Digestive proteases of three carps Catla catla, Labeo rohita and Hypophthalmichthys molitrix: partial characterization and protein hydrolysis efficiency

Characteristics and functional efficacy of digestive proteases of Catla catla, catla, Labeo rohita, rohu and Hypophthalmichthys molitrix, silver carp were studied. Total protease activity was significantly (P < 0.05) higher in rohu (1.219 ± 0.059 U mg protein⁻¹ min⁻¹) followed by silver carp (1.084 ± 0.061 U mg  protein⁻¹ min⁻¹), and catla (0.193 ± 0.006 U mg  protein⁻¹ min⁻¹). Trypsin activity of silver carp and rohu was 89–91% higher than catla. Chymotrypsin activity was significantly (P < 0.05) higher in silver carp compared with rohu and catla. The protease activity of rohu and silver carp displayed bell‐shaped curves with maximum activity at pH 9; whereas in catla, maximum activity was found between pH 8 and 11. Inhibition of protease activity with soybean trypsin inhibitor (SBTI), phenylmethylsulfonyl fluoride (PMSF) revealed the presence of serine proteases and inhibition of activity with N‐α‐p‐tosyl‐L‐lysine‐chloromethyl ketone (TLCK) and N‐tosyl‐L‐phenylalanychloromethane (TPCK) indicated the presence of trypsin‐like and chymotrypsin‐like enzymes in all these three carps. SDS‐PAGE showed the presence of several protein bands ranging from 15.3 to 121.9 kDa in enzyme extracts of catla, rohu and silver carp. The substrate SDS‐PAGE evidenced the presence of various protease activity bands ranging from 21.6–93.7, 21.6–63.8 and 26.7–98.5 kDa for catla, rohu and silver carp respectively. In pH‐stat hydrolysis of Chilean fishmeal showed significantly (P < 0.05) higher degree of hydrolysis compared with soybean meal, silver cup (a commercial fish feed of Mexico) and wheat flour, with enzyme preparations of three fishes. The rate of hydrolysis was significantly (P < 0.05) higher in silver carp compared with others.     

Ontogenic changes in the digestive enzyme patterns and characterization of proteases in Indian major carp Cirrhinus mrigala

Digestive enzymes of Cirrhinus mrigala (Ham.) were studied during ontogenic development. Specific amylase activity was detected in first feeding fish. The enzyme activity decreased up to day‐18 and then it increased with the age of fish to reach the highest level on day‐34. Protease activity was 28.61 ± 8.90 mU mg protein^?1 min^?1 on day‐4 and increased with the age throughout the study period. Trypsin activity was 31.86 ± 1.12 mU mg protein^?1 min^?1 on day‐4. The activity decreased up to day‐10 and from day‐12 onwards increased up to day‐26. Chymotrypsin activity was 14.56 ± 2.74 mU mg protein^?1 min^?1on day‐4 and constantly increased up to day‐26. A significant increase in lipase activity was observed between days‐24 and 34. SDS‐PAGE and substrate SDS‐PAGE showed the diversity of protein (17.4–127.8 kDa) and protease activity bands (16.6–88.8 kDa) during ontogenesis. Soybean trypsin inhibitor, phenyl methyl sulphonyl fluoride, N‐α‐p‐tosyl‐l ‐lysine chloromethylketone and N‐tosyl‐l ‐phenylalanine chloromethylketone inhibited the protease activity up to 79.72–97.21, 65.55–94.83, 45.41–75.31 and 40.78–64.72%, respectively. Inhibition study in substrate SDS‐PAGE revealed the abundance of serine proteases and the presence of isoforms of trypsin and chymotrypsin. Ethylenediamine‐tetraacetate showed 5.56–22.78% inhibition of metal ion‐specific enzyme activity.     

