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Protease development in Dover sole [Solea solea (L.)]
Aquaculture, 53 (1986) 253-262 Elsevier Science Publishers B.V., Amsterdam - Printed in The Netherlands
PROTEASE DEVELOPMENT
J. CLARK’,
K.R. MURRAYI
IN DOVER SOLE [SOLEA
SOLEA
253
(L.) ]
and J.R.STARK’
of Brewing and Biological Sciences and z Department of Chemical and Process Engineering, Heriot-Watt (Great Britain)
’ Department
University, Edinburgh
(Accepted 6 February 1986)
ABSTRACT Clark, J., Murray, K.R. and Stark, J.R., 1986. Protease development in Dover sole [Solea solea (L.)]. Aquaculture, 53: 253-262. An examination of the changes in distribution of exo- and endoproteases has been made in Dover sole intestinal extracts from 24-day larvae to adult fish. Pepsin-like activity was not detectable in 24-day-old animals but was first measured in appreciable amounts at 200 days. All other proteases were present at 24 days and increased in value as the fish matured. The significance of these results to the release of nutrients from fish diets is discussed.
INTRODUCTION
The seedling production of larval fish, bivalves and crustaceans for mass production relies still on live diets such as algae, rotifers (Brachionus plicutilis) and planktonic Crustacea, e.g., Artemia. The culture of these food organisms is not only expensive and labour intensive but is also subject to fluctuations in both the availability, and the nutritional quality of the natural resource (Seidel and Simpson, 1984). The problems, therefore, inherent in the production of live feeds and the manipulation of their nutritional profile has stimulated interest in the development of an artificial diet which meets certain criteria. It has been suggested that the digestive systems of the early larval stages in some species are not fully developed (Tanaka et al., 1972; Kawai and Ikeda, 1973) and therefore the diet should be adapted to meet the changing life stages of the larvae. To acquire information for future development of large-scale supplies of microparticulate diets it is necessary to study the digestive enzyme complement of commercially viable species such as Dover sole Solea solea (L.) (Clark et al., 1984) so that the digestive capacity can be directly related to the nutritional requirements of early larvae. Even more important is the capability of the larvae to digest and assimilate the microparticulate diet which may have an outer wall which is resistant to hydrolysis and dissolution in the alimentary canal. 0044-8486/861$03.50
o 1986 Elsevier Science Publishers B.V.
254
The initial survey of digestive enzymes present in juvenile Dover sole has concentrated on carbohydrases (Clark et al., 1984) and proteases (Clark et al., 1985a). This has led to further studies, particularly on elastase (Clark et al., 1985b), as part of an overall research programme on marine larval feed technology. This paper reports the development of seven proteolytic enzymes in Dover sole aged from 24 days to adult. MATERIALS
AND METHODS
Fish All sole were’supplied by Seafresh Farms (Scotland) Ltd., Hunterston, and were aged 24, 49, 80 and 200 days; adult fish were 2-group. All fish were reared at 20°C from hatching and were starved overnight to evacuate the gut. Fish were weaned from Artemia nauplii on to a dry pelleted diet at 60 days and from 80 days upward had been fed on artificial diets for at least 3 days prior to analysis of the guts and their contents (so that there were no contaminating enzymes from live diets in any of the preparations). In all cases the gut could be easily excised from frozen whole fish; guts were weighed and homogenised in distilled water to give a 1:lO homogenate. Dilutions were carried out as required. The lumen contents were included in all homogenates which were a combination of 90-100 fish, 40-60 fish, and 10 fish for the 24, 49 and 80 and 200-day-old fish gut homogenates, respectively. With the adult a single fish was used for the preparation of the homogenate. Chemicals All chemicals were supplied by the Sigma Chemical Co. unless otherwise stated. Enzyme assays The buffers used in all enzyme assay procedures were 0.1 M HCl (pH l.O), glycine-HCl (pH l.O-1.7), citrate-phosphate (pH 1.7-7.8), glycine-NaOH (pH 7.8-10.1) and phosphate-NaOH (pH 0.1-11.2). All enzyme assays were carried out at the appropriate optimum conditions of pH and temperature (37°C) as indicated by Clark et al. (1985a). TotaZpro tease Total proteolytic activity was estimated by a modification of the casein hydrolysis method of Kunitz (1947). Mixtures consisting of a 1% (W/V) aqueous solution of casein (0.5 ml), intestinal homogenate (0.1 ml) and buffer (0.4 ml) were incubated for 1 h. Ice-cold 5% (w/u) trichloroacetic
255
acid (1.5 ml) was added and the mixture left at 2°C for 30 min. The samples were then centrifuged at 2OOOg for 10 min and the absorbance of the supernatant solution recorded at 280 nm. Tyrosine was used as standard and the units expressed as pg tyrosine liberated per min/g protein. The protein content of the homogenates was determined by the method of Lowry et al. (1951) as modified by Miller (1959). Trypsin
