, which is highly homologous to other fish trypsins. The k cat /K m of the enzyme for Boc-Val-Pro-Arg-MCA was 2.60 ± 0.07 s ⁻¹ mM ⁻¹ . Purified trypsin also hydrolysed fish muscle proteins, suggesting its effectiveness in degradation of food proteins.
Partitioned trypsin from spleen of albacore tuna (Thunnus alalunga) by ATPS was evaluated for its potential applications in laundry detergents and in the recovery of carotenoprotein from Pacific white shrimp (Litopenaeus vannamei) shells. The partitioned trypsin was extremely stable toward various surfactants and bleach agents and showed excellent stability and compatibility with commercial liquid and solid detergents. Additionally, partitioned trypsin showed an efficient hydrolysis and recovery of carotenoprotein from Pacific white shrimp shells. The carotenoprotein recovery was maximized by the hydrolysis of shrimp shells using 0.8 trypsin units/g shrimp shells at 25 °C for 45 min and shrimp shells/buffer ratio of 1:2 (w/v). Carotenoprotein consisted of 72.4% protein, 18.8% lipid, 7.1% ash, 1.6% chitin, and 73.3 µg total astaxanthin/g sample. It was rich in essential amino acids. When the hydrolytic activities of albacore tuna and bovine trypsins used in the extraction of carotenoprotein in Pacific white shrimp shells were compared, the recovery efficacy of protein and pigment by albacore tuna trypsin was similar to that achieved by bovine trypsin. These results suggest that albacore tuna trypsin could be used as an ideal choice for application in detergent formulations and for extraction of carotenoproteins.
In the present study, the alkaline protease has been extracted from the fish processing waste using ammonium sulphate fractionation followed by ion-exchange chromatography on sephadex G-25 and on DEAE column with a 4.0 fold increase in purification of yield 7.7%. The molecular weight of the purified protease was found to be 33 kDa as determined by SDS-PAGE. The optimum temperature was found to be 30 °C at pH 8. The activation energy (Ea) for casein hydrolysis and temperature quotient (Q10) was found to be 38.25 kJ/mol and 1.65, respectively. The kinetic constants km, Vmax, kcat, and kcat/km and thermodynamic parameters ΔH*, ΔS*, ΔG*, ΔG*E-S, and ΔG*E-T revealed high affinity of the fish protease for casein. Using CD spectroscopy it was found that the fish protease has 32.7% alpha-helical, 32.8% β-turn and 34.5% random coil. 3D structure of target protein was predicted by homology modeling. Ramachandran plot revealed that the total residues in favored, allowed and outlier regions are 96.6%, 2.3%, and 1.1% residues. The biological function of the modeled fish protease was predicted by COACH based on the I-TASSER model, suggests that the fish protease may be exploited as biocatalyst in various industrial applications and processes. AutoDock 4.2.6. was used to study the protein-ligand interactions, which may lead to the discovery of novel semisynthetic enzymes from renewable biowaste.
Two trypsins (A and B) from the liver of albacore tuna (Thunnus alalunga) were purified to homogeneity using a series of column chromatographies including Sephacryl S-200, Sephadex G-50 and Diethylaminoethyl-cellulose. Purity was increased to 80.35- and 101.23-fold with approximately 3.1 and 19.2% yield for trypsins A and B, respectively. The molecular weights of trypsins A and B were estimated to be 21 and 24kDa, respectively, by SDS-PAGE and size exclusion chromatography. Both trypsins showed only one band on native-PAGE. Trypsins A and B exhibited the maximal activity at 60°C and 55°C, respectively, and had the same optimal pH at 8.5 using N(α)-p-Tosyl-L-arginine methyl ester hydrochloride (TAME) as a substrate. Stabilities of both trypsins were well maintained at a temperature up to 50°C and in the pH range of 7.0 to 11.0 and were highly dependent on the presence of calcium ion. The inhibition test demonstrated strong inhibition by soybean trypsin inhibitor and TLCK. Activity of both trypsins continuously decreased with increasing NaCl concentration (0-30%). The N-terminal amino acid sequence of 20 residues of the two trypsin isoforms had homology when compared to those of other fish trypsins.
An antibacterial peptide was purified from an acidified gill extract of the pufferfish Takifugu pardalis. The acidified gill extract was put through a Sep-Pak C18 solid phase extraction cartridge using a stepwise gradient and divided into a flow-through (F.T.) and 60% methanol fraction (RM 60). Among the eluents, RM 60 had potent antibacterial activity against Bacillus subtilis KCTC 1021. RM 60 was partially purified on a cationic-exchange column (SP-5PW) by a linear gradient, and the antibacterial peptide was then further purified, using a series of cationic-exchange and C_{18} reversed-phase HPLC columns. For characterization of the purified peptide, its molecular weight and amino acid sequence were analyzed by MALDI-TOF MS and Edman degradation. The molecular weight of the peptide was about 1171.6 Da. The amino acid sequence of the peptide was partially determined as: STKEKAPRKQ. A comparison of the N-terminal amino acid sequence of the purified peptide with that of other known polypeptides revealed high homology with the N-terminus of the histone H3 protein, which belongs to the histone H3 family. Thus, this peptide was designated as a puffer fish gill (PFG)-related antimicrobial peptide. This is the report to describe an antimicrobial function for the N-terminus of histone H3 of an animal species. The findings suggest that this peptide plays a significant role in the innate defense system of the pufferfish.
