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Metabolism in marine flatfish—I. Carbohydrate digestion in Dover sole (Solea solea L.)
Comp. Biochem. Physiol. Vol. 77B, No. 4, pp. 821 827, 1984 Printed in Great Britain
0305-0491/84 $3.00+0.00 ~.~ 1984PergamonPress Ltd
METABOLISM IN MARINE FLATFISH--I. C A R B O H Y D R A T E DIGESTION IN DOVER SOLE (SOLEA SOLEA L.) J. CLARK, J. E. MCNAUGHTONand J. R. STARK Department of Brewing and Biological Sciences, Heriot-Watt University, Edinburgh, Scotland. (Tel.: 031-225-8432) (Received 7 September 1983) A b s t r a e t - - l . A survey of the carbohydrases in the digestive tract of juvenile and adult Dover sole indicated the presence of strong amylase and maltase activities. 2. The highest levels of these enzymes were present in the foregut region. 3. Chitinase and chitobiase activities were also detected and shown to be optimal at pH 9.5 and 5.0 respectively. 4. Other carbohydrases were either absent from gut homogenates or were only present at very low levels. 5. The relationship between these results and the nutritional requirements of Dover sole is discussed and comparisons made with studies on other fish.
INTRODUCTION
The farming of marine fish is presently based on the use of live food organisms during the first weeks after hatching and, while the most promising development would be to use artificial diets from first feeding, considerable problems exist. These difficulties are reviewed by Girin (1979) and are principally concerned with survival, although growth rates are also of importance. A marine flatfish, the plaice, (Pleuronectes platessa L.) has been reared from eggs to metamorphosis on artificial diets (Adron et al., 1974) in studies which indicated a survival rate of about 20% compared with about 38% in control larvae fed Artemia nauplii under similar conditions. It has also been demonstrated by Gatesoupe et al. (1977) that sole larvae can be reared on artificial diets straight from first feeding but again many problems must be solved before the exclusive use of artificial diets can become commercially viable. A second, although not so important, economic consideration is the requirement that fish have for relatively expensive, high protein diet. As is indicated in recent reviews (Pfeffer, 1982; Walton and Cowey, 1982) it is well recognised that fish obtain most of their energy from protein and fat and have a limited ability to utilise carbohydrate, therefore any developments which allowed the use of diets with higher carbohydrate content would obviously be of economic significance. It is against this background that studies on the digestive enzymes in marine flatfish were undertaken to obtain a fuller understanding of factors which may be of importance in the formulation of diets. In the present paper the carbohydrases in the digestive tract of Dover sole (Solea solea L.) have been examined and the results compared with studies on other fish species. Farmed Dover sole have a good commercial potential since the fish is rarely caught in the wild and, with its high quality food protein, it is a readily marketable product. However, the above mentioned problems concerning diet and nutrition are somewhat
accentuated by the fact that sole is an extremely fastidious feeder. This gives rise to difficulties in weaning from a live (Artemia) diet to an artificial diet and this type of problem in Dover sole and in other species of fish has prompted research into feeding stimulants (De Groot, 1969; Mackie et al. 1979). For these reasons, the ongrowing of sole to marketable size has only recently become of commercial importance. Although a considerable amount of work has been carried out on fish nutrition in general (Hastings and Dickie, 1972) and many studies have been made on both live and artificial diets (see for example Dye, 1980 and Myers, 1979 respectively) there is less information on the biochemical aspects of digestive processes in fish. However information is available on rainbow trout (see for example Bergot, 1979, Spannhof and Plantikow, 1983 and Inaba et al., 1963) and on some flatfish species in Japanese waters (Yasunaga, 1972). The biochemistry of digestion in Dover sole has so far been largely neglected and the following study therefore sets out, as its initial objective, to examine the enzymes present in the digestive tract of juvenile and adult sole and to correlate these findings to the nutritional requirements of this fish. MATERIALS AND METHODS
Juvenile and adult samples were supplied by Seafresh Farms, Hunterston, Scotland. 0-group juveniles weighed between 15-34g and were 50-150ram long whereas adult fish were 2-group (average weight 400 g and length 300 ram). All fish had been continuously fed on artificial diets for at least 3 weeks (so there were no contaminating enzymes from live diets in any of the preparations). Preparation of extracts The majority of these studies were carried out on juvenik fish using homogenates of the whole digestive tract. FoJ examination of different parts of the intestine the alimentary canal of adult fish was divided into stomach foregut, midgut, hindgut and rectum. Due to the lack ol
821
822
J. CLARK et al.
regional differentiation such divisions were arbitrary, being made on the basis of percentage of total gut length as suggested by Braber and De Groot (1973). All fish were starved for 3 hr before being killed by a blow to the head followed by deep-freezing. After partial thawing the intestine could be readily excised, weighed and homogenized with distilled water to give a 1:10 homogenate. Most analyses were carried out by using a further dilution of this sample to give a 1:40 homogenate. The lumen contents, present in the gut, were included in all homogenates unless otherwise stated. When required, pure gut homogenate was prepared by rinsing out an open digestive tract with distilled water and then homogenizing the cleaned intestine.
Chemicals Unless otherwise stated all chemicals were supplied by Sigma Chemical Co.