Trypsin-like activity was measured by recording enzymic hydrolysis of 10’ M p-toluene-sulphonyl-L-a&nine methyl ester (TAME) at 247 nm using a modification of the method of Hummel (1959). The substrate was supplied by BDH Chemicals Ltd. (Broom Road, Poole, Great Britain) and assays were carried out at pH 8.0. The extract used was obtained by centrifuging (2000g) a 1:40 aqueous homogenate of gut tissue and assaying the supernatant for enzyme activity. Activity was expressed as enzyme units per mg protein, 1 enzyme unit being equivalent to a 0.001 change in absorbance per min. Chymo trypsin
Chymotrypsin-like activity was measured using 0.00107 M benzoyl-ltyrosine ethyl ester (BTEE) and recording the change in absorbance at 253 nm as described by Hummel (1959) at pH 7.6. The substrate was supplied by BDH Chemicals. Elas tase
Elastase-like activity was measured using elastin-orcein (Sachar et al., 1955) by a modification of the method described in the Worthington Manual (1972). Elastin-orcein (10 mg), pH 10.1 buffer (0.9 ml) and 1:lO whole gut homogenate (0.1 ml) were mixed by continuous rotation in a Spiromix roller (Denley Ltd., Sussex, Great Britain) at 40 rev./min. The mixture was centrifuged (2000g) and the absorbance read at 590 nm against an appropriate elastin-orcein blank which had been incubated for the same time. Activity was expressed relative to the adult value as 100 since it is not possible to express hydrolysis of this product in absolute units. Leucine
aminopeptidase
Leucine aminopeptidase activity was measured by recording the hydrolysis of 0.125 M L-leucinamide at 238 nm using a modification of the method of Mitz and Schleuter (1958) at pH 8.3. Enzyme extracts consisted of the supernatant solution treated as for the trypsin assay and the unit of enzyme activity defined as the hydrolysis of 1 pmole of substrate per min.
256
Carboxypep tidase A and B Carboxypeptidase activity was measured by recording the hydrolysis 0.001 M hippuryl-L-phenylalanine at 254 nm (Folk and Schirmer, 1963) pH 7.5. The tissue extract and definition of enzyme activity were as scribed above for leucine aminopeptidase. An identical assay procedure used for carboxypeptidase B in which 0.001 M hippuryl-L-arginine was substrate (Folk et al., 1960).
of and dewas the
RESULTS
At all pH values the protease levels, as measured by the hydrolysis of casein, showed a general increase with the age of the fish (Fig. 1); however, several differences were manifest. Pepsin-like activity (measured by hydrolysis of casein at pH 2.0) could not be detected in fish aged 24, 49 or 80 days, but by 200 days the activity was 0.8 units/mg protein rising to a value of 2 units/mg protein in the adult fish. Neutral and alkaline proteases were readily detected in fish of all ages. At pH 7.6, gut homogenates from
0
1
2
3
4
5 6 PH
7
8
9
10 11
Fig. 1. Protease development in Dover sole. Hydrolysis of casein by homogenates whole intestine (and contents) from fish of different ages.
of
257
24day-old fish showed 0.59 units/mg protein compared with 4.50 units in adult fish; this constitutes an eight-fold increase in neutral protease activity between 24-day-old fish and adult fish. At pH 10.1, alkaline protease values rose from 0.71 units/mg protein in 24-day-old fish to 8.90 units/mg protein in adult fish; a la-fold increase in activity between the 24-day-old fish and the adult fish. The ratios of neutralalkaline protease also changed as the fish aged, from 1: 1 in 24-day-old fish to 1:2 in the adult. Levels of trypsin and chymotrypsin as measured with specific synthetic substrates (Fig. 2) increased from day 24 to day 200, but there was very little difference in enzyme levels thereafter. At all life stages the levels of chymotrypsin exceeded that of trypsin, with a pronounced rise in chymotryptic activity between 80 and 200 days. The ratio of chymotrypsin:trypsin was also seen to increase in favour of chymotrypsin as the fish aged, aithough trypsin activity increased eight-fold between 24 days and adult fish (1.76 units/mg protein compared to 14 units/mg protein); this differs from chymotrypsin in which there was only a four-fold increase in activity, from 11.3 units/mg protein in 24-day-old sole to 42.0 units/mg protein in adult sole. chymotrypsin M trypsin -
Fish age (days) Fig. 2. Activities of trypsin and chymotrypsin
in Dover sole of different ages.
Fig. 3. shows the relative activity of an elastase-like enzyme in Dover sole of different ages. It can be seen that although activity was highest in adult sole, there was very little difference in relative activity from 80 days onward; the enzyme extract of 80day-old fish in fact had 93% of the activity of the adult fish gut preparation. There was, however, a sharp increase in activity from day 24 up to day 80, and it can be seen that between day 24 and adulthood there was an eight-fold increase in the relative activity of the elastase-like enzyme. Leucine aminopeptidase activity (Fig. 4) did not increase much between 24- and 49-day-old fish, but the level rose quickly to a maximum value of 22.6 units/mg protein for 200day-old fish. Although activity fell slightly in
80
200
Adult
Fish age (days) Fig. 3. Relative activity of elastase in Dover sole of different ages.