Characteristics of endogenous proteases of shrimp, Acetes vulgaris (AP) and Macrobrachium lanchesteri (MP) as well as the changes in proteolytic activity during Kapi production were investigated. Maximal activity of AP and MP was found at pH 7, 60C and pH 8, 60C, respectively. Activity of both proteases decreased with increasing NaCl concentration (0–30%). Both extracts were strongly inhibited by N‐ethylmaleimide‐phenylmethane‐sulfonyl fluoride (PMSF) and soybean trypsin inhibitor (SBTI), suggesting that major proteases belonged to serine proteases. This was coincidental with high trypsin activity toward BAPNA and chymotrypsin activity toward BTEE. Proteolytic activity, trypsin and chymotrypsin were detectable throughout Kapi fermentation. The activity was decreased when salting was implemented. Nevertheless, activities increased continuously with increasing fermentation time. During Kapi production, proteins underwent degradation as indicated by the formation of oligopeptides and disappearance of myosin heavy chain and actin. Therefore, both endogenous and microbial proteases were more likely involved in proteolysis of shrimp during Kapi production.
Practical Applications
Kapi, traditional salted shrimp paste, is usually used as a condiment to enhance the palatability of many Thai foods. Recently, small shrimp Acetes Vulgaris and Macrobrachium lanchesteri have become the new alternative raw materials for Kapi production because of their availability. During fermentation, protein hydrolysis is induced by endogenous proteases in shrimp as well as those produced by halophilic bacteria. Those changes medicated by proteolysis can be associated with the final characteristics of Kapi.
An antibacterial peptide from skin extract of the catfish Silurus asotus was purified and characterized. The acidified skin extract was put through a Sep-Pak C18 solid phase extraction cartridge using a stepwise gradient and divided into flow-through (F.T.), 10% methanol-elute (RM10), 60% methanolelute (RM60), and 100% methanol-elute (RM100) fractions. RM10, RM60, and RM 100 showed antimicrobial activity against Escherichia coli D31. On the other hand, the F.T. fraction did not show antimicrobial activity. Among the various fractions, RM 60 had the highest activity. RM 60 was partially purified on a cation exchange column (CM52) by a stepwise gradient. The ammonium acetate (pH 5.15) 0.02 M ? 0.8 M fraction showed antimicrobial activity. Then an antimicrobial peptide was purified using a 0.6M fraction with strong antibacterial activity through a series of five C18 reversed-phase HPLC columns. For the characterization of the purified peptide, the molecular weight and amino acid sequence were analyzed by MALDI-TOF MS and Edman degradation. The molecular weight of this peptide was about 4182.1 [M+H]?. The amino acid sequence of this peptide was partially determined as follows: PALXXKARREAKVKF. These findings suggest that this peptide plays a significant role in the innate defense system of catfish skin.
This study explores the feasibility of utilizing enzymes derived from fish wastes as a low cost depilatory agent. These enzymes are eco-friendly alternative to the conventional lime-sulphide dehairing process. The crude enzyme extract was partially purified with a yield of 60% (Specific protease activity is 4683 U/mg of protein). Optimum pH and temperature for enhanced protease activity of partially purified protease were 8.0 and 30◦C, respectively. The partially purified protease was evaluated for its dehairing efficiency of goat skins. It was observed that the release of proteoglycans and saccharides in the spent liquor after dehairing was 4300 μg/mL and 28 μg/mL, respectively. In the wet blue stage, the enzyme treated leathers exhibited shrinkage temperature of about 110±2◦C, which was found to be on par with that of control leathers. The morphological features were examined through histological and scanning electron microscopic analysis, which reveals a complete removal of hair in the skin matrix. The organoleptic properties and visual assessment of crust leathers were carried out. The physical strength of the crust leathers developed from the enzyme treatment has met the UNIDO norms. Moreover, a significant reduction in the pollution loads was observed in terms of total solids (81%), biological oxygen demand (90%), and chemical oxygen demand (88%) in the dehairing waste stream.