Analytical procedures The protein content of homogenates was determined by the method of Miller (1959) using bovine serum albumin (25-100 #g) as standard. Reducing sugars were estimated by the Somogyi-Nelson procedure as described by Robyt and Whelan (1968). The buffers used were either citratephosphate, glycine-HC1 or glycine-NaOH. For survey purposes substrate (10mg) was incubated with homogenate (1 ml) at 37°C for 15hr. Toluene was added to prevent bacterial growth. Samples were spotted on to chromatograms developed in ethyl acetate:pyridine:water (10:4:3 by vol.) and the products revealed using the silver nitrate procedure of Trevelyan et al. (1950). Appropriate standards were included. Amylase activity was measured in the presence and absence of calcium ions in digests containing 1~o soluble starch (5 ml), 0.1 M citrate phosphate buffer pH 6.5 (1 ml), 0.1 M calcium chloride (1 ml) or 0.1 M EDTA (1 ml) and a 1:40 intestine homogenate (3 ml). Digests were incubated for times ranging from 2 hr to 24 hr before taking samples (1 ml) or smaller volumes as appropriate for estimation of reducing sugars. Similar digests were used for examination of the pH-activity profile except that water (1 ml) was used in place of calcium chloride or EDTA solutions. The units of amylase activity were expressed as #moles of maltose equivalents released per rain per gram protein. The action pattern of amylase attack on starch was examined by paper chromatography and also by estimating the release of reducing sugars and the reduction in iodine staining power at the same time intervals. This was carried out by incubating 1~ starch solution (4 ml) with a 1:10 homogenate (4ml). Samples (0.1 ml) were taken at intervals and the volume made up to 1 ml with water before estimating reducing sugars. At the same time-intervals, samples (0.1 ml) were mixed with water (10ml) and 0.1 ml of a solution containing iodine (0.2~o) and potassium iodide (2~o). The iodine staining colour was read at 600 nm. The iodine staining power was plotted against the production of reducing sugars expressed as percentage conversion of the starch substrate. Glucosidases were examined using p-nitrophenyl glycosides (3 mg) in buffer (1 ml) with homogenate (1 ml) for times ranging from 1-24 hr. The digests were then heated at 100°C for 2 min cooled in ice and centrifuged to remove precipitated material. The supernatant solution was treated with 1 M potassium bicarbonate solution (1 ml) and the absorbance read at 420nm against an appropriate blank containing heat-inactivated homogenate. The hydrolysis of disaccharides (maltose, sucrose, lactose and cellobiose) was examined chromatographically as well as by means of glucose oxidase procedure. For this purpose a 1~o aqueous solution of the disaccharide (0.5 ml) was incubated with a 1 : 10 gut homogenate (0.5 ml) at 37°C for times ranging from 20 min-5 hr. The digests were heated for 2min at 100°C, centrifuged and the supernatant solution
analysed by the glucose oxidase procedure as described in the Boehringer Mannheim Peridochrom kit No. 676543. The hydrolysis of chitin was examined using chitin azure (10mg) suspended in buffer (1 ml) and 1:10 homogenate (0.5 ml). The mixture was placed in a sealed centrifuge tube on a Spiramix rotating shaker to keep the solid substrate in suspension. After a suitable time interval the digest was centrifuged and the absorbance of the supernatant solution read at 545 nm against appropriate blanks for each pHvalue. Chitobiase activity was investigated using pnitrophenyl fl-glucosaminide as described above under "glucosidases" and also by paper chromatography using N,N'-diacetylchitobiose as substrate. Chromatofocusing was used to examine the ~-glucosidase and chitobiose activities. A 6ml sample of 1:10 gut homogenate was applied to the column (1 x 25cm) containing 15 ml of a slurry of Polybuffer Exchanger 94 (Pharmacia) which had been equilibrated with 25 mM imidazole-HC1 buffer pH 7.4. The column was then eluted with Polybuffer 74-HC1 (pH 5.0) at a rate of about 20 ml/hr. Fractions (6 ml) were collected and analysed for enzyme activities using pnitrophenyl e-glucoside or p-nitrophenyl /~-glucosaminide as previously described. RESULTS
Survey of activities present The hydrolytic activity against different substrates are s h o w n in Table 1. These results are compiled from reducing sugar m e a s u r e m e n t s a n d from analysis by paper c h r o m a t o g r a p h y . The substrates which were readily hydrolysed all c o n t a i n e d ~-glucosidic linkages ( p - n i t r o p h e n y l ~-glucoside, maltose, maltotriose, starch a n d glycogen); the only exceptions to this being p - n i t r o p h e n y l fl-glucosaminide which was easily hydrolysed a n d chitin and chitin azure which were also attacked a l t h o u g h at a slower rate. All other substrates were either not hydrolysed or were only hydrolysed to a very limited extent. This general p a t t e r n of activity was the same for b o t h adult and juvenile D o v e r sole homogenates.
Amylase activity H o m o g e n a t e s of b o t h juvenile a n d adult Dover sole intestine acted on starch with the p r o d u c t i o n of reducing sugars. The o p t i m u m p H for this activity was at p H 6.7 (Fig. 1). A n e x a m i n a t i o n o f the products of starch hydrolysis by p a p e r chrom a t o g r a p h y are s h o w n in Table 2. In the initial phases of action, a series of sugars were formed with R glucose-values of 1.00, 0.63, 0.41, 0.24 and 0.13 c o r r e s p o n d i n g to authentic samples of glucose and the maltosaccharide series of sugars. As the action progressed, less of the oligosaccharides r e m a i n e d a n d more glucose was formed. The m o d e of action of amylase was further studied by g r a p h i n g the production o f reducing sugars the reduction in iodine staining power o f the starch substrate. The result o f this experiment is s h o w n in Fig. 2. The rate of release of reducing sugars by the intestinal amylase from D o v e r sole is illustrated in Fig. 3 which indicates the effect of calcium chloride and also shows t h a t the reaction rates becomes slower as the hydrolysis of the starch substrate proceeds. Fig. 4(a) show the rate of release o f reducing sugars from h o m o g e n a t e s of different parts of the intestinal tract of an adult D o v e r sole. The m a x i m u m activity was found in the foregut region. The effect of centrifugation on the
Metabolism in marine flatfish--I
823
Table 1. Carbohydrases in the alimentary canal of Dover sole Substrate Hydrolysis Methyl ~t-glucopyranoside Methyl fl-glucopyranoside p.Nitrophenyl-N-acetyl-fl-glucosaminide + + p-Nitrophenyl ~-glucopyranoside + + + p-Nitrophenyl fl-glucopyranoside + Maltose + + + Cellobiose + Sucrose Lactose + N,N'-diacetyl chitobiose + Isomaltose + Melibiose Raft]nose Maltotriose + + + Starch + + + Glycogen + + + Pullulan Laminarin Inulin Xylan Agar Alginic acid Sodium carboxymethyl cellulose Cellulose Pectin Chitin azure + Chitin + - = no hydrolysis, + = a trace of hydrolysis. +, + + and + + + indicate increasing extents of hydrolysis.
Table 2 Products of hydrolysis of starch by an intestinal homogenate from Dover sole Incubation time Product 15 min 1 hr 2 hr 24 hr
activity o f the amylase indicated that approximately half o f the activity was lost when the solid material was r e m o v e d f r o m intestinal homogenates. A t t e m p t s to purify the a-amylase by c h r o m a t o f o c u s i n g were not successful due to the lack of stability of the enzyme.
Chitobiase and chitinase
Glucosidases Using the a p p r o p r i a t e p - n i t r o p h e n y l derivatives, a a n d fl-glucosidase activities were examined. Both activities were optimal at a b o u t p H 5.7 but the a-glucosidase was m o r e t h a n twice as active as the fl-glucosidase. F u r t h e r m o r e the c h r o m a t o g r a p h i c studies indicated t h a t there was only very slight hydrolysis o f cellobiose (4 0-fl-D-glucosylglucopyranoside). The latter fact was verified by estimating the glucose released from cellobiose using the glucose oxidase method. As is shown in Fig. 5 a n initial
Glucose
+ +
Maltose + + Maltotriose + + Maltotetraose + + Maltopentaose + indicates absence of product. +, + +, + + + and + + + product.
+ + +
+ + +
+ + + +
+ + + + + + +
+ + + + -
-
-
+ indicate increasing amounts of
release of glucose d u r i n g the first h o u r was followed by only very limited glucose p r o d u c t i o n during a subsequent four h o u r period. This c o m p a r e s with a steady p r o d u c t i o n o f glucose from maltose. In view o f this the fl-glucosidase a n d cellobiase activities were not studied further. The a-glucosidase activity was examined by c h r o m a t o f o c u s i n g a n d as shown in Fig. 6 which indicates t h a t there are multiple forms of this enzyme. The relative a m o u n t s of a-glucosidase in the intestine was measured using p - n i t r o p h e n y l a-glucoside and, as is s h o w n in Fig. 4(b), the maxi m u m activity was present in the foregut region. The relative a m o u n t s of enzyme in each region were in parallel with the a m o u n t s o f a-amylase in these regions.