Study of digestive proteinases and proteinase inhibitors of Daphnia carinata

Quantification of proteases activities and their class structure have been studied in a cladoceran, Daphnia carinata. Protease activity ranged from 0.28 to 0.55 Unit mg⁻¹ protein min⁻¹ with an average value of 0.42±0.06 Unit mg⁻¹ protein min⁻¹. Chymotrypsin activity was more than twofold higher (0.49±0.09 Unit mg⁻¹ protein min⁻¹) than the trypsin activity (0.21±0.02 Unit mg⁻¹ protein min⁻¹). Protease activity and reduction of activity in bands of samples treated with specific inhibitors were documented in photometric assay and substrate SDS–PAGE. Proteinase activity against azocasein was inhibited (91.4±1.5%) with SBTI. PMSF reduced the enzyme activity by 53.1±6.5%, and the azocasein hydrolysis was reduced up to 64.6±3.8% by the specific inhibitor of trypsin, TLCK. In the present investigation, the molecular weight of various activity bands ranged from 16.3 to 51.1 kDa. The molecular weights of several protein bands are similar to protease activity zones. The knowledge of digestive enzyme profiles of fish food organisms generated in the present study may assist in the formulation of age-specific feed.     

Identification and partial characterization of selected proteolytic enzymes in the digestive system of giant freshwater Prawn Macrobrachium rosenbergii (De Man) Postlarvae

Biochemical assays and substrate SDS-PAGE were conducted to partially characterize and identify various types of proteases present in the digestive tract of PL₁₅ giant freshwater prawn ( Macrobrachium rosenbergii ). Casein hydrolytic assay of the enzyme extracts showed major proteolytic activities at pH 3.0, 6.0 and 9.0, while assay of preincubated enzyme extracts with phenylmethylsulphonyl fluoride (PMSF), a serine protease inhibitor produced a 33.17% reduction in alkaline protease activity. When specific inhibitors tosyl-lysine chloromethyl ketone and tosyl-phenylalanine chloromethyl ketone were used, they resulted in a reduction in activity of proteases in the enzyme extracts by 82.41% and 55.03%, respectively, confirming the presence of trypsin and chymotrypsin, while ethylenediamine tetraacetic acid produced protease activity reduction in 33.92% showing the presence of metalloproteases in the digestive tract of the prawn. Further characterization of the alkaline proteases using SDS-PAGE technique, after incubating the extract in the presence or absence of specific inhibitors, produced six bands corresponding to molecular masses of between 13.48 and 136.1 kDa; two trypsin bands of 13.48 and 36.4 kDa, three chymotrypsin bands in the range of 23.0–73.4 kDa and one for metalloprotease of 136.1 kDa, all of which were identified from a zymogram. This study suggests that protein digestion in M. rosenbergii is initiated by an acid protease followed by a combination of action of alkaline proteases: trypsin, chymotrypsin and metalloproteases.     

In vitro assay of plant protease inhibitors from four different sources on digestive proteases of rohu, Labeo rohita (Hamilton), fingerlings

The in vitro inhibitory effect of protease inhibitors from four seed extracts (soybean, grasspea, black gram and horse gram) on digestive proteases of rohu was assessed by enzyme inhibition assay and substrate sodium dodecyl sulphate‐polyacrylamide gel electrophoresis. High proteolytic activity was detected in the intestinal extract of rohu (Labeo rohita) fingerlings at two different pH ranges (8–8.5 and 10–11). That protein digestion occurs mainly in the alkaline condition in this fish without a stomach is evident from very high trypsin activity (0.95±0.04 benzoyl‐dl ‐arginine‐p‐nitroanilide U mg protein⁻¹) in the intestine. In case of grass pea seed, more than 50% inhibition of alkaline protease activity was recorded when the ratio of inhibitor to enzyme was 9.41 μg U⁻¹. More than 40% inhibition of protease activity was recorded in case of horse gram seed when the ratio of inhibitor to enzyme was 5.51 μg U⁻¹. Black gram at 11.0 μg U⁻¹ and soybean seed proteins at 62.75 μg U⁻¹ resulted in 50% and more than 30% inhibition of digestive protease activity in rohu fingerlings respectively. A plot of the inhibition values obtained by changing the relative concentrations of enzyme/inhibitor resulted in different dose–response curves for different protein sources. The use of substrate gel electrophoresis allowed the visualization of the aforementioned differences in inhibition. Each seed extract produced a characteristic profile of protease inhibition. It is concluded that protease inhibitors present in plant protein sources adversely affect the digestive proteases in fish and hence there is a need to eliminate/reduce the amount of such inhibitors through proper processing before incorporation into aquafeeds.     