IntroductionProteasesTransglutaminasePolyphenoloxidaseTrimethylamine-N-Oxide DemethylaseLipaseReferences
Trypsin was purified to homogeneity from hepatopancreas of royal red prawn by ammonium sulphate (400g L−1-600g L−1 saturation) precipitation, Benzamidine affinity column and MonoQ column chromatography. Trypsin was purified to 91.0-fold with a yield of 12.1% and showed a single band on native-PAGE. Trypsin had a molecular weight of 27 kDa as estimated by SDS-PAGE. The optimal pH and temperature for Boc-Val-Pro-Arg-MCA hydrolysis were 9.0 and 50°C, respectively, while purified enzyme was stable to heat treatment up to 50°C and over a pH range of 7.0-11.0. Trypsin activity was strongly inhibited by soybean trypsin inhibitor, N-p-tosyl-L-lysine chloromethyl ketone (TLCK) and Pefabloc SC and was partially inhibited by ethylenediaminetetraacetic acid (EDTA). Apparent Km value of trypsin was 0.28 µM and kcat value was 656.44s−1 for Boc-Val-Pro-Arg-MCA. The N-terminal amino acid sequence of 20 residues of trypsin was IVGGTVATPYEFPYQISFQD, which was highly homologous with those from other species of prawn. Purified trypsin also showed high collagenolytic activity toward prawn and shrimp collagens, suggested that it’s possible to use for collagens hydrolysis.
This chapter is on the major proteinases (also known as proteolytic enzymes or proteases) that are produced by the digestive glands of marine animals. Like the proteinases from plants, animals, and microorganisms, digestive proteinases from marine animals are hydrolytic in their action, and catalyze the cleavage of peptide bonds with the participation of water molecules as reactants. In terms of current food industry (and other industrial) applications, proteinases are by far the most important and most widely used group of enzymes (1, 2). They are used to improve product handling characteristics and texture of cereals and baked goods, enhance the drying as well as the quality of egg products, tenderize meat, recover proteins/peptides from bones, and hydrolyze blood proteins. Proteinases are used for the production of protein hydrolysates, reduction of stickwater vis-cosity, and for roe processing. They are used to make pulses and rennet pud-dings, and in cheesemaking/cheese ripening. Proteinases are also used for biomedical applications to reduce tissue inflammation, dissolve blood clots, pro-mote wound healing, activate hormones, diagnose candidiasis, and to aid or fa-cilitate digestion (3, 4). There is great demand for enzymes with the right combination of prop-erties for a plethora of industrial applications. Industrial proteinases are mostly derived from microorganisms, and to a lesser extent from plant and animal sources (3). So far, there is only very limited use of marine proteinases by industry. The reasons for the rather limited use of marine digestive pro-teinases include the relative paucity of basic information on these enzymes, the cyclical nature of the source material (which precludes supply in a steady manner), and the stereotypical attitude of the general public toward the source material: fish offal. However, marine animals comprise several thousands of very diverse species that subsist under different habitat conditions (5, 6).
Some of these differences are in terms of parameters such as temperature, pressure, salinity, light intensity, and aeration. Over the years, marine animals have adapted to different environmental conditions, and these adaptations, to-gether with inter- and intraspecies genetic variations, have resulted in diges-tive proteinases with certain unique properties compared with their counterpart enzymes from land animals, plants, or microorganisms (6–9). Some of the distinctive features of marine digestive proteinases include a higher catalytic efficiency at low reaction temperatures, lower thermostabil-ity, cold stability, and substantial catalytic activity/stability at neutral to alka-line pH (6, 9, 10). Homologous digestive proteinases from marine animals may also differ from one another in their response to specific inhibitors, for example, α-macroglobulin from beef serum inhibited sheephead and bluefish trypsins to different extents (11, 12). Proteinases from the same species may also display season-dependent differences in properties. For example, a thy-roid proteinase from winter turbot has different iodoacetate sensitivity than the enzyme from spring turbot. Digestive proteinases from marine animals may also differ in their sensitivity to pressure (12, 13), in their ability to hy-drolyze native protein substrates (14–16), or in their sensitivities to pH, acids, alkali, salts, urea, detergents, and other materials.
Worldwide sales of industrial enzymes were estimated at about $1.5 bil-lion for 1998 (17). It is suggested that some of the unique properties of marine enzymes may be exploited in various food applications, and thereby, obtain a share of the lucrative industrial enzymes market to increase profits for the fish-ing industry. For example, the higher catalytic activity at low reaction temper-atures may be used to process foods at low temperature to reduce energy costs and destruction of heat-labile essential food components (9). The lower ther-mostability of marine digestive proteinases (compared with their homologues from other animals, plants, and microorganisms), would permit their ready in-activation by milder heat treatments, while their ability to denature native pro-tein substrates may be advantageous in fruit juice manufacture, for the inactivation of undesirable endogenous enzymes such as polyphenol oxidases (PPO) or pectin-methyl esterase (PME). Some marine enzymes are currently being used as food process aids (18–21), and this use of marine en-zymes is expected to increase due to the possibilities afforded by recombinant DNA technology.