As indicated in Table 1 b o t h chitin a n d chitin azure were hydrolysed but only if the p H was on the alkaline side of neutrality. The hydrolysis o f p - n i t r o p h e n y l N-acetylglucosaminide a n d N , N ' diacetylchitobiose occurred only in more acid solutions. These observations were studied q u a n titatively as indicated in Fig. 7. The o p t i m u m values were at p H 9.5 for chitinase a n d p H 5.0 for chitobiase. The chitobiase activity was m u c h easier to study t h a n the chitinase because of the sensitivity o f the p - n i t r o p h e n o l assay. Using c h r o m a t o f o c u s i n g (as previously described) to chitobiase activity could also be separated into isoenzymes with isoelectric points 1'0
o'BL
(b)
100
Iodine Stain
"" \ \
(A6oo) I""..~ (a,
BO
Retative Activity
0.6t-~ , ~.. .
~
60
40
20
0 pH
Fig. 1. Activity-pH curves for hydrolysis of starch (0--0) and p-nitrophenyl •-glucoside ( 0 O) by intestinal homogenates. C.B.P.77/4B--L
"%""""""i ..................i'" 20 40 60
BO
°Hydrolysis of starch Fig. 2. Hydrolysis of starch (release of reducing sugars) and reduction in iodine stain (a) Dover sole intestinal extract, (b) theoretical curve for an exo-enzyme (. . . . . ) and (c) for an endo-enzyme ( ......... ).
824
J. CLARK et al. [
7ooI
,°1
/-"
t
600 0
250 1
J
//-I /
500
/'~
/ 300
200 0
.E cu "5 L Q_
,,~
0 200
150. 0
1
2
/+
3
5
hme (h)
~
Fig. 5. Hydrolysis of disaccharides by juvenile Dover sole intestinal homogenate measured by the glucose oxidase method using maltose ( ' - - - ' L cetlobiose ( A - -A), sucrose (&--- &) and lactose ( 0 -©),
100-
'G
~
so2"~
2'o
~o
go
8b
lOO
Time (mm) Fig. 3. Release of reducing sugars from starch by Dover sole intestinal homogenates in the presence of calcium chloride ( 0 - - 0 ) and with EDTA ( I l l - - U ) .
(a)
I~electric pts (pH)
Lla/
20-
~'~
](b) 5-6 Ifrl 55 I"'
(b)
_
_
_
_
1-6 A~20 12
at pH 6.6, 5.7 and 5.6. Furthermore it was shown that on centrifugation of the gut homogenates almost all the activity was in the supernatant solution and only traces of enzyme remained attached to the mucosa.
0-80.1NiNaCI
04
O t h e r activities
Figure 5 shows the very limited extent of hydrolysis of lactose and sucrose as compared to maltose. As in the case of cellobiose, a short quick release of a small amount of glucose is followed by a barely measurable production of glucose.
a°y.
~o
~-
io
go
lOO 12o
Fraction No. Fig. 6. Chromatofocusing of Dover sole intestinal homogenate showing multiple forms of ~.-glucosidase. Fractions were analysed using p-nitrophenyl :~-glucoside as substrate and the release of p-nitrophenol measured at 420 nm.
,0lb
200.
oi.
'I//
1oo// o
i;
~o
Time (min)
3'o
o
ib
~o
~'o
Time (rain3
Fig. 4. Rate of hydrolysis of (a) starch and (b) phenyl c~-glucoside by homogenates of"Dover sole stomach (O ©) foregut ( 0 - - 0 ) midgut (A A) and hindgut ( A - - A ) .
Metabolism in marine flatfish--I
The extremely minute traces of lactase and sucrase activities detected in gut homogenates from Dover sole is in contrast to the situation in most mammals where it has been shown for example that the digestive tracts of rat, rabbit, sheep and humans all readily hydrolyse maltose, sucrose and lactose (Prosser and Brown, 1961). In a similar study on the digestive enzymes of the scallop Pecten maximus a much wider range of carbohydrate substrates were hydrolysed (Stark and Walker, 1983).
100" 80' Relafive
60
Acfivify
40
20
Amylase and ~-glucosidase activities 2
4
6
8
10
12
pH Fig. 7. Activity--pH curves for hydrolysis of chitin azure
(O
O) (0
825
and p-nitrophenyl N-acetylglucosaminide 0 ) by homogenates of Dover sole intestine.
DISCUSSION Previous studies on the digestive enzymes in fish include reports on both freshwater and marine species. Olatunde and Ogunbiji (1977) working on freshwater species from Lake Kainja, Nigeria have demonstrated the presence of amylase activity in the tropical catfish Entropicus niloticus and Schilbe mystus but were unable to detect any cellulase, lactase, sucrase or maltase activities in the digestive tract of these species. However, in earlier studies, Sarbahi (1951) reported sucrase and maltase activities in goldfish. Amylase activity has been demonstrated in the bream Abramis brama from a freshwater reservoir in Russia and the activity of this enzyme varied inversely with the age of the fish (Kuzmina, 1980). Thus, although amylase activity has been found in a variety of different species of fish, variations in the levels of glycosidases seem to occur. Kawai and Ikeda (1971) examined the enzymes present in carp (Cyprinus carpio ), ayu (Plecoglossus altivelis) and red sea bream (Pagrus major) and compared these with the carbohydrases present in the digestive system of rainbow trout. These fish all possessed fairly strong amylase and c~-glucosidase (maltase) activities in their digestive systems but fl-glucosidase, ~-galactosidase, fl-fructosidase and cellulase were absent. This was similar to the enzyme profile found in the pyloric caecum of the salmon Oncorhynchus keta reported by Ushiyama et al. (1965).
Survey of enzymic activities The survey of enzymic activities in the present study (Table 1) indicated that homogenates of Dover sole intestine contained relatively strong amylase and maltase activities. This was shown by the release of reducing sugars from starch and glycogen and the production of glucose from maltose and maltotriose. Many other carbohydrases were either totally absent or were present only in trace amounts. The exceptions to this were chitinase and chitobiase activities; the presence of these enzymes was indicated by the hydrolysis of chitin and chitin azure in the case of chitinase and N,N'-diacetylchitobiose and p nitrophenyl-n-acetyl-fl-glucosaminide in the case of chitobiase.