25
leucine aminopeptidase
22 ‘E 3
5 0
24
49
80
200
Adult
Fish age (days) Fig. 4. Activity of leucine aminopeptidase
in Dover sole of different ages.
I
24
49
80
200
Adult
Fish age (days) Fig. 5. Activities of carboxypeptidases
A and B in Dover sole of different ages
259
adult fish, there was still almost a three-fold rise in activity from 7.5 units/ mg protein for 24-day-old fish to 20.0 units/mg protein for adult fish. The level of carboxypeptidase A was always higher than that of carboxypeptidase B (Fig. 5); carboxypeptidase A levels did not increase much from day 24 to day 49, but there was then a rapid rise in activity up to 200 days. Between 200 days and adult fish there was very little difference in activity. There was, overall, a three-fold increase in the level of carboxypeptidase A, from 8.4 units/mg protein in 24-day-old fish to 23.0 units/mg protein in adult fish. Carboxypeptidase B, on the other hand, exhibited a gradual increase in activity from day 24 to day 200 whereupon there seemed to be a plateau of activity. There was, however, a seven-fold increase in activity from 24-day-old fish (1.4 units/mg protein) to adult fish (10.5 units/mg protein). DISCUSSION
In the present studies, fish of an age suitable for dissection of the gut have been chosen. In work on other species (e.g., Dabrowski, 1979, 1982; Ragyanszki, 1980) analysis of protease in fish younger than 24 days has been carried out, but in our experience, with Dover sole, the use of whole fish homogenates (for specimens which are too small for dissection) results in high blank values and data which are difficult to reproduce. Furthermore, with whole fish homogenates, it is not always possible to be certain that a particular enzyme has originated from the digestive tract. The development of enzymes in several different species of fish has been studied but the techniques used have differed considerably. Kuzmina (1980), in the work on the bream, A brumis bruma, has indicated that there is an inverse relationship between amylase activity and the age of the fish. However, most other studies show that the general trend is for there to be an increase of enzyme activity after the fish reaches about 30 days old and progressing into adulthood. Kawai and Ikeda (1973) suggested that the problems of feeding at an early age may be due to low contents of digestive enzymes and hence there have been a number of attempts to investigate the enzymic content of fish from the egg stage through hatching. Alliot (1981) has reviewed the development of digestive enzymes in teleosts and has indicated that these enzymes exist soon after hatching, and in work on the European sea bass, Dicentrurchus lubrux, Alliot et al. (1977) found that the trypsin activity increased slowly between 0 and 5 days, decreased from 5 to 15 days and thereafter remained quite stable up to 30 days. Using similar methods, these authors (Alliot et al., 1980) have examined the activities of a number of enzymes, including leucine aminopeptidase and trypsin, in sole larvae from O-30 days. In the present experiments, the general trend of development of enzymes (Fig. 1) indicates that there is late development of pepsin-like activity (pH 2.0). This has been noted in other fish (Kawai and Ikeda, 1973) and is evidently dependent on the development of an active stomach zone in the in-
260
testine. This has been shown by the fact that in Chimaera which are stomachless, there are no pepsin-like enzymes (Barrington, 1957). In very young fish the relative amount of neutral and alkaline proteases is about the same but as the fish age the latter activity becomes more predominant. Previous studies (Clark et al., 1985a) have suggested that there are a number of enzyme activities at pH 7-8, including trypsin, chymotrypsin and collagenase, whereas at pH 10 there is a single major contribution from an elastase-like enzyme (Clark et al., 1985b). With the exception of pepsin-like activity, where the very acidic pH gives a degree of specificity, casein is a non-specific substrate. Assays for each of the other enzymes were therefore carried out using specific substrates. Chymotrypsin activity increased with the age of the fish (Fig. 2) but the rate of increase was far less pronounced with trypsin. Tanaka et al. (1972) reported that in carp fry, trypsin activity hardly increased in activity during transition from larvae to juveniles. Also in studies on carp, Ragyanszki (1980) reported chymotrypsin activity to be from two to nine times higher than trypsin during the period of 0 to 12 days of age. In the present work all activities showed a general increase with age of the fish but the details differed somewhat. Elastase activity increased much more rapidly during the period between 24 and 80 days (Fig. 3) than did trypsin or chymotrypsin (Fig. 4). Leucine aminopeptidase rose rapidly between 49 and 200 days and then plateaued whereas the rise with the two carboxypeptidases was more gradual and continuous. The problems associated with feeding of certain flatfish larvae including Dover sole have been discussed by