Proteolytic activities of splenic extract from three tuna species including skipjack tuna (Katsuwonus pelamis), yellowfin tuna (Thunnus albacores) and tongol tuna (Thunnus tonggol) were studied. Optimal activity of splenic extract from all tuna species was at pH 9.0 and 55C when casein was used as a substrate. Among all species tested, yellowfin tuna showed the highest activity, followed by skipjack tuna and tongol tuna. The proteolytic activity was strongly inhibited by soybean trypsin inhibitor, TLCK and partially inhibited by ethylenediaminetetraacetic acid. E-64, N-ethylmaleimide, iodoacetic acid, TPCK and pepstatin A showed no inhibition. The effect of NaCl and CaCl2 on proteolytic activity was also investigated. Activities continuously decreased as NaCl concentration increased, and no activity remained in the presence of 30% NaCl. On the other hand, activities increased as CaCl2 concentration increased. The highest activity was obtained in the presence of 1 mM CaCl2. SDS-substrate gel electrophoresis revealed that major proteinases in splenic extract from different tuna species were different in apparent molecular weights and sensitivity to TLCK. Although the major activity bands of all species were strongly inhibited by soybean trypsin inhibitor, varying sensitivity to TLCK probably implied the differences in binding characteristic of enzyme to substrate and/or inhibitors. The results suggest that major proteinases in spleen of all tuna species were trypsin-like serine proteinases.
This paper describes simple methods for diversified utilization of fish stomach and intestines for food, feed and biotechnological purposes. A high concentration of pepsin (9 g/litre) was obtained by ultrafiltration of the aqueous phase from a cod-stomach silage preserved with formic acid. A concentrate of trypsin-like enzymes could be obtained by ultrafiltration of fish sauce produced by salt fermentation of cod intestines. The permeate from the ultrafiltration contained the major part of proteinous material (peptides and amino acids), and had a palatable taste similar to traditional fish sauce. The results show that 7 litres of enzyme concentrate and 50 litres of fish sauce can be produced from 100 kg cod intestines within 16 days of fermentation at 27°C.
Proteolytic enzyme are widely used as food processing aids. In recent years, proteolytic enzymes from marine organisms have been used as process aids. Other possible uses of enzymes form marine animals in the food industry are based on the unique properties of these enzymes. Examples of unique properties include ease of heat denaturation, high molecular activity at low temperatures and ability to catalyze hydrolysis of native proteins. The possible advantage of using an enzyme form a marine organism in specific applications rather than a conventional plant, animal or microbial source is discussed.
Trypsin was purified from the pyloric caeca of rainbow trout (Oncorhynchus mykiss) by (NH4)2SO4 fractionation, hydrophobic interaction chromatography, and affinity chromatography. The isolated enzyme had a single band on SDS-PAGE with an estimated molecular mass of 25.7 kDa. The trypsin was stable at pH 5-11 for 30 min at 30-degrees-C, and its maximal activity against alpha-benzoyl-L-arginine p-nitroanilide (L-BAPNA) was between pH 9 and 10. The enzyme was stable up to about 40 and 50-degrees-C for 30 min, at pH 5.4 and 8.0, respectively, but the thermal stability was highly calcium dependent (10-15 mM). Maximal activity of the enzyme against L-BAPNA was around 60-degrees-C. The catalytic efficiency (k(cat)/K(M)) of the trout trypsin was about 12-15 fold higher than that of bovine trypsin, when measured at 10-20-degrees-C, with L-BAPNA as a substrate, and its caseinolytic activity, at these temperatures, was about 1.6-2 times higher than that of the bovine enzyme, reflecting adaption of the activities of the fish trypsin to low temperatures.
Two trypsins, designated as trypsin A and trypsin B, have been purified from the hepatopancreas of carp. The purification procedures consisted of ammonium sulfate fractionation, and chromatographies on DEAE-Sephacel, Ultrogel AcA54 and Q-Sepharose. Trypsin A was purified to homogeneity with the molecular mass of approximately 28 kDa, while trypsin B gave two close bands of 28.5 kDa and 28 kDa on sodium dodecylsulfate polyacrylamide gel electrophoresis both under reducing and non-reducing conditions. On native-PAGE, both trypsin A and trypsin B showed a single band. Trypsin A and trypsin B revealed optimum temperature of 40°C and 45°C, respectively, and shared the same optimum pH 9.0 using Boc-Phe-Ser-Arg-MCA as substrate. Both enzymes were effectively inhibited by trypsin inhibitors and their susceptibilities were similar. The NH2-terminal amino acid sequences of trypsin A and trypsin B were determined to 37th and 40th amino acid residue, respectively. Their sequences were very homologous, but not identical to that of a trypsin-type serine proteinase from carp muscle and these of other trypsins. Immunoblotting test using the antibody raised against trypsin A cross-reacted with trypsin B positively.