The characterisation of the amylase activity in homogenates of Dover sole intestine was based on two experiments. The reducing sugars produced in the initial phases of hydrolysis of starch (Table 2) formed a homologous series indicating that the process was one of random hydrolysis. As a reaction proceeded glucose became the predominant sugar indicating that amylase action was being followed by hydrolysis of lower maltosaccharides by an ct-glucosidase (or maltase). These results were substantiated by the second experiment, shown in Fig. 2 in which the iodine staining power of the starch substrate is measured at different stages during the hydrolysis. Endo- or random attack on starch causes a rapid reduction in the molecular weight of the polymer and in its iodine staining properties (since long runs of ct-l,4-1inked glucose residues are required for iodine binding). Exo-hydrolysis, on the other hand, results in a rapid production of reducing sugars as each cleavage produces a free sugar but, because the main part of the molecule remains intact, there is a very slow reduction in iodine stain. The theoretical curves for exo- and endo-acting amylases are shown in Fig. 2b and 2c respectively. The curve for Dover sole amylase (a) can be explained by assuming an initial random attack followed by hydrolysis of the oligosaccharides formed to produce reducing sugars at a faster rate than would be expected from an endo-amylase acting on its own. The curve (a) is therefore displaced to the right of (c). The pH profiles (Fig. 1) indicated that the amylase had optimal activity at pH 6.7 whereas a-glucosidase was optimal at pH 5.7; this would suggest that these two activities originated from different enzyme proteins. Amylases from the digestive tracts of fish would appear to differ depending on species. For example the amylase from the salmon, Oncorhynchus keta had a pH optimum of 8.5 (Ushiyama et al., 1965) whereas in studies on amylases from three species of flatfish the pH optimum in each case was 7.5 (Yasunaga, 1972). Amylase from Tilapia mossambica, on the other hand, was optimal at pH 6.7 (Nagase, 1964). The amylase from Dover sole was more than twice as active in the presence of calcium chloride than in its . absence (Fig. 3) (EDTA was added to chelate any endogenous calcium ions). Attempts to purify this enzyme by chromatofocusing resulted in total loss of activity. However, the a-glucosidase activity was readily resolved into isoenzymes by this procedure (Fig. 7). The morphology of the digestive system of Dover sole shows a simple structure which penetrates deeply into the body cavity. The lack of distinguishable oesophagus and stomach regions in the digestive tract
826
J. CLARK et al.
of sole has been described by De G r o o t (1969). It was thus impossible, in the present study, to clearly define the different sections of the gut and an arbitrary division had to be made. The analysis of enzymes from different parts of the gut (Fig. 4a, b) indicated a similar pattern in distribution between c~-amylase and c~-glucosidase activities. In each case, the amount of amylase and ~-glucosidase activities followed the order: foregut > hind-gut > mid-gut > stomach. In similar studies, Kawai and Ikeda (1971) demonstrated marked amylase activity in the distal portion of the carp intestine when this was divided into three equal portions by length. High levels of amylase were observed in the mid-gut of the flatfish Limanda yokohamae (Yasunaga, 1972). The studies of Ugole (1960), K u z ' m i n a (1977) and Spannhof and Plantikow (1983) have indicated that the intestinal mucosa is of significance in amylolytic digestion of starch. It was therefore of interest to examine the effect of centrifugation of homogenates on the amylase activity. In the present study with Dover sole, the level of c~-amylase was halved when a centrifuged homogenate was used in place of a whole homogenate containing particulate material. It is therefore evident that in Dover sole the intestinal membrane either binds some of the ~-amylase, as is believed to occur in land animals (Dalqvist, 1978), or contributes in some other way to amylase action. Other activities
As can be seen from Table 1, several substrates were not attacked by Dover sole intestinal homogenares. These included the polysaccharides laminarin, cellulose, xylan and pullulan. In addition, the disaccharides sucrose, lactose and cellobiose were only hydrolysed very slightly as judged by paper chromatography. A study of the rate of hydrolysis of these disaccharides compared with that of maltose was carried out using a glucose oxidase method (Fig. 5). Whole homogenates were used in these experiments and it is evident that whilst maltose undergoes rapid and steady hydrolysis, the other disaccharides are hydrolysed only to a very limited extent even after prolonged incubation. Hydrolysis o f chitin
Chitinase occurs in the digestive system of certain fishes and other vertebrates (Fange and Grove, 1979) and has been examined in a number of different species of fishes (Yoshida and Sara, 1970). In the present study chitinase and chitobiase activities were detected in the digestive system of Dover sole. Chitin itself is a hard and insoluble substrate which is difficult to use in enzyme assays and chitobiose is expensive. Hence, other than for the initial survey, the artificial substrates chitin azure and p-nitrophenyl N-acetyl-/3-glucosaminide were used. Chitinase was optimal at 9.5 whilst the chitobiase showed maximum activity at pH 5.0. (Fig. 7). Further studies will be necessary to ascertain whether these enzymes are produced by intestinal bacteria or by the digestive system of the Dover sole itself. However the present studies indicated that chitobiase existed as isoenzymes which appeared in the non-particulate fraction of homogenates. In conclusion the present studies indicate that
homogenates of Dover sole intestine have the ability to hydrolyse starch-type polysaccharides and chitin to their respective monosaccharide units. The amylase/c~-glucosidase system will convert starch, (which may be present as such in artificial diets) to glucose; but in the natural environment glycogen, which is a constituent of crustaceans (Kjolberg et al,, 1963) forms part of the diet. Whilst the chitinase-chitobiase system can, at least qualitatively, hydrolyse the shells of crustaceans to N-acetylglucosamine it is not known to what extent this occurs on a quantitative basis. Further work is in progress to study the factors that are of significance in the synthesis of these enzymes and in particular to examine the role of digestive enzymes during the earlier periods of the life cycle of the Dover sole. Acknowledgements--This work was supported by an SERC Marine Technology Grant and forms part of the research programme of the Aquaculture Enginering Group, HeriotWatt University. The authors also wish to thank Mr S. T. Hull and his staff (Seafresh Farms Ltd) for their help.