Howell (1973) who emphasises the need for a diet which can be reliably produced. Whilst the brine shrimp Arternia salina L. nauplii can readily be stored and hatched, it has to be imported and the reliability of stocks may be in doubt. In this study the pH profile of hydrolysis against casein indicated that at pH 9.5-10 there was maximum attack on the protein material particularly when extracts of the digestive tract of older fish were examined. However, this pH value is unlikely to exist in the lumen under normal physiological conditions and therefore the levels of trypsin and chymotrypsin must be considered of prime importance. Further studies are in progress on the action of different levels of enzymes on a variety of types of artificial feeds in order to correlate the present work to the digestive capabilities of the fish. ACKNOWLEDGEMENT
The authors wish to acknowledge the SERC (Marine Technology Directorate) for the support of this work. REFERENCES Alliot, E., 1981. Nutrition des poissons. III. Evolution de quelques actkit& digestives au tours du dhveloppement larvaire des tUost6ens. M. Fontaine (Editor), Editions du
261 Centre National de la Recherche Scientifique, Paris. A&es Colloq. CNERNA, pp. 7988. Alliot, E., Pastoureaud, A. and Trellu, J., 1977. Evolution des activites enzymatiques dans le tube digestif au tours de la vie larvaire du bar (Dicentrarchus Zabrax). Variations des proteinogrammes et des zymogrammes. 3rd Meeting of the ICES Working Group on Mariculture, Brest, France, lo-13 May 1977. A&es Colloq. CNEXO, 4: 85-91. Alliot, E., Pastoureaud, A. and Trellu, J., 1980. Evolution des activites enzymatiques dans le tractus digestif au tours de la vie larvaire de la Sole. Variations des proteinogrammes et des zymogrammes. Biochem. Syst. Ecol., 8: 441-445. Barrington, E.J.W., 1957. The alimentary canal and digestion. In: M.E. Brown (Editor), The Physiology of Fishes, Vol. 1, Academic Press, New York, NY, pp. 109-161. Clark, J., MacNaughton, J.E. and Stark, J.R., 1984. Metabolism in marine flatfish. I. Carbohydrate digestion in Dover sole (Solea soleo L.). Comp. Biochem. Physiol. B, 77: 821-827. Clark, J., MacDonald, N.L. and Stark, J.R., 1985a. Metabolism in marine flatfish. II. Protein digestion in Dover sole (Solea solea L.). Comp. Biochem. Physiol. B, 81: 217-222. Clark, J., MacDonald, N.L. and Stark, J.R., 198513. Metabolism in marine flatfish. III. Measurement of elastase activity in the digestive tract of Dover sole (Soiea solea L.). Comp. Biochem. Physiol. B, 81: 695-700. Dabrowski, K.B., 1979. The role of proteolytic enzymes in fish digestion. In: E. Styczynska-Jurewicz, T. Backiel, E. Jaspers and G. Persoone (Editors), Cultivation Of Fish Fry and Its Live Food. Eur. Maricult. Sot., Bredene, Belgium, Spec. Publ. No. 4, pp. 102-126. Dabrowski, K.B., 1982. Proteolytic enzyme activity decline in starving fish alevins and larvae. Environ. Biol. Fish, 7 : 73-76. Folk, J.E. and Schirmer, E.W., 1963. The porcine pancreatic carboxypeptidase A system. J. Biol. Chem., 238: 3883-3889. Folk, J.E., Piez, K.A., Carroll, W.R. and Gladner, J., 1960. Carboxypeptidase B. IV. Purification and characterisation of the porcine enzyme. J. Biol. Chem., 235: 22722276. Howell, B.R., 1973. Problems associated with the feeding of certain flatfish larvae. Inf. Tee. Inst. Invest. Pesq., 14: 109-116. Hummel, B.C.W., 1959. A modified spectrophotometric determination of chymotrypsin, trypsin and thrombin. Can. J. Biochem. Physiol., 37: 1393-1399. Kawai, S. and Ikeda, S., 1973. Studies on digestive enzymes of fishes. IV. Development of the digestive enzymes of carp and black sea bream after hatching. Bull. Jpn. Sot. Sci. Fish., 39: 877-881. Kunitz, M., 1947. Crystalline soybean trypsin inhibitor. II. General properties. J. Gen. Physiol., 30: 291-310. Kuzmina, V.V., 1980. Seasonal and age-related changes in a-amylase activity in the bream, Abramis brama. J. Ichthyol. (U.S.S.R.), 20: 105-110. Lowry, O.H., Rosebrough, N.J. Farr, A.L. and Randall, R.J., 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem., 193: 265-275. Miller, G.L., 1959. Protein determination for large numbers of samples. Anal. Chem., 31: 964. Mitz, M.A. and Schleuter, R.J., 1958. Direct spectrophotometric measurement of the peptide bond: application to the determination of acylase I. Biochim. Biophys. Acta, 27: 168. Ragyanszki, M., 1980. Preliminary investigations on the proteolytic digestive enzymes in carp fry. Aquacult. Hung. (Szarvas), 2: 27-30. Sachar, L.A., Winter, K.K., Sicher, N. and Frankel, S., 1955. Photometric method for estimation of elastase activity. Proc. Sot. Exp. Biol. Med., 80: 323.
262 Seidel, CR. and Simpson, K.L., 1984. Rapid differentiation of Artemia ssp. populations by thin-layer isoelectric focusing. Aquacult. Eng., 3: 303-316. Tanaka, M., Kawai, S. and Yamamoto, S., 1972. On the development of the digestive system and changes in activities of digestive enzymes during larval and juvenile stages in ayu. Bull. Jpn. Sot. Sci. Fish., 38: 1143-1152. Worthington Manual, 1972. Worthington Biochem. Corp., Freehold, NJ, U.S.A.