ABSTRACTA 38.5 kDa alkaline protease from pyloric caeca of tambaqui (Colossoma macropomumj, a tropical freshwater fish, was partially purified in three steps: thermal treatment (45Cfor 30 min), salting-out (ammonium sulfate at 40–80% of saturation) and gel filtration (Sephadex G-75), The purification and yield were 51.2-fold and 40%, respectively. The effects of pH, temperature, inhibitors, and substrates on proteolytic activities of partially purified enzyme were investigated. The optimum pH was 9.5, while the optimum temperature was 60C. This alkaline proteolytic activity remained unaltered after 30 min incubation at 55C. Active site inhibition provided additional evidence that this activity is attributed to a trypsin-like enzyme.
An alkaline protease was extracted from the viscera (intestine) of Nile tilapia, Oreochromis niloticus, the second most important fish in Brazilian aquaculture. This enzyme is usually discarded among the tons of waste produced by its processing. The enzyme was purified in three steps: heat treatment, ammonium sulphate fractionation and Sephadex G-75 gel filtration, presenting an yield and purification of 30% and 22-fold, respectively, and showing a single band by SDS-PAGE (23.5 kDa). This enzyme showed Km for the hydrolysis of benzoyl-dl-arginine-p-nitroanilide (BAPNA) equal to 0.755 ± 0.008 mM, an optimum temperature at 50 °C, was stable for 30 min at 50 °C, and optimum pH of 8.0. The protease was strongly inhibited by Al3+ and Cd2+, followed by Cu2+, Hg2+, Zn2+ and Co2+. Inhibition by PMSF and specific trypsin inhibitors provided additional evidences that this activity can be attributed to a trypsin-like enzyme.
Maximum amidolytic and esterolytic activities of crayfish hepatopancreatic trypsins occurred in a pH range of 7.5–8.5. Activity of trypsin A increased by addition of 0.5 mM of Ca2+ ions; however, other trypsins were not affected by this concentration. Kinetic properties of crayfish trypsins toward esterolytic reaction were similar, but those for amidolytic reaction were different. Activation energies for esterolytic reaction were approximately 6.4–9.0 kcal/mole, while those for amidolytic reaction were between 5.9 and 6.9 kcal/mole.
Trypsin was purified from the pyloric ceca of walleye pollock (Theragra chalcogramma) by gel filtration on Sephacryl S-200 and Sephadex G-50. The final enzyme preparation was nearly homogeneous in sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) and the molecular mass of the enzyme was estimated to be 24 kDa by SDS–PAGE. Trypsin activity was effectively inhibited by serine protease inhibitors, such as soybean trypsin inhibitor and TLCK. Trypsin had maximal activities at around pH 8.0 and 50 °C for the hydrolysis of Nα-p-tosyl-l-arginine methyl ester hydrochloride. Trypsin was unstable above 30 °C and below pH 5.0, and was stabilized by calcium ions. Walleye pollock trypsin was more thermally unstable than trypsin from the Temperate Zone fish and Tropical Zone fish. The N-terminal amino acid sequence of the trypsin, IVGGYECTKHSQAHQVSLNS, was found, and the sequential identity between the walleye pollock trypsin and Frigid Zone fish trypsin was higher (85–100%) than with Temperate Zone fish trypsin (75–90%), Tropical Zone fish trypsin (75–85%), or mammalian trypsin (60–65%).
Trypsins, TR-S and TR-P, from the viscera of true sardine (Sardinops melanostictus) and from the pyloric ceca of arabesque greenling (Pleuroprammus azonus), respectively, were purified by gel filtration and anion-exchange chromatography. Final enzyme preparations were nearly homogeneous in sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) and the molecular weights of both enzymes were estimated to be 24,000 Da by SDS–PAGE. The N-terminal amino acid sequences of the TR-S, IVGGYECKAYSQPWQVSLNS, and TR-P, IVGGYECTPHTQAHQVSLNS, were found. The TR-S and TR-P had maximal activities at around pH 8.0 for hydrolysis of Nα-p-tosyl-l-arginine methyl ester. Optimum temperature of the TR-S and TR-P were 60 and 50 °C, respectively. The TR-S and TR-P were unstable at above 50 and 30 °C, respectively, and below pH 5.0. Both TR-S and TR-P were stabilized by calcium ion.
Two isozymes of trypsin (TR-I and TR-II) were purified from the viscera of Japanese anchovy (Engraulis japonica) by gel filtration and anion-exchange chromatography. Final enzyme preparations were nearly homogeneous in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and the molecular weights of both enzymes were estimated to be 24,000 Da by SDS-PAGE. The N-terminal amino acid sequences of the TR-I, IVGGYECQAHSQPHTVSLNS, and TR-II, IVGGYECQPYSQPHQVSLDS, were found. Both TR-I and TR-II had maximal activities at around pH 8.0 and 60C for hydrolysis of Nα-p-tosyl-L-arginine methyl ester hydrochloride. The TR-I and TR-II were unstable at above 50C and below pH 5.0 and were stabilized by calcium ion.