REFERENCES
Adron J. W., Blair A. and Cowey C. B. (1974) Rearing of plaice (Pleuronectes platessa) larvae to metamorphosis using an artificial diet. Fish Bull. Cal([i 72, 352 357. Bergot F. (1979) Effects of dietary carbohydrates and of their mode of distribution on glycaemia in rainbow trout. Comp. Biochem. Physiol. 64A, 543-547. Braber L. and De Groot S. J. (1973) On the morphology of the alimentary tract of flatfishes (Pleuronectiformes). J. Fish. Biol. 5, 147 153. Dalqvist A. (1978) Disturbances of the digestion and absorption of carbohydrates. In Intern. Rev. Biochem. (Edited by Manners D. J.), Vol. 16, pp. 178-207. University Press, Baltimore. De Groot S. J. (1969) Digestive system and sensorial factors in relation to the feeding behaviour of flatfish (Pleuronectiformes). J. Cons. Int. Explor. Mer. 32, 385 395. Dye J. E. (1980) The production and efficient use of freshl~ hatched brine shrimp nauplii (Artemia) in the larval rearing of marine fish at the hatcheries of the British White Fish Authority. In The Brine Shrimp Artemia. Ecology, Culturing, Use in Aquaculture (Edited by Persoone G., Soreloos P., Roels O. and Jaspers E.), Vol. 3. pp. 271-276. Universa Press, Wetterem, Belgium. Fange R. and Grove D. (1979) Digestion. In Fish Physiology (Edited by Hoar W. S., Randall D. J. and Brett J. R.), Vol. VIII, pp. 161-261. Academic Press, New York. Gatesoupe F. J., Girin M. and Luquet P. (1977) Recherche d'une alimentation artificielle adapt@ a l'elevage des stades larvaires des poissons. II. Application a l'616vage larvaire du bar et de la sole. 3rd Meeting (?f the LC.E.S. Working Group on Mariculture (Brest). Actes de Colloques du C.N.E.X.O. 4, 59-66. Girin M. (1979) Feeding problems and the technology of rearing marine fish larvae. In Finfish Nutrition and FishJbed technology (Edited by Halver J. E. and Tiews K.), Vol. 1, pp. 36~366. Heeneman, Berlin. Hastings W. H. and Dickie L. M. (1972) Feed formulation and evaluation. In Fish Nutrition (Edited by Halver J. E.), pp. 327-374. Academic Press, New York. Inaba D., Ogino C., Takamatsu T., Ueda T. and Kurokawa K. (1963) Digestibility of dietary components in fishes. II. Digestibility of dietary protein and starch in rainbow trout. Bull. Jap. Soc. scient. Fish 29, 242 244. Kawai S. and Ikeda S. (1971) Studies on digestive enzymes
Metabolism in marine flatfish--I of fishes. I. Carbohydrases in digestive organs of several fishes. Bull. Jap. Soc. scient. Fish. 37, 333-337. Kjolberg O., Manners D. J. and Wright A. (1963) The molecular structure of some glycogens. Comp. Biochem. Physiol. 8, 353-365. Kuzmina V. V. (1977) Peculiarities of the membrane digestion in freshwater teleosts. Vop. Ikhyol. 17, 111-119. Kuzmina V. V. (1980) Seasonal and age-related changes in a-amylase activity in the bream, Abramis brama. J. h'hthyol. 20, 105-110. Mackie A. M., Adron J. W. and Grant P. T. (1979) Chemical nature of feeding stimulants for juvenile Dover sole (Solea solea L.). J. Fish. Biol. 16, 701-708. Meyers S. P. (1979) Formulation of water-stable diets for larval fishes. In Finfish Nutrition and Fishfeed Technology, Vol. II (Edited by Halver J. E. and Tiews K.), pp. 13-20. Heeneman, Berlin. Miller G. L. (1959) Protein determination for large numbers of samples. Analyt. Chem. 31, 964. Nagase G. (1964) Contribution to the physiology of digestion in Tilapia mossambica Peters: digestive enzymes and the effect of diet on their activity. Z. vergl. Physiol. 49, 270-284. Olatunde A. A. and Ogunbiji O. A. (1977) Digestive enzymes in the alimentary tracts of three tropical catfish. Hydrobiologia 56, 21-24. Pfeffer E. (1982) Utilization of dietary protein by salmonid fish. Comp. Biochem. Physiol. 73B, 51-57. Prosser C. L. and Brown F. A. (1961) Feeding and digestion. In Comparative Animal Physiology (2nd edition) p. 120. W. B. Saunders Company (Philadelphia and London).
827
Robyt J. F. and Whelan W. J. (1968) The fl-amylases. In Starch and its Derivatives (Edited by Radley J. A.), pp. 477-497. Chapman & Hall, London. Sarbahi D. S. (1951) Digestive enzymes in goldfish. Biol. Bull. 100, 244--257. Spannhof L. and Plantikow H, (1983) Studies on carbohydrate digestion in rainbow trout. Aquaculture 30, 95-108. Stark J. R. and Walker R. S. (1983) Carbohydrate digestion in Pecten maximus. Comp. Biochem. Physiol. 76B, 173-177. Trevelyan W. E., Procter D. P. and Harrison J. S. (1950) Detection of sugars on paper chromatograms. Nature, Lond. 166, 444-445. Ugolev A. M. (1960) Influence of the surface of the small intestine on enzymatic hydrolysis of starch by enzymes. Nature, Lond. 188, 588-589. Ushiyama H., Fujimori T., Shibata T. and Yoshimura K. (1965) Studies on the carbohydrases in the pyloric caeca of the salmon. Bull. Fac. Fish. Hokkaido Univ. 16, 183-188. Walton M. J. and Cowey C. B. (1982) Aspects of intermediary metabolism in salmonid fish. Comp. Biochem. Physiol. 73B, 59-79. Yasunaga Y. (1972) Studies on the digestive functions of fishes III. Activity of the digestive enzymes: protease and amylase of some flatfish species. Bull. Tokai Reg. Fish Res. Lab. 71, 169-175. Yoshida Y. and Sera H. (1970) On chitinolytic activities in the digestive tract of seven species of fishes and the mastication and digestion of foods by them. Bull. Nauka. Reg. Fish. Res. Lab. 36, 751-754.
0305-0491/84 $3.00+0.00 ~.~ 1984PergamonPress Ltd
METABOLISM IN MARINE FLATFISH--I. C A R B O H Y D R A T E DIGESTION IN DOVER SOLE (SOLEA SOLEA L.) J. CLARK, J. E. MCNAUGHTONand J. R. STARK Department of Brewing and Biological Sciences, Heriot-Watt University, Edinburgh, Scotland. (Tel.: 031-225-8432) (Received 7 September 1983) A b s t r a e t - - l . A survey of the carbohydrases in the digestive tract of juvenile and adult Dover sole indicated the presence of strong amylase and maltase activities. 2. The highest levels of these enzymes were present in the foregut region. 3. Chitinase and chitobiase activities were also detected and shown to be optimal at pH 9.5 and 5.0 respectively. 4. Other carbohydrases were either absent from gut homogenates or were only present at very low levels. 5. The relationship between these results and the nutritional requirements of Dover sole is discussed and comparisons made with studies on other fish.