PROTEASE DEVELOPMENT
J. CLARK’,
K.R. MURRAYI
IN DOVER SOLE [SOLEA
SOLEA
253
(L.) ]
and J.R.STARK’
of Brewing and Biological Sciences and z Department of Chemical and Process Engineering, Heriot-Watt (Great Britain)
’ Department
University, Edinburgh
(Accepted 6 February 1986)
ABSTRACT Clark, J., Murray, K.R. and Stark, J.R., 1986. Protease development in Dover sole [Solea solea (L.)]. Aquaculture, 53: 253-262. An examination of the changes in distribution of exo- and endoproteases has been made in Dover sole intestinal extracts from 24-day larvae to adult fish. Pepsin-like activity was not detectable in 24-day-old animals but was first measured in appreciable amounts at 200 days. All other proteases were present at 24 days and increased in value as the fish matured. The significance of these results to the release of nutrients from fish diets is discussed.
INTRODUCTION
The seedling production of larval fish, bivalves and crustaceans for mass production relies still on live diets such as algae, rotifers (Brachionus plicutilis) and planktonic Crustacea, e.g., Artemia. The culture of these food organisms is not only expensive and labour intensive but is also subject to fluctuations in both the availability, and the nutritional quality of the natural resource (Seidel and Simpson, 1984). The problems, therefore, inherent in the production of live feeds and the manipulation of their nutritional profile has stimulated interest in the development of an artificial diet which meets certain criteria. It has been suggested that the digestive systems of the early larval stages in some species are not fully developed (Tanaka et al., 1972; Kawai and Ikeda, 1973) and therefore the diet should be adapted to meet the changing life stages of the larvae. To acquire information for future development of large-scale supplies of microparticulate diets it is necessary to study the digestive enzyme complement of commercially viable species such as Dover sole Solea solea (L.) (Clark et al., 1984) so that the digestive capacity can be directly related to the nutritional requirements of early larvae. Even more important is the capability of the larvae to digest and assimilate the microparticulate diet which may have an outer wall which is resistant to hydrolysis and dissolution in the alimentary canal. 0044-8486/861$03.50
o 1986 Elsevier Science Publishers B.V.
254
The initial survey of digestive enzymes present in juvenile Dover sole has concentrated on carbohydrases (Clark et al., 1984) and proteases (Clark et al., 1985a). This has led to further studies, particularly on elastase (Clark et al., 1985b), as part of an overall research programme on marine larval feed technology. This paper reports the development of seven proteolytic enzymes in Dover sole aged from 24 days to adult. MATERIALS
AND METHODS
Fish All sole were’supplied by Seafresh Farms (Scotland) Ltd., Hunterston, and were aged 24, 49, 80 and 200 days; adult fish were 2-group. All fish were reared at 20°C from hatching and were starved overnight to evacuate the gut. Fish were weaned from Artemia nauplii on to a dry pelleted diet at 60 days and from 80 days upward had been fed on artificial diets for at least 3 days prior to analysis of the guts and their contents (so that there were no contaminating enzymes from live diets in any of the preparations). In all cases the gut could be easily excised from frozen whole fish; guts were weighed and homogenised in distilled water to give a 1:lO homogenate. Dilutions were carried out as required. The lumen contents were included in all homogenates which were a combination of 90-100 fish, 40-60 fish, and 10 fish for the 24, 49 and 80 and 200-day-old fish gut homogenates, respectively. With the adult a single fish was used for the preparation of the homogenate. Chemicals All chemicals were supplied by the Sigma Chemical Co. unless otherwise stated. Enzyme assays The buffers used in all enzyme assay procedures were 0.1 M HCl (pH l.O), glycine-HCl (pH l.O-1.7), citrate-phosphate (pH 1.7-7.8), glycine-NaOH (pH 7.8-10.1) and phosphate-NaOH (pH 0.1-11.2). All enzyme assays were carried out at the appropriate optimum conditions of pH and temperature (37°C) as indicated by Clark et al. (1985a). TotaZpro tease Total proteolytic activity was estimated by a modification of the casein hydrolysis method of Kunitz (1947). Mixtures consisting of a 1% (W/V) aqueous solution of casein (0.5 ml), intestinal homogenate (0.1 ml) and buffer (0.4 ml) were incubated for 1 h. Ice-cold 5% (w/u) trichloroacetic
255
acid (1.5 ml) was added and the mixture left at 2°C for 30 min. The samples were then centrifuged at 2OOOg for 10 min and the absorbance of the supernatant solution recorded at 280 nm. Tyrosine was used as standard and the units expressed as pg tyrosine liberated per min/g protein. The protein content of the homogenates was determined by the method of Lowry et al. (1951) as modified by Miller (1959). Trypsin