The amino acid sequence of pancreatic trypsin from the spiny Pacific dogfish (Squalus acanthias) has been determined and compared with the sequences of bovine and porcine trypsin. Dogfish trypsin contains one less amino acid residue (222) than the other two enzymes. Two-thirds of the residues in corresponding positions in dogfish and bovine trypsin are identical and the sequences ofall three enzymes are homologous. Of the 223 amino acid residues of bovine trypsin, 77 are replaced without significant changes in function. Seven replacements, all conservative, occur in the interior of the protein; the remainder are on the surface. All residues known to be components of the active site of bovine trypsin are present in corresponding positions in dogfish trypsin. Comparison of the three enzymes suggests calcium binding sites in dogfish trypsin. A corrected sequence of bovine trypsin identifies residue 67 as Asn and residues 84-87 as Ser-Asn-Thr-Leu.
In the absence of changes in functional mRNA levels, stimulation of the pancreas with caerulein, a peptide analog of cholecystokinin, has been previously shown to increase the synthesis of anionic but not cationic trypsinogen. To look for structure-function correlations, a high-yield, full-length cDNA library has been constructed from canine pancreatic poly(A)+ mRNA. Full-length clones coding for the two major trypsinogen isoenzyme forms have been identified by colony hybridization and verified by in vitro translation of hybrid-selected mRNA in the presence of microsomal membranes and an optimal redox potential. Disulfide-bonded translation products were separated and identified by two-dimensional isoelectric focusing-sodium dodecyl sulfate-gel electrophoresis. Nucleotide sequence analysis allowed us to deduce the amino acid sequences for the anionic and cationic forms of canine trypsinogen, which contain 232 and 231 residues, respectively (77% amino acid identity), and the 15-residue amino terminal signal sequences (53% amino acid identity) associated with the two presecretory forms. Measurements of relative and absolute mRNA levels, when related to relative protein synthesis values, indicated that the translational efficiency of anionic trypsinogen mRNA exceeded that of cationic trypsinogen mRNA by 1.5- to 2.9-fold under basal conditions. Analysis of the 5' noncoding regions of trypsinogen mRNAs revealed a striking conservation of sequence (10 of 12 bases) between dog and rat anionic trypsinogen forms. This contrasted markedly with the divergence of the 5' noncoding regions observed between dog anionic and cationic trypsinogen mRNAs.
The amino acid sequence of porcine trypsin has been determined by sequenator analysis of the reduced and S-pyridylethylated protein and of eight suitably chosen peptide fragments. The fragments were the products of cleavage by autolysis, cyanogen bromide, 2-(2-nitrophenylsulfenyl)-3-methyl-3-bromoindolenine, hydroxylamine, and trypsin, respectively. All but the last 2 of the 223 amino residues were uniquely placed by these analyses. Comparison of this sequence with that of bovine trypsin indicated 82% identity, corresponding to a unit evolutionary period of approximately 3 million years. Of the 41 amino acid substitutions, 36 are on the surface of the bovine enzyme and 5 in the interior. The latter are of the conservative type. All residues known to be components of the active site of bovine trypsin are present in identical positions in porcine trypsin, but the porcine enzyme does not possess the calcium binding site identified in the bovine enzyme.
Using an improved method of gel electrophoresis, many hitherto unknown
proteins have been found in bacteriophage T4 and some of these have been
identified with specific gene products. Four major components of the
head are cleaved during the process of assembly, apparently after the
precursor proteins have assembled into some large intermediate
structure.
Two fish acid proteinases designated acid proteinase I and II were found and isolated by (NH4)2SO4 fractionation, CM-cellulose chromatography and gel filtration on Sephadex G-100. The final preparations were judged nearly homogeneous by multiple criteria. The molecular criteria. The molecular weights of the enzymes I and II were determined by the sedimentation equilibrium method to be 37 000 and 33 400, respectively. The sedimentation coefficients (S0 20, w) were 3.06 and 3.09, respectively. Enzymes I and II contained similar amino acid composition except for the contents of histidine, arginine, threonine, serine and proline. Enzymes I and II differed from each other in optimal pH and stability at pH 7. Each enzyme could scarcely hydrolyze a synthetic pepsin substrate, N-acetyl-L-phenylalanyl-3,5-diiodo-L-tyrosine (APDT). Both of the enzymes were inhibited by acid proteinase specific reagents: pepstatin, diazoacetyl-DL-norleucine methyl ester (DAN), 1,2-epoxy-3-(p-nitrophenoxy) propane (EPNP) and p-bromophenacyl bromide. These results indicate fish enzymes are similar to mammalian pepsin and microbial acid proteinases in their active site structure having two different carboxyl groups, although they differ in regard to a number of molecular and enzymatic properties.