INTRODUCTION
The farming of marine fish is presently based on the use of live food organisms during the first weeks after hatching and, while the most promising development would be to use artificial diets from first feeding, considerable problems exist. These difficulties are reviewed by Girin (1979) and are principally concerned with survival, although growth rates are also of importance. A marine flatfish, the plaice, (Pleuronectes platessa L.) has been reared from eggs to metamorphosis on artificial diets (Adron et al., 1974) in studies which indicated a survival rate of about 20% compared with about 38% in control larvae fed Artemia nauplii under similar conditions. It has also been demonstrated by Gatesoupe et al. (1977) that sole larvae can be reared on artificial diets straight from first feeding but again many problems must be solved before the exclusive use of artificial diets can become commercially viable. A second, although not so important, economic consideration is the requirement that fish have for relatively expensive, high protein diet. As is indicated in recent reviews (Pfeffer, 1982; Walton and Cowey, 1982) it is well recognised that fish obtain most of their energy from protein and fat and have a limited ability to utilise carbohydrate, therefore any developments which allowed the use of diets with higher carbohydrate content would obviously be of economic significance. It is against this background that studies on the digestive enzymes in marine flatfish were undertaken to obtain a fuller understanding of factors which may be of importance in the formulation of diets. In the present paper the carbohydrases in the digestive tract of Dover sole (Solea solea L.) have been examined and the results compared with studies on other fish species. Farmed Dover sole have a good commercial potential since the fish is rarely caught in the wild and, with its high quality food protein, it is a readily marketable product. However, the above mentioned problems concerning diet and nutrition are somewhat
accentuated by the fact that sole is an extremely fastidious feeder. This gives rise to difficulties in weaning from a live (Artemia) diet to an artificial diet and this type of problem in Dover sole and in other species of fish has prompted research into feeding stimulants (De Groot, 1969; Mackie et al. 1979). For these reasons, the ongrowing of sole to marketable size has only recently become of commercial importance. Although a considerable amount of work has been carried out on fish nutrition in general (Hastings and Dickie, 1972) and many studies have been made on both live and artificial diets (see for example Dye, 1980 and Myers, 1979 respectively) there is less information on the biochemical aspects of digestive processes in fish. However information is available on rainbow trout (see for example Bergot, 1979, Spannhof and Plantikow, 1983 and Inaba et al., 1963) and on some flatfish species in Japanese waters (Yasunaga, 1972). The biochemistry of digestion in Dover sole has so far been largely neglected and the following study therefore sets out, as its initial objective, to examine the enzymes present in the digestive tract of juvenile and adult sole and to correlate these findings to the nutritional requirements of this fish. MATERIALS AND METHODS
Juvenile and adult samples were supplied by Seafresh Farms, Hunterston, Scotland. 0-group juveniles weighed between 15-34g and were 50-150ram long whereas adult fish were 2-group (average weight 400 g and length 300 ram). All fish had been continuously fed on artificial diets for at least 3 weeks (so there were no contaminating enzymes from live diets in any of the preparations). Preparation of extracts The majority of these studies were carried out on juvenik fish using homogenates of the whole digestive tract. FoJ examination of different parts of the intestine the alimentary canal of adult fish was divided into stomach foregut, midgut, hindgut and rectum. Due to the lack ol
821
822
J. CLARK et al.
regional differentiation such divisions were arbitrary, being made on the basis of percentage of total gut length as suggested by Braber and De Groot (1973). All fish were starved for 3 hr before being killed by a blow to the head followed by deep-freezing. After partial thawing the intestine could be readily excised, weighed and homogenized with distilled water to give a 1:10 homogenate. Most analyses were carried out by using a further dilution of this sample to give a 1:40 homogenate. The lumen contents, present in the gut, were included in all homogenates unless otherwise stated. When required, pure gut homogenate was prepared by rinsing out an open digestive tract with distilled water and then homogenizing the cleaned intestine.
Chemicals Unless otherwise stated all chemicals were supplied by Sigma Chemical Co.
Analytical procedures The protein content of homogenates was determined by the method of Miller (1959) using bovine serum albumin (25-100 #g) as standard. Reducing sugars were estimated by the Somogyi-Nelson procedure as described by Robyt and Whelan (1968). The buffers used were either citratephosphate, glycine-HC1 or glycine-NaOH. For survey purposes substrate (10mg) was incubated with homogenate (1 ml) at 37°C for 15hr. Toluene was added to prevent bacterial growth. Samples were spotted on to chromatograms developed in ethyl acetate:pyridine:water (10:4:3 by vol.) and the products revealed using the silver nitrate procedure of Trevelyan et al. (1950). Appropriate standards were included. Amylase activity was measured in the presence and absence of calcium ions in digests containing 1~o soluble starch (5 ml), 0.1 M citrate phosphate buffer pH 6.5 (1 ml), 0.1 M calcium chloride (1 ml) or 0.1 M EDTA (1 ml) and a 1:40 intestine homogenate (3 ml). Digests were incubated for times ranging from 2 hr to 24 hr before taking samples (1 ml) or smaller volumes as appropriate for estimation of reducing sugars. Similar digests were used for examination of the pH-activity profile except that water (1 ml) was used in place of calcium chloride or EDTA solutions. The units of amylase activity were expressed as #moles of maltose equivalents released per rain per gram protein. The action pattern of amylase attack on starch was examined by paper chromatography and also by estimating the release of reducing sugars and the reduction in iodine staining power at the same time intervals. This was carried out by incubating 1~ starch solution (4 ml) with a 1:10 homogenate (4ml). Samples (0.1 ml) were taken at intervals and the volume made up to 1 ml with water before estimating reducing sugars. At the same time-intervals, samples (0.1 ml) were mixed with water (10ml) and 0.1 ml of a solution containing iodine (0.2~o) and potassium iodide (2~o). The iodine staining colour was read at 600 nm. The iodine staining power was plotted against the production of reducing sugars expressed as percentage conversion of the starch substrate. Glucosidases were examined using p-nitrophenyl glycosides (3 mg) in buffer (1 ml) with homogenate (1 ml) for times ranging from 1-24 hr. The digests were then heated at 100°C for 2 min cooled in ice and centrifuged to remove precipitated material. The supernatant solution was treated with 1 M potassium bicarbonate solution (1 ml) and the absorbance read at 420nm against an appropriate blank containing heat-inactivated homogenate. The hydrolysis of disaccharides (maltose, sucrose, lactose and cellobiose) was examined chromatographically as well as by means of glucose oxidase procedure. For this purpose a 1~o aqueous solution of the disaccharide (0.5 ml) was incubated with a 1 : 10 gut homogenate (0.5 ml) at 37°C for times ranging from 20 min-5 hr. The digests were heated for 2min at 100°C, centrifuged and the supernatant solution
analysed by the glucose oxidase procedure as described in the Boehringer Mannheim Peridochrom kit No. 676543. The hydrolysis of chitin was examined using chitin azure (10mg) suspended in buffer (1 ml) and 1:10 homogenate (0.5 ml). The mixture was placed in a sealed centrifuge tube on a Spiramix rotating shaker to keep the solid substrate in suspension. After a suitable time interval the digest was centrifuged and the absorbance of the supernatant solution read at 545 nm against appropriate blanks for each pHvalue. Chitobiase activity was investigated using pnitrophenyl fl-glucosaminide as described above under "glucosidases" and also by paper chromatography using N,N'-diacetylchitobiose as substrate. Chromatofocusing was used to examine the ~-glucosidase and chitobiose activities. A 6ml sample of 1:10 gut homogenate was applied to the column (1 x 25cm) containing 15 ml of a slurry of Polybuffer Exchanger 94 (Pharmacia) which had been equilibrated with 25 mM imidazole-HC1 buffer pH 7.4. The column was then eluted with Polybuffer 74-HC1 (pH 5.0) at a rate of about 20 ml/hr. Fractions (6 ml) were collected and analysed for enzyme activities using pnitrophenyl e-glucoside or p-nitrophenyl /~-glucosaminide as previously described. RESULTS
Survey of activities present The hydrolytic activity against different substrates are s h o w n in Table 1. These results are compiled from reducing sugar m e a s u r e m e n t s a n d from analysis by paper c h r o m a t o g r a p h y . The substrates which were readily hydrolysed all c o n t a i n e d ~-glucosidic linkages ( p - n i t r o p h e n y l ~-glucoside, maltose, maltotriose, starch a n d glycogen); the only exceptions to this being p - n i t r o p h e n y l fl-glucosaminide which was easily hydrolysed a n d chitin and chitin azure which were also attacked a l t h o u g h at a slower rate. All other substrates were either not hydrolysed or were only hydrolysed to a very limited extent. This general p a t t e r n of activity was the same for b o t h adult and juvenile D o v e r sole homogenates.