Characterization of protease activity in developing discus Symphysodon aequifasciata larva

The activity of different protease classes was monitored in developing discus (Symphysodon spp.) larvae using a combination of biochemical assays and substrate SDS–PAGE techniques. Results showed the presence of alkaline proteases of serine proteases such as trypsin with a significant increase in activity levels detected beginning 3 days after hatching. Other alkaline proteases such as metallo‐proteases and chymotrypsin, a type of serine protease, were only detected in older larval stages, at around 20–30 days after hatching. Acidic protease activity was very low during the first 20–25 days of development before showing a significant (P < 0.01) rise. This is despite the formation of a stomach observed 10 days after hatching. Based on the development of the protein digestive system observed, the use of microdiets to replace Artemia should be considered 25 days after hatching.     

Changes in digestive enzyme activity during initial ontogeny of bay snook Petenia splendida

Several samples of P. splendida larvae were obtained from eggs until day 60 after hatching (dah) to determine acid and alkaline proteases, trypsin, chymotrypsin, leucine aminopeptidase, α-amylase, lipase, and acid and alkaline phosphatase activities using biochemical techniques. Additionally, SDS–PAGE alkaline protease zymogram and PAGE acid protease zymogram were carried out to identify active isoforms during larviculture. Alkaline protease and chymotrypsin were present at the moment of hatching, increased gradually reaching the maximum values at 35 dah. Trypsin and leucine aminopeptidase activities were low from hatching, increasing gradually as larvae grew. Alkaline protease zymogram showed four zymogens, which appears at different days, remaining present until the end of the larviculture (95.2 kDa at 11 dah, 26.4 kDa at 9 dah, 21.4 kDa at 3 dah, and 23.3 kDa at hatching). Pepsin activity was present at day 7 after hatching and increased progressively until the end of the larviculture. Acid protease zymogram only showed one zymogen (0.65 rf), which appear at 6 dah. Lipase was high at the time of hatching and increased until 15 dah, after which decreased gradually. Amylase was high from the beginning and until 15 dah and then decreased rapidly to almost nothing onward. Alkaline and acid phosphatases presented a high activity at the egg stage, fell slightly during the first feeding and increased again from 20 to 30 dah. Results obtained in this study show that larvae can be fed artificial diets starting on day 10 after hatching.     

Differential inhibition of digestive proteases by tannin in two size groups of rohu (Labeo rohita,Hamilton): A biochemical and zymography study

This study was intended to evaluate tannin mediated inhibition of digestive proteases in two different size groups (fingerlings, F, 4.5 ± 0.7 cm; advanced fingerlings, AF, 18.2 ± 1.6 cm) of rohu, Labeo rohita. Graded levels (50, 100, 150 and 200 nM) of tannin (Gallotannin, 99% purity) were added to the enzyme extracts (30°C, 1 hr) prior to determination of enzyme activities. Changes in the activity of trypsin, chymotrypsin and total protease in relation to the control sets were determined through biochemical assay of enzymes and SDS‐PAGE zymography. The study revealed that tannin significantly inhibited trypsin, chymotrypsin and total protease activities in a dose dependent manner as evident from the regression equations. The degree of the inhibition appeared to be significantly higher for the F in contrast to the AF (F_5,66 = 282.311; p < 0.0001). Trypsin activities were reduced by 8.04 ± 0.19% to 52.68 ± 0.72% and 5.61 ± 0.22% to 39.46 ± 0.19% in F and AF, respectively. The reduction in Chymotrypsin activities ranged between 16.11 ± 0.03% to 38.02 ± 0.27% in F and 6.31 ± 0.07% to 22.80 ± 0.32% in AF. Total protease activities were reduced by 10.9 ± 0.07% to 49.60 ± 0.32% in L. rohitaF, whereas, it ranged between 5.19 ± 0.06% to 32.60 ± 0.13% in AF. On a comparative scale, the difference in tannin induced inhibition in F and AF were more prominent for trypsin and chymotrypsin than total protease. Further, nine protease activity bands (15.9–69 kD) with different electromobility were noticed in both the size groups. Subsequent densitometry analysis revealed that the average densities of the protease activity bands were gradually decreased with increasing level of tannin exposure (50–200 nM). The study might indicate adaptive tolerance to tannin in larger size groups and emphasizes the need for removal of tannin in the plant feedstuffs, especially for feeding the fingerlings of L. rohita.     