1. Two trypsin-like enzymes, assayed by their amidase activity with N-alpha-benzoyl-DL-arginine-p-nitroanilide (DL-BAPNA) as the substrate, were isolated from the gut of the arctic fish capelin (Mallotus villosus). 2. Purification involved affinity chromatography (Benzamidine-CH-Sepharose 4B) of the 30 to 70% (NH4)2SO4 precipitation fraction of a crude extract of the gut, followed by DEAE-Sephadex chromatography, yielding two enzymes, designated Enzyme I and II. 3. Both enzymes had MW of about 28,000 as determined by SDS-electrophoresis. Their isoelectric points were 5.6-5.9 (Enzyme I) and 5.1-5.3 (Enzyme II) and they had similar amino acid composition. 4. Both enzymes were inhibited by standard trypsin inhibitors including the serine protease inhibitor phenylmethyl sulphonyl fluoride (PMSF), but not by the chymotrypsin inhibitor L-1-tosylamide-2-phenylethyl chloromethyl ketone (TPCK). 5. The enzymes had a pH optimum of 8-9 and their stability was not affected by CaCl2. Low pH (2.3) caused an initial rapid loss of enzyme activity, followed by relatively slow decomposition of the activity remaining after 1 hr at 4 degrees C. 6. The enzymes had an apparent temperature optimum of 42 degrees C, resulting from rapid self digestion at higher temperatures.
Pancreatic cDNA libraries from Atlantic salmon (Salmo salar) were constructed and screened with salmon trypsin-specific probes. Five clones containing near full-length transcripts were selected for further characterization. Comparison of deduced amino acid sequences revealed that all variants possessed the canonical serine protease catalytic triad, consisting of histidine, aspartic acid and serine residues, a substrate-binding pocket with aspartic acid at the bottom, and 12 cysteine residues comprising six disulphide bridges. Translation in vitro of one of the trypsin clones produced a protein with the expected molecular mass of 24.5 kDa. Three of the Atlantic salmon trypsins (SalTRP-I, SalTRP-IA and SalTRP-IB) possessed very similar sequences and may represent allelic variants encoded by the same gene focus; however, existence as tetraploid loci or isoloci where disomic inheritance is incomplete may also exist in Atlantic salmon and cannot be excluded. Two other trypsin clones (SalTRP-II and SalTRP-III) are probably encoded by separate gene loci. Analysis of genomic DNA by Southern blotting and hybridization to a trypsin probe showed a complex pattern, indicative of a large number of gene loci for trypsin in Atlantic salmon. The charged amino acid distribution showed that four of the Atlantic salmon trypsin clones encoded anionic forms of the enzyme, while the fifth clone represented a cationic variant. Multiple alignments of the Atlantic salmon trypsin sequences with trypsin, chymotrypsin and elastase from different species placed all Atlantic salmon sequences approximately equidistant from trypsins of other species. Interestingly, the distance between the anionic and cationic variants from Atlantic salmon was similar to the distance between salmon and mammalian trypsins, revealing an early separation of these two types of trypsin, possibly prior to the derivation of fish during evolution. A structural model based on X-ray diffraction studies of the salmon trypsin protein was very similar to that of the mammalian enzyme. All residues which differ in charge between anionic and cationic trypsins were located at exposed regions of the proteins.
The cDNAs encoding two different anionic forms of Atlantic cod trypsinogen have been isolated and sequenced. The nucleotide sequences include the 5'-noncoding and 3'-noncoding regions in addition to preproenzymes of 241 amino acids. These consist of hydrophobic signal peptides, activation hexapeptides and trypsins of 222 amino acid residues. The cod trypsins contain all the major structural features common to trypsins such as the catalytic triad His57, Asp102 and Ser195. Furthermore, the obligatory Asp189 and the six disulphide bonds are conserved. Eight amino acid residues are different between the isozymes, leading to a difference of four charges. Both cod trypsins are one-amino-acid-residue shorter than most mammalian trypsins as a result of deletion of proline at position 152, and have a high methionine content. In addition, the cod preproenzyme signal and activation peptides differ markedly from their mammalian analogues. The amino acid identity between the cod and bovine trypsins is approximately 60%.
The spectrophotometric procedure proposed by Schwert and Takenaka for the assay of chymotrypsin and trypsin has been modified and extended to include the application to N-benzoyl-L-tyrosine ethyl ester and α-p-toluenesulphonyl-L-arginine methyl ester. The greater degree of sensitivity and specificity thus achieved permits the determination of traces of chymotrypsin in the presence of relatively large amounts of trypsin and vice versa. A similar spectrophotometric procedure for the assay of thrombin is described.