Amylase activity H o m o g e n a t e s of b o t h juvenile a n d adult Dover sole intestine acted on starch with the p r o d u c t i o n of reducing sugars. The o p t i m u m p H for this activity was at p H 6.7 (Fig. 1). A n e x a m i n a t i o n o f the products of starch hydrolysis by p a p e r chrom a t o g r a p h y are s h o w n in Table 2. In the initial phases of action, a series of sugars were formed with R glucose-values of 1.00, 0.63, 0.41, 0.24 and 0.13 c o r r e s p o n d i n g to authentic samples of glucose and the maltosaccharide series of sugars. As the action progressed, less of the oligosaccharides r e m a i n e d a n d more glucose was formed. The m o d e of action of amylase was further studied by g r a p h i n g the production o f reducing sugars the reduction in iodine staining power o f the starch substrate. The result o f this experiment is s h o w n in Fig. 2. The rate of release of reducing sugars by the intestinal amylase from D o v e r sole is illustrated in Fig. 3 which indicates the effect of calcium chloride and also shows t h a t the reaction rates becomes slower as the hydrolysis of the starch substrate proceeds. Fig. 4(a) show the rate of release o f reducing sugars from h o m o g e n a t e s of different parts of the intestinal tract of an adult D o v e r sole. The m a x i m u m activity was found in the foregut region. The effect of centrifugation on the
Metabolism in marine flatfish--I
823
Table 1. Carbohydrases in the alimentary canal of Dover sole Substrate Hydrolysis Methyl ~t-glucopyranoside Methyl fl-glucopyranoside p.Nitrophenyl-N-acetyl-fl-glucosaminide + + p-Nitrophenyl ~-glucopyranoside + + + p-Nitrophenyl fl-glucopyranoside + Maltose + + + Cellobiose + Sucrose Lactose + N,N'-diacetyl chitobiose + Isomaltose + Melibiose Raft]nose Maltotriose + + + Starch + + + Glycogen + + + Pullulan Laminarin Inulin Xylan Agar Alginic acid Sodium carboxymethyl cellulose Cellulose Pectin Chitin azure + Chitin + - = no hydrolysis, + = a trace of hydrolysis. +, + + and + + + indicate increasing extents of hydrolysis.
Table 2 Products of hydrolysis of starch by an intestinal homogenate from Dover sole Incubation time Product 15 min 1 hr 2 hr 24 hr
activity o f the amylase indicated that approximately half o f the activity was lost when the solid material was r e m o v e d f r o m intestinal homogenates. A t t e m p t s to purify the a-amylase by c h r o m a t o f o c u s i n g were not successful due to the lack of stability of the enzyme.
Chitobiase and chitinase
Glucosidases Using the a p p r o p r i a t e p - n i t r o p h e n y l derivatives, a a n d fl-glucosidase activities were examined. Both activities were optimal at a b o u t p H 5.7 but the a-glucosidase was m o r e t h a n twice as active as the fl-glucosidase. F u r t h e r m o r e the c h r o m a t o g r a p h i c studies indicated t h a t there was only very slight hydrolysis o f cellobiose (4 0-fl-D-glucosylglucopyranoside). The latter fact was verified by estimating the glucose released from cellobiose using the glucose oxidase method. As is shown in Fig. 5 a n initial
Glucose
+ +
Maltose + + Maltotriose + + Maltotetraose + + Maltopentaose + indicates absence of product. +, + +, + + + and + + + product.
+ + +
+ + +
+ + + +
+ + + + + + +
+ + + + -
-
-
+ indicate increasing amounts of
release of glucose d u r i n g the first h o u r was followed by only very limited glucose p r o d u c t i o n during a subsequent four h o u r period. This c o m p a r e s with a steady p r o d u c t i o n o f glucose from maltose. In view o f this the fl-glucosidase a n d cellobiase activities were not studied further. The a-glucosidase activity was examined by c h r o m a t o f o c u s i n g a n d as shown in Fig. 6 which indicates t h a t there are multiple forms of this enzyme. The relative a m o u n t s of a-glucosidase in the intestine was measured using p - n i t r o p h e n y l a-glucoside and, as is s h o w n in Fig. 4(b), the maxi m u m activity was present in the foregut region. The relative a m o u n t s of enzyme in each region were in parallel with the a m o u n t s o f a-amylase in these regions.
As indicated in Table 1 b o t h chitin a n d chitin azure were hydrolysed but only if the p H was on the alkaline side of neutrality. The hydrolysis o f p - n i t r o p h e n y l N-acetylglucosaminide a n d N , N ' diacetylchitobiose occurred only in more acid solutions. These observations were studied q u a n titatively as indicated in Fig. 7. The o p t i m u m values were at p H 9.5 for chitinase a n d p H 5.0 for chitobiase. The chitobiase activity was m u c h easier to study t h a n the chitinase because of the sensitivity o f the p - n i t r o p h e n o l assay. Using c h r o m a t o f o c u s i n g (as previously described) to chitobiase activity could also be separated into isoenzymes with isoelectric points 1'0
o'BL
(b)
100
Iodine Stain
"" \ \
(A6oo) I""..~ (a,
BO
Retative Activity
0.6t-~ , ~.. .
~
60
40
20
0 pH
Fig. 1. Activity-pH curves for hydrolysis of starch (0--0) and p-nitrophenyl •-glucoside ( 0 O) by intestinal homogenates. C.B.P.77/4B--L
"%""""""i ..................i'" 20 40 60
BO
°Hydrolysis of starch Fig. 2. Hydrolysis of starch (release of reducing sugars) and reduction in iodine stain (a) Dover sole intestinal extract, (b) theoretical curve for an exo-enzyme (. . . . . ) and (c) for an endo-enzyme ( ......... ).
824
J. CLARK et al. [
7ooI
,°1
/-"
t
600 0
250 1
J
//-I /
500
/'~
/ 300
200 0
.E cu "5 L Q_
,,~
0 200
150. 0
1
2
/+
3
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hme (h)
~
Fig. 5. Hydrolysis of disaccharides by juvenile Dover sole intestinal homogenate measured by the glucose oxidase method using maltose ( ' - - - ' L cetlobiose ( A - -A), sucrose (&--- &) and lactose ( 0 -©),
100-
'G
~
so2"~
2'o
~o
go
8b
lOO
Time (mm) Fig. 3. Release of reducing sugars from starch by Dover sole intestinal homogenates in the presence of calcium chloride ( 0 - - 0 ) and with EDTA ( I l l - - U ) .
(a)
I~electric pts (pH)
Lla/
20-
~'~
](b) 5-6 Ifrl 55 I"'
(b)
_
_
_
_
1-6 A~20 12
at pH 6.6, 5.7 and 5.6. Furthermore it was shown that on centrifugation of the gut homogenates almost all the activity was in the supernatant solution and only traces of enzyme remained attached to the mucosa.