Trypsin-like activity was measured by recording enzymic hydrolysis of 10’ M p-toluene-sulphonyl-L-a&nine methyl ester (TAME) at 247 nm using a modification of the method of Hummel (1959). The substrate was supplied by BDH Chemicals Ltd. (Broom Road, Poole, Great Britain) and assays were carried out at pH 8.0. The extract used was obtained by centrifuging (2000g) a 1:40 aqueous homogenate of gut tissue and assaying the supernatant for enzyme activity. Activity was expressed as enzyme units per mg protein, 1 enzyme unit being equivalent to a 0.001 change in absorbance per min. Chymo trypsin
Chymotrypsin-like activity was measured using 0.00107 M benzoyl-ltyrosine ethyl ester (BTEE) and recording the change in absorbance at 253 nm as described by Hummel (1959) at pH 7.6. The substrate was supplied by BDH Chemicals. Elas tase
Elastase-like activity was measured using elastin-orcein (Sachar et al., 1955) by a modification of the method described in the Worthington Manual (1972). Elastin-orcein (10 mg), pH 10.1 buffer (0.9 ml) and 1:lO whole gut homogenate (0.1 ml) were mixed by continuous rotation in a Spiromix roller (Denley Ltd., Sussex, Great Britain) at 40 rev./min. The mixture was centrifuged (2000g) and the absorbance read at 590 nm against an appropriate elastin-orcein blank which had been incubated for the same time. Activity was expressed relative to the adult value as 100 since it is not possible to express hydrolysis of this product in absolute units. Leucine
aminopeptidase
Leucine aminopeptidase activity was measured by recording the hydrolysis of 0.125 M L-leucinamide at 238 nm using a modification of the method of Mitz and Schleuter (1958) at pH 8.3. Enzyme extracts consisted of the supernatant solution treated as for the trypsin assay and the unit of enzyme activity defined as the hydrolysis of 1 pmole of substrate per min.
256
Carboxypep tidase A and B Carboxypeptidase activity was measured by recording the hydrolysis 0.001 M hippuryl-L-phenylalanine at 254 nm (Folk and Schirmer, 1963) pH 7.5. The tissue extract and definition of enzyme activity were as scribed above for leucine aminopeptidase. An identical assay procedure used for carboxypeptidase B in which 0.001 M hippuryl-L-arginine was substrate (Folk et al., 1960).
of and dewas the
RESULTS
At all pH values the protease levels, as measured by the hydrolysis of casein, showed a general increase with the age of the fish (Fig. 1); however, several differences were manifest. Pepsin-like activity (measured by hydrolysis of casein at pH 2.0) could not be detected in fish aged 24, 49 or 80 days, but by 200 days the activity was 0.8 units/mg protein rising to a value of 2 units/mg protein in the adult fish. Neutral and alkaline proteases were readily detected in fish of all ages. At pH 7.6, gut homogenates from
0
1
2
3
4
5 6 PH
7
8
9
10 11
Fig. 1. Protease development in Dover sole. Hydrolysis of casein by homogenates whole intestine (and contents) from fish of different ages.
of
257
24day-old fish showed 0.59 units/mg protein compared with 4.50 units in adult fish; this constitutes an eight-fold increase in neutral protease activity between 24-day-old fish and adult fish. At pH 10.1, alkaline protease values rose from 0.71 units/mg protein in 24-day-old fish to 8.90 units/mg protein in adult fish; a la-fold increase in activity between the 24-day-old fish and the adult fish. The ratios of neutralalkaline protease also changed as the fish aged, from 1: 1 in 24-day-old fish to 1:2 in the adult. Levels of trypsin and chymotrypsin as measured with specific synthetic substrates (Fig. 2) increased from day 24 to day 200, but there was very little difference in enzyme levels thereafter. At all life stages the levels of chymotrypsin exceeded that of trypsin, with a pronounced rise in chymotryptic activity between 80 and 200 days. The ratio of chymotrypsin:trypsin was also seen to increase in favour of chymotrypsin as the fish aged, aithough trypsin activity increased eight-fold between 24 days and adult fish (1.76 units/mg protein compared to 14 units/mg protein); this differs from chymotrypsin in which there was only a four-fold increase in activity, from 11.3 units/mg protein in 24-day-old sole to 42.0 units/mg protein in adult sole. chymotrypsin M trypsin -
Fish age (days) Fig. 2. Activities of trypsin and chymotrypsin
in Dover sole of different ages.
Fig. 3. shows the relative activity of an elastase-like enzyme in Dover sole of different ages. It can be seen that although activity was highest in adult sole, there was very little difference in relative activity from 80 days onward; the enzyme extract of 80day-old fish in fact had 93% of the activity of the adult fish gut preparation. There was, however, a sharp increase in activity from day 24 up to day 80, and it can be seen that between day 24 and adulthood there was an eight-fold increase in the relative activity of the elastase-like enzyme. Leucine aminopeptidase activity (Fig. 4) did not increase much between 24- and 49-day-old fish, but the level rose quickly to a maximum value of 22.6 units/mg protein for 200day-old fish. Although activity fell slightly in
80
200
Adult
Fish age (days) Fig. 3. Relative activity of elastase in Dover sole of different ages.
25
leucine aminopeptidase
22 ‘E 3
5 0
24
49
80
200
Adult
Fish age (days) Fig. 4. Activity of leucine aminopeptidase
in Dover sole of different ages.