Trypsin from pyloric caeca of Monterey sardine was purified by fractionation with ammonium sulfate, gel filtration, affinity and ionic exchange chromatography. Fraction 102, obtained from ionic exchange chromatography, generated one band in sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and isoelectric focusing. The molecular mass of the isolated trypsin was 25 kDa and showed esterase-specific activity on Nalpha-p-tosyl-L-arginine methyl ester (TAME) that was 4.5 times greater than amidase-specific activity on N-benzoyl-L-arginine-p-nitroanilide. The purified enzyme was partially inhibited by the serine-protease phenyl-methyl-sulfonyl fluoride (PMSF) inhibitor and fully inhibited by the soybean trypsin inhibitor (SBTI) and benzamidine, but was not inhibited by the metallo-protease inactivator EDTA or the chymotrypsin inhibitor tosyl-L-phenylalanine chloromethyl-ketone. The optimum pH for activity was 8.0 and maximum stability was observed between pH 7 and 8. A marked loss in stability was observed below pH 4 and above pH 11. Activity was optimum at 50 degrees C and lost activity at higher temperatures. The kinetic trypsin constants K(m) and k(cat) were 0.051 mM and 2.12 s(-1), respectively, while the catalytic efficiency (k(cat)/K(m)) was 41 s(-1) mM(-1). General characteristics of the Monterey sardine trypsin resemble those of trypsins from other fish, especially trypsins from the anchovy Engraulis japonica and Engraulis encrasicholus and the sardine Sardinops melanostica.
A trypsin fraction was isolated from the pyloric ceca of New Zealand farmed chinook salmon (Oncorhynchus tshawytscha) by ammonium sulfate fractionation, acetone precipitation and affinity chromatography. The chinook salmon enzyme hydrolyzed the trypsin-specific synthetic substrate benzoyl-DL-arginine-p-nitroanilide (DL-BAPNA), and was inhibited by the general serine protease inhibitor phenyl methyl sulfonyl fluoride (PMSF), and also by the specific trypsin inhibitors - soybean trypsin inhibitor (SBTI) and benzamidine. The enzyme was active over a broad pH range (from 7.5 to at least pH 10.0) at 25 degrees C and was stable from pH 4.0 to pH 10.0 when incubated at 20 degrees C, with a maximum at pH 8.0. The optimum temperature for the hydrolysis of DL-BAPNA by the chinook salmon enzyme was 60 degrees C, however, the enzyme was unstable at temperatures above 40 degrees C. The molecular mass of the chinook salmon trypsin was estimated as 28 kDa by SDS-PAGE.
Two anionic trypsins (A and B) were purified to homogeneity from yellowfin tuna (Thunnus albacores) spleen by a series of column chromatographies including Sephacryl S-200, Sephadex G-50 and DEAE-cellulose. Purity was increased to 70.6- and 91.5-fold with approximately 2.8% and 15.6% yield for trypsin A and B, respectively. The apparent molecular weight of both trypsins was estimated to be 24 kDa by size exclusion chromatography and SDS-PAGE. Both trypsin A and B appeared as a single band on native-PAGE. Trypsin A and B exhibited the maximal activity at 55 and 65 degrees C, respectively, and had the same optimal pH at 8.5 using TAME as a substrate. Both trypsins were stable to heat treatment up to 50 degrees C and in the pH range of 6.0 to 11.0. Both trypsin A and B were stabilized by calcium ion. The activities were inhibited effectively by soybean trypsin inhibitor, TLCK and partially inhibited by EDTA, but were not inhibited by E-64, N-ethylmaleimide, iodoacetic acid, TPCK and pepstatin A. Activity of both trypsins continuously decreased with increasing NaCl concentration (0-30%). Apparent Km and Kcat of trypsin A and B for TAME were 0.2-0.33 mM and 66.7-80 S(-1), respectively. The N-terminal amino acid sequences of trypsin A, IVGGYECQAHSQPHQVSLNA, and trypsin B, IVGGYECQAHSQPPQVSLNA, indicated the high homology between both enzymes.
Trypsin from tongol tuna (Thunnus tonggol) spleen was purified to 402-fold by ammonium sulfate precipitation, followed by a series of chromatographic separations. The molecular mass of trypsin was estimated to be 24 kDa by size-exclusion chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Trypsin appearing as a single band on native PAGE showed the maximal activity at pH 8.5 and 65 degrees C. It was stable in a wide pH range of 6-11 but unstable at the temperatures greater than 50 degrees C. The enzyme required calcium ion for thermal stability. The activity was strongly inhibited by 1.0 g/L soybean trypsin inhibitor and 5 mM TLCK and partially inhibited by 2 mM ethylenediaminetetraacetic acid. Activity was lowered with an increasing NaCl concentration (0-30%). The enzyme had a Km for Nalpha-p-tosyl-L-arginine methyl ester hydrochloride of 0.25 mM and a Kcat of 200 s-1. The N-terminal amino acid sequence of trypsin was determined as IVGGYECQAHSQPHQVSLNA and was very homologous to other trypsins.
A modified spectrophotometric determination of chymotrypsin, trypsin, and thrombin
- Hummel
Purification and characterisation of trypsin from the pyloric caeca of rainbow trout (Oncorhynchus mykiss)
- Krisjansson