Digestive proteinases and peptidases in the hepatopancreas of the southern brown shrimp (Farfantepenaeus subtilis) in two sub‐adult stages

The aim of this study was to examine proteinases and peptidases from the hepatopancreas of two sub‐adult stages of Farfantepenaeus subtilis: SAS₆ (5.93 ± 0.69 g wet weight) and SAS₁₃ (13.26 ± 0.60 g wet weight). Trypsin and chymotrypsin activity was higher in the extract from the SAS₆ individuals (P < 0.05). The highest activity among aminoacyl‐β‐naphthylamide substrates was found using alanine‐, arginine‐, leucine‐ and lysine‐β‐naphthylamide. There was a positive correlation between the recommended concentration of essential amino acids in penaeid shrimp feed and aminopeptidase activity in both sub‐adult stages. Proteolytic activity of F. subtilis was strongly inhibited by specific trypsin inhibitors. The optimal temperature for trypsin, chymotrypsin and leucine aminopeptidase activity was between 45 and 55 °C. Six and seven bands were found in caseinolytic zymograms for SAS₆ and SAS₁₃ respectively. All bands were inhibited by phenylmethylsulfonyl fluoride in both sub‐adult stages. The use of tosyl‐lysine‐chloromethyl‐ketone and benzamidine caused strong inhibition of the proteolytic bands. Trypsin and chymotrypsin activity was the main difference observed between the protease pattern of SAS₆ and SAS₁₃F. subtilis.     

Effect of cortisol and triiodothyronine bath treatments on the digestive enzyme profile and growth of Catla catla larvae during ontogenic development

Digestive enzyme profile is a good indicator of the nutritional and health status of the fish. The present investigation aims to evaluate the effect of exogenous bath treatment of hormones, cortisol and triiodothyronine, on the digestive enzyme activities and growth of carp Catla catla (Ham.) during ontogenic development. Catla larvae (4 days old) were given bath treatment with cortisol (hydrocortisone, 0.2 mg L^?1), 3,5,3′‐triiodothyronine (T3, 2.5 mg L^?1) and a combination of cortisol and T3 for 30 min. Digestive enzyme profile was recorded on every third day and was continued for 30 days. Larvae were fed with live food for initial 14 days and then weaned to mix feeding of live food and prepared diet. Significantly (P < 0.05) higher amylase, total protease, trypsin, chymotrypsin, lipase, chitinase and chitinobiase activities were found in the hormone‐treated groups compared to the control one during ontogenic development. Among the treated groups, amylase activity was highest in cortisol‐treated larvae. Total protease, trypsin, chymotrypsin, lipase, chitinase and chitinobiase activities were significantly (P < 0.05) higher in larvae exposed at combined treatment of cortisol and T3 compared to the other two groups in most sampling days. Average length, weight and specific growth rate of treated larvae were higher compared to the control one. The combined bath treatment of cortisol and triiodothyronine influenced the digestive enzyme activities of catla larvae and thereby enhanced the growth at early developmental stage. This helps the larvae to overcome the problems associated with early developmental stage.     

Development of Digestive Enzymes in California Halibut <Emphasis Type="Italic">Paralichthys californicus</Emphasis> Larvae

California halibut Paralichthys californicus is an important commercial species with high aquaculture potential in Baja California Sur, México. To optimize the feeding process using live prey and/or inert diets, we evaluated alkaline proteases, pepsin, trypsin, chymotrypsin, leucine aminopeptidase, lipase, α-amylase, and acid and alkaline phosphatase activities on starved larvae and larvae fed live prey. Highest activities were observed for alkaline protease, trypsin, chymotrypsin, leucine aminopeptidase, and alkaline phosphatase in feeding larvae than starved larvae on day 4 after hatching. At day 5, a sizeable increase in all enzymatic activities was detected in feeding larvae. Alkaline protease, trypsin, chymotrypsin, and alkaline phosphatase decreases progressively from day 5 until day 18. At day 18, a slight pepsin activity was observed. This was considered an indicator of the start of digestive system maturation. We concluded that total enzymatic equipment for this species is complete between day 18 and 30 after hatching. Based on this evidence, early weaning from live prey to inert feed would be possible at this time.     

Activity,molecular mass and hydrolysis on baker's yeast protein of extracellular proteases from the putative probiotic bacteria Microbacterium sp. strain 8L and Exiguobacterium mexicanum strain 8N

The bacteria Microbacterium sp. 8L and Exiguobacterium mexicanum 8N are known to improve the culture of Artemia franciscana using baker's yeast as food. Using spectrophotometry, sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS‐PAGE), substrate‐SDS‐PAGE and pH‐stat in vitro‐digestibility assays, the activity, molecular mass and hydrolysis on baker's yeast protein of proteases from extracellular polymeric substances (EPS) of the strains 8L and 8N along with the pathogenic strains Microbacterium sp. 8R and Vibrio parahaemolyticus 588 CECT (Vp) were studied. The EPSs of 8L and 8R showed one activity band, on which the serine inhibitor phenylmethylsulphonyl fluoride (PMSF) had no effect. The EPSs of 8N showed four bands; two were unaffected by PMSF, whereas one was affected, and the other was partially affected. The EPSs of Vp showed two bands, one partially inhibited by PMSF. No inhibitory effects from 1‐chloro‐3‐tosylamido‐7‐amino‐2‐heptanone (trypsin inhibitor) were observed in the protease bands of the studied bacteria. The EPSs of 8L and 8N showed a similar degree of hydrolysis (pH‐stat). The EPSs of 8L had the lowest Dice index of similarity of yeast protein profiles at 1 h of reaction. We conclude that the strain 8L could benefit A. franciscana by providing bacterial proteases for digestion of baker's yeast.     

Partial characterisation of digestive proteases of the Mayan cichlid Cichlasoma urophthalmus

The characterisation of digestive proteases in native freshwater fish such as the Mayan cichlid Cichlasoma urophthalmus provides scientific elements that may be used to design balanced feed that matches with the digestive capacity of the fish. The purpose of this study was to characterise the digestive proteases, including the effect of the pH and the temperature on enzyme activity and stability, as well as the effect of inhibitors using multienzymatic extracts of the stomach and intestine of C. urophthalmus juveniles. Results showed that the optimum activities of the acid and alkaline proteases occurred at pH values of 3 and 9, respectively, whereas their optimum temperatures were 55 and 65 °C, respectively. The acid proteases were most stable at pH values of 2–3 and at temperatures of 35–45 °C, whereas the alkaline proteases were most stable at pH values of 6–9 and at 25–55 °C. The inhibition assays recorded a residual activity of 4 % with pepstatin A for the acid proteases. The inhibition of the alkaline proteases was greater than 80 % with TPCK, TLCK, EDTA and ovalbumin, and of 60 and 43.8 % with PMSF and SBT1, respectively. The results obtained in this study make it possible to state that C. urophthalmus has a sufficiently complete digestive enzyme machinery to degrade food items characteristic of an omnivorous fish species, although specimens showed a tendency to carnivory.     

Ontogenic development of enzymatic activity and digestive system in Jullien's golden carp (Probarbus jullieni Sauvage, 1880)

Ontogenic development of the main enzymes and histological structure of digestive organs were studied in Jullien's golden carp (Probarbus jullieni) from hatching until 50 days after hatching (DAH). The larval fish were produced by artificial insemination and fed only Moina sp. till end of experiment. Body weight (mg) and total length (cm) of Jullien's golden carp increased exponentially and linearly. The results indicate the fish weight grew fast with increasing rate, while length increased at a constant rate over the studied period. Up‐regulation of acid protease was observed in newly hatched larvae and the specific activity gradually decreased with time. Trypsin specific activity was relatively stable within the first 35 DAH, while fluctuations in chymotrypsin were observed. Among these three proteolytic enzymes, acid proteases exihibited relatively high specific activity in newly hatched larvae, suggesting a role in yolk protein degradation. Alkaline proteases became more prominent with age and correlated with an abrupt decrease in acid proteases. Increased lipase‐specific activity appeared within 3 DAH and then gradually decreased with time, indicating the capacity to digest yolk lipid reserve. Amylase and cellulase‐specific activities changed in a similar manner, and the sensitivity to time was higher in amylase than in cellulase. The digestive organs and accessory organs developed around 3–5 DAH. However, intestinal histology was almost fully developed around 18 DAH. These findings should be useful for deciding the preferred timing for weaning, as well as on developing artificial diets referenced to the physiological changes of digestive enzymes.     

Partial characterization of digestive proteases in tropical gar Atractosteus tropicus juveniles

Tropical gar (Atractosteus tropicus) is an economically and socially important freshwater species from Southeastern Mexico, with a high aquaculture potential. With this in mind, the purpose of this study was to characterize the digestive proteases of tropical gar juveniles through biochemical and electrophoretic analyses. Twenty specimens with an average weight of 73.6 ± 12.7 g were used to obtain stomach and intestinal tissue from which multienzymatic extracts were prepared. The general activities of the acid and alkaline proteases were evaluated, as well as the specific activities of trypsin, chymotrypsin, leucine aminopeptidase and carboxypeptidase A. The effect of the pH and temperature on the proteases was also analyzed, together with the composition of the multienzymatic extracts using protease inhibitors and electrophoretic tests. Results showed that A. tropicus have a functional stomach in which protein hydrolysis starts with pepsin and which contains endo- and exopeptidases (trypsin, chymotrypsin, leucine aminopeptidase and carboxypeptidase A) and proteases that are resistant to high temperatures (45 and 55 °C for alkaline and acid proteases, respectively) and pH values. Using zymogram technique, we found two acid protease isoforms (0.35 and 0.71 rf) and five alkaline protease isoforms (83.7, 43.7, 27.5, 24.0 and 19.4 kDa), which decrease or disappear with the different inhibitors. Thus, this species is considered to be a carnivore capable of adapting to its environment by consuming different types of proteins from preys and also could adapt rapidly to consume a compound diet with different animal protein sources.     

Characterization of digestive enzymes protease and alpha‐amylase activities in the thick‐lipped grey mullet (Chelon labrosus,Risso 1827)

Activities of pepsin, trypsin, chymotrypsin, alpha‐amylase and lipase, as well as their optima and stability to pH and temperature, were determined in digestive extracts of thick‐lipped grey mullet, Chelon labrosus, of three different sizes: Group 1 (45.2 ± 3.0 g), Group 2 (180.9 ± 4.2 g), and Group 3 (328.5 ± 43.3 g). SDS‐PAGE zymograms were also used to assess the role of serine proteases in the digestive tract of C. labrosus. On the other hand, possible changes in the digestive enzyme profile of C. labrosus during development were observed, with a comparatively lower pepsin activity, higher activities of alkaline proteases and alpha‐amylase and no lipase activity recorded in pre‐adult specimens. It is suggested that these variations are linked to the changes in diet composition with age, moving from a partly carnivorous to a more herbivorous feeding habit.     

Isolation,Purification, and Biochemical Characterization of Trypsin from Indian Mackerel (Rastralliger kanagurta)

Trypsin from viscera of Indian mackerel (Rastralliger kanagurta) was purified by ammonium sulphate precipitation and chromatographic techniques such as size exclusion, ion exchange, and affinity chromatography, with a 14.4-fold increase in specific activity and 18.7% recovery. The molecular weight of the trypsin was estimated to be approximately 26 kDa using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Purified trypsin showed amidase-specific activity which was determined using benzoyl-dl-arginine-p-nitroanilide (BAPNA). The optimum pH and temperature for isolated trypsin activity were 9.0 and 50°C, respectively. The purified trypsin was strongly inhibited by soybean trypsin inhibitor (SBTI) and N-p-tosyl-1-lysine chloromethyl ketone (TLCK). Purified trypsin showed almost 40% recovery at high NaCl concentration (30%). The N-terminal amino acid sequence of the first 10 amino acids of purified trypsin was IVGGYESQPH. The Michaelis-Menten constant (K_m) and catalytic constant (K_cat) of purified trypsin were 0.430 mM and 0.77 s^?1, respectively, determined using BAPNA as a substrate. Purified trypsin showed digestion of casein similar to bovine trypsin by the fluorometric method.     

Partial purification and characterization of extracellular metalloproteases with caseinolytic, aminopeptidolytic and collagenolytic activities from Vibrio anguillarum

Abstract. Proteases produced by Vibrio anguillarum were isolated from culture supernatant by ultrafiltration, gel chromatography and ion exchange chromatography. The enzyme(s) were shown to be collagenolytic when assayed with native collagen substrates. In addition, the enzyme(s) hydrolysed azocasein, azocollagen, the collagenase substrate 4-phenylazobenzyloxycarbonyl-Pro-Leu-Gly-Pro-D-Arg and the aminopeptidase substrate L-Leu-pNA effectively. Separation of the proteases by Mono Q ion exchange chromatography and native polyacrylamide gel electrophoresis revealed four distinct protein bands containing caseinase activity. However, only two of the bands showed aminopeptidase activity. The aminopeptidase activities could be separated from the caseinase activities by isoelectric focusing. Secreted proteases of different serotypcs of V. anguillarum showed a heterogeneous caseinolytic pattern. The molecular mass of the major enzyme was estimated at 35kDa as determined by its mobility on SDS-polyacrylamide gels. Serine protease inhibitors like PMSF, TPCK, TLCK and benzamidine had no inhibitory effects on the proteolytic activity when tested with azocasein as substrate. However, the enzyme was strongly inhibited by metal chelators like EDTA and 1, 10-phenanthroline. Also, normal salmon scrum and purified α₂-macroglobulin from salmon serum strongly inhibited the caseinolytic activity of the enzyme.     

Development of digestive enzymes in larvae of Mayan cichlid Cichlasoma urophthalmus

The development of digestive enzymes during the early ontogeny of the Mayan cichlid (Cichlasoma urophthalmus) was studied using biochemical and electrophoretic techniques. From yolk absorption (6?days after hatching: dah), larvae were fed Artemia nauplii until 15?dah, afterward they were fed with commercial microparticulated trout food (45% protein and 16% lipids) from 16 to 60?dah. Several samples were collected including yolk-sac larvae (considered as day 1 after hatching) and specimens up to 60?dah. Most digestive enzymes were present from yolk absorption (5?C6?dah), except for the specific acid proteases activity (pepsin-like), which increase rapidly from 8?dah up to 20?dah. Three alkaline proteases isoforms (24.0, 24.8, 84.5?kDa) were detected at 8?dah using SDS?CPAGE zymogram, corresponding to trypsin, chymotrypsin and probably leucine aminopeptidase enzymes, and only one isoform was detected (relative electromobility, Rf?=?0.54) for acid proteases (pepsin-like) from 3?dah onwards using PAGE zymogram. We concluded that C. urophthamus is a precocious fish with a great capacity to digest all kinds of food items, including artificial diets provided from 13?dah.     

Antioxidative Activitiy of Protein Hydrolysate from the Muscle of Common Kilka (Clupeonella cultriventris caspia) Prepared Using the Purified Trypsin from Common Kilka Intestine

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.