I
24
49
80
200
Adult
Fish age (days) Fig. 5. Activities of carboxypeptidases
A and B in Dover sole of different ages
259
adult fish, there was still almost a three-fold rise in activity from 7.5 units/ mg protein for 24-day-old fish to 20.0 units/mg protein for adult fish. The level of carboxypeptidase A was always higher than that of carboxypeptidase B (Fig. 5); carboxypeptidase A levels did not increase much from day 24 to day 49, but there was then a rapid rise in activity up to 200 days. Between 200 days and adult fish there was very little difference in activity. There was, overall, a three-fold increase in the level of carboxypeptidase A, from 8.4 units/mg protein in 24-day-old fish to 23.0 units/mg protein in adult fish. Carboxypeptidase B, on the other hand, exhibited a gradual increase in activity from day 24 to day 200 whereupon there seemed to be a plateau of activity. There was, however, a seven-fold increase in activity from 24-day-old fish (1.4 units/mg protein) to adult fish (10.5 units/mg protein). DISCUSSION
In the present studies, fish of an age suitable for dissection of the gut have been chosen. In work on other species (e.g., Dabrowski, 1979, 1982; Ragyanszki, 1980) analysis of protease in fish younger than 24 days has been carried out, but in our experience, with Dover sole, the use of whole fish homogenates (for specimens which are too small for dissection) results in high blank values and data which are difficult to reproduce. Furthermore, with whole fish homogenates, it is not always possible to be certain that a particular enzyme has originated from the digestive tract. The development of enzymes in several different species of fish has been studied but the techniques used have differed considerably. Kuzmina (1980), in the work on the bream, A brumis bruma, has indicated that there is an inverse relationship between amylase activity and the age of the fish. However, most other studies show that the general trend is for there to be an increase of enzyme activity after the fish reaches about 30 days old and progressing into adulthood. Kawai and Ikeda (1973) suggested that the problems of feeding at an early age may be due to low contents of digestive enzymes and hence there have been a number of attempts to investigate the enzymic content of fish from the egg stage through hatching. Alliot (1981) has reviewed the development of digestive enzymes in teleosts and has indicated that these enzymes exist soon after hatching, and in work on the European sea bass, Dicentrurchus lubrux, Alliot et al. (1977) found that the trypsin activity increased slowly between 0 and 5 days, decreased from 5 to 15 days and thereafter remained quite stable up to 30 days. Using similar methods, these authors (Alliot et al., 1980) have examined the activities of a number of enzymes, including leucine aminopeptidase and trypsin, in sole larvae from O-30 days. In the present experiments, the general trend of development of enzymes (Fig. 1) indicates that there is late development of pepsin-like activity (pH 2.0). This has been noted in other fish (Kawai and Ikeda, 1973) and is evidently dependent on the development of an active stomach zone in the in-
260
testine. This has been shown by the fact that in Chimaera which are stomachless, there are no pepsin-like enzymes (Barrington, 1957). In very young fish the relative amount of neutral and alkaline proteases is about the same but as the fish age the latter activity becomes more predominant. Previous studies (Clark et al., 1985a) have suggested that there are a number of enzyme activities at pH 7-8, including trypsin, chymotrypsin and collagenase, whereas at pH 10 there is a single major contribution from an elastase-like enzyme (Clark et al., 1985b). With the exception of pepsin-like activity, where the very acidic pH gives a degree of specificity, casein is a non-specific substrate. Assays for each of the other enzymes were therefore carried out using specific substrates. Chymotrypsin activity increased with the age of the fish (Fig. 2) but the rate of increase was far less pronounced with trypsin. Tanaka et al. (1972) reported that in carp fry, trypsin activity hardly increased in activity during transition from larvae to juveniles. Also in studies on carp, Ragyanszki (1980) reported chymotrypsin activity to be from two to nine times higher than trypsin during the period of 0 to 12 days of age. In the present work all activities showed a general increase with age of the fish but the details differed somewhat. Elastase activity increased much more rapidly during the period between 24 and 80 days (Fig. 3) than did trypsin or chymotrypsin (Fig. 4). Leucine aminopeptidase rose rapidly between 49 and 200 days and then plateaued whereas the rise with the two carboxypeptidases was more gradual and continuous. The problems associated with feeding of certain flatfish larvae including Dover sole have been discussed by Howell (1973) who emphasises the need for a diet which can be reliably