0-80.1NiNaCI
04
O t h e r activities
Figure 5 shows the very limited extent of hydrolysis of lactose and sucrose as compared to maltose. As in the case of cellobiose, a short quick release of a small amount of glucose is followed by a barely measurable production of glucose.
a°y.
~o
~-
io
go
lOO 12o
Fraction No. Fig. 6. Chromatofocusing of Dover sole intestinal homogenate showing multiple forms of ~.-glucosidase. Fractions were analysed using p-nitrophenyl :~-glucoside as substrate and the release of p-nitrophenol measured at 420 nm.
,0lb
200.
oi.
'I//
1oo// o
i;
~o
Time (min)
3'o
o
ib
~o
~'o
Time (rain3
Fig. 4. Rate of hydrolysis of (a) starch and (b) phenyl c~-glucoside by homogenates of"Dover sole stomach (O ©) foregut ( 0 - - 0 ) midgut (A A) and hindgut ( A - - A ) .
Metabolism in marine flatfish--I
The extremely minute traces of lactase and sucrase activities detected in gut homogenates from Dover sole is in contrast to the situation in most mammals where it has been shown for example that the digestive tracts of rat, rabbit, sheep and humans all readily hydrolyse maltose, sucrose and lactose (Prosser and Brown, 1961). In a similar study on the digestive enzymes of the scallop Pecten maximus a much wider range of carbohydrate substrates were hydrolysed (Stark and Walker, 1983).
100" 80' Relafive
60
Acfivify
40
20
Amylase and ~-glucosidase activities 2
4
6
8
10
12
pH Fig. 7. Activity--pH curves for hydrolysis of chitin azure
(O
O) (0
825
and p-nitrophenyl N-acetylglucosaminide 0 ) by homogenates of Dover sole intestine.
DISCUSSION Previous studies on the digestive enzymes in fish include reports on both freshwater and marine species. Olatunde and Ogunbiji (1977) working on freshwater species from Lake Kainja, Nigeria have demonstrated the presence of amylase activity in the tropical catfish Entropicus niloticus and Schilbe mystus but were unable to detect any cellulase, lactase, sucrase or maltase activities in the digestive tract of these species. However, in earlier studies, Sarbahi (1951) reported sucrase and maltase activities in goldfish. Amylase activity has been demonstrated in the bream Abramis brama from a freshwater reservoir in Russia and the activity of this enzyme varied inversely with the age of the fish (Kuzmina, 1980). Thus, although amylase activity has been found in a variety of different species of fish, variations in the levels of glycosidases seem to occur. Kawai and Ikeda (1971) examined the enzymes present in carp (Cyprinus carpio ), ayu (Plecoglossus altivelis) and red sea bream (Pagrus major) and compared these with the carbohydrases present in the digestive system of rainbow trout. These fish all possessed fairly strong amylase and c~-glucosidase (maltase) activities in their digestive systems but fl-glucosidase, ~-galactosidase, fl-fructosidase and cellulase were absent. This was similar to the enzyme profile found in the pyloric caecum of the salmon Oncorhynchus keta reported by Ushiyama et al. (1965).
Survey of enzymic activities The survey of enzymic activities in the present study (Table 1) indicated that homogenates of Dover sole intestine contained relatively strong amylase and maltase activities. This was shown by the release of reducing sugars from starch and glycogen and the production of glucose from maltose and maltotriose. Many other carbohydrases were either totally absent or were present only in trace amounts. The exceptions to this were chitinase and chitobiase activities; the presence of these enzymes was indicated by the hydrolysis of chitin and chitin azure in the case of chitinase and N,N'-diacetylchitobiose and p nitrophenyl-n-acetyl-fl-glucosaminide in the case of chitobiase.
The characterisation of the amylase activity in homogenates of Dover sole intestine was based on two experiments. The reducing sugars produced in the initial phases of hydrolysis of starch (Table 2) formed a homologous series indicating that the process was one of random hydrolysis. As a reaction proceeded glucose became the predominant sugar indicating that amylase action was being followed by hydrolysis of lower maltosaccharides by an ct-glucosidase (or maltase). These results were substantiated by the second experiment, shown in Fig. 2 in which the iodine staining power of the starch substrate is measured at different stages during the hydrolysis. Endo- or random attack on starch causes a rapid reduction in the molecular weight of the polymer and in its iodine staining properties (since long runs of ct-l,4-1inked glucose residues are required for iodine binding). Exo-hydrolysis, on the other hand, results in a rapid production of reducing sugars as each cleavage produces a free sugar but, because the main part of the molecule remains intact, there is a very slow reduction in iodine stain. The theoretical curves for exo- and endo-acting amylases are shown in Fig. 2b and 2c respectively. The curve for Dover sole amylase (a) can be explained by assuming an initial random attack followed by hydrolysis of the oligosaccharides formed to produce reducing sugars at a faster rate than would be expected from an endo-amylase acting on its own. The curve (a) is therefore displaced to the right of (c). The pH profiles (Fig. 1) indicated that the amylase had optimal activity at pH 6.7 whereas a-glucosidase was optimal at pH 5.7; this would suggest that these two activities originated from different enzyme proteins. Amylases from the digestive tracts of fish would appear to differ depending on species. For example the amylase from the salmon, Oncorhynchus keta had a pH optimum of 8.5 (Ushiyama et al., 1965) whereas in studies on amylases from three species of flatfish the pH optimum in each case was 7.5 (Yasunaga, 1972). Amylase from Tilapia mossambica, on the other hand, was optimal at pH 6.7 (Nagase, 1964). The amylase from Dover sole was more than twice as active in the presence of calcium chloride than in its . absence (Fig. 3) (EDTA was added to chelate any endogenous calcium ions). Attempts to purify this enzyme by chromatofocusing resulted in total loss of activity. However, the a-glucosidase activity was readily resolved into isoenzymes by this procedure (Fig. 7). The morphology of the digestive system of Dover sole shows a simple structure which penetrates deeply into the body cavity. The lack of distinguishable oesophagus and stomach regions in the digestive tract
826
J. CLARK et al.
of sole has been described by De G r o o t (1969). It was thus impossible, in the present study, to clearly define the different sections of the gut and an arbitrary division had to be made. The analysis of enzymes from different parts of the gut (Fig. 4a, b) indicated a similar pattern in distribution between c~-amylase and c~-glucosidase activities. In each case, the amount of amylase and ~-glucosidase activities followed the order: foregut > hind-gut > mid-gut > stomach. In similar studies, Kawai and Ikeda (1971) demonstrated marked amylase activity in the distal portion of the carp intestine when this was divided into three equal portions by length. High levels of amylase were observed in the mid-gut of the flatfish Limanda yokohamae (Yasunaga, 1972). The studies of Ugole (1960), K u z ' m i n a (1977) and Spannhof and Plantikow (1983) have indicated that the intestinal mucosa is of significance in amylolytic digestion of starch