produced. Whilst the brine shrimp Arternia salina L. nauplii can readily be stored and hatched, it has to be imported and the reliability of stocks may be in doubt. In this study the pH profile of hydrolysis against casein indicated that at pH 9.5-10 there was maximum attack on the protein material particularly when extracts of the digestive tract of older fish were examined. However, this pH value is unlikely to exist in the lumen under normal physiological conditions and therefore the levels of trypsin and chymotrypsin must be considered of prime importance. Further studies are in progress on the action of different levels of enzymes on a variety of types of artificial feeds in order to correlate the present work to the digestive capabilities of the fish. ACKNOWLEDGEMENT
The authors wish to acknowledge the SERC (Marine Technology Directorate) for the support of this work. REFERENCES Alliot, E., 1981. Nutrition des poissons. III. Evolution de quelques actkit& digestives au tours du dhveloppement larvaire des tUost6ens. M. Fontaine (Editor), Editions du
261 Centre National de la Recherche Scientifique, Paris. A&es Colloq. CNERNA, pp. 7988. Alliot, E., Pastoureaud, A. and Trellu, J., 1977. Evolution des activites enzymatiques dans le tube digestif au tours de la vie larvaire du bar (Dicentrarchus Zabrax). Variations des proteinogrammes et des zymogrammes. 3rd Meeting of the ICES Working Group on Mariculture, Brest, France, lo-13 May 1977. A&es Colloq. CNEXO, 4: 85-91. Alliot, E., Pastoureaud, A. and Trellu, J., 1980. Evolution des activites enzymatiques dans le tractus digestif au tours de la vie larvaire de la Sole. Variations des proteinogrammes et des zymogrammes. Biochem. Syst. Ecol., 8: 441-445. Barrington, E.J.W., 1957. The alimentary canal and digestion. In: M.E. Brown (Editor), The Physiology of Fishes, Vol. 1, Academic Press, New York, NY, pp. 109-161. Clark, J., MacNaughton, J.E. and Stark, J.R., 1984. Metabolism in marine flatfish. I. Carbohydrate digestion in Dover sole (Solea soleo L.). Comp. Biochem. Physiol. B, 77: 821-827. Clark, J., MacDonald, N.L. and Stark, J.R., 1985a. Metabolism in marine flatfish. II. Protein digestion in Dover sole (Solea solea L.). Comp. Biochem. Physiol. B, 81: 217-222. Clark, J., MacDonald, N.L. and Stark, J.R., 198513. Metabolism in marine flatfish. III. Measurement of elastase activity in the digestive tract of Dover sole (Soiea solea L.). Comp. Biochem. Physiol. B, 81: 695-700. Dabrowski, K.B., 1979. The role of proteolytic enzymes in fish digestion. In: E. Styczynska-Jurewicz, T. Backiel, E. Jaspers and G. Persoone (Editors), Cultivation Of Fish Fry and Its Live Food. Eur. Maricult. Sot., Bredene, Belgium, Spec. Publ. No. 4, pp. 102-126. Dabrowski, K.B., 1982. Proteolytic enzyme activity decline in starving fish alevins and larvae. Environ. Biol. Fish, 7 : 73-76. Folk, J.E. and Schirmer, E.W., 1963. The porcine pancreatic carboxypeptidase A system. J. Biol. Chem., 238: 3883-3889. Folk, J.E., Piez, K.A., Carroll, W.R. and Gladner, J., 1960. Carboxypeptidase B. IV. Purification and characterisation of the porcine enzyme. J. Biol. Chem., 235: 22722276. Howell, B.R., 1973. Problems associated with the feeding of certain flatfish larvae. Inf. Tee. Inst. Invest. Pesq., 14: 109-116. Hummel, B.C.W., 1959. A modified spectrophotometric determination of chymotrypsin, trypsin and thrombin. Can. J. Biochem. Physiol., 37: 1393-1399. Kawai, S. and Ikeda, S., 1973. Studies on digestive enzymes of fishes. IV. Development of the digestive enzymes of carp and black sea bream after hatching. Bull. Jpn. Sot. Sci. Fish., 39: 877-881. Kunitz, M., 1947. Crystalline soybean trypsin inhibitor. II. General properties. J. Gen. Physiol., 30: 291-310. Kuzmina, V.V., 1980. Seasonal and age-related changes in a-amylase activity in the bream, Abramis brama. J. Ichthyol. (U.S.S.R.), 20: 105-110. Lowry, O.H., Rosebrough, N.J. Farr, A.L. and Randall, R.J., 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem., 193: 265-275. Miller, G.L., 1959. Protein determination for large numbers of samples. Anal. Chem., 31: 964. Mitz, M.A. and Schleuter, R.J., 1958. Direct spectrophotometric measurement of the peptide bond: application to the determination of acylase I. Biochim. Biophys. Acta, 27: 168. Ragyanszki, M., 1980. Preliminary investigations on the proteolytic digestive enzymes in carp fry. Aquacult. Hung. (Szarvas), 2: 27-30. Sachar, L.A., Winter, K.K., Sicher, N. and Frankel, S., 1955. Photometric method for estimation of elastase activity. Proc. Sot. Exp. Biol. Med., 80: 323.
262 Seidel, CR. and Simpson, K.L., 1984. Rapid differentiation of Artemia ssp. populations by thin-layer isoelectric focusing. Aquacult. Eng., 3: 303-316. Tanaka, M., Kawai, S. and Yamamoto, S., 1972. On the development of the digestive system and changes in activities of digestive enzymes during larval and juvenile stages in ayu. Bull. Jpn. Sot. Sci. Fish., 38: 1143-1152. Worthington Manual, 1972. Worthington Biochem. Corp., Freehold, NJ, U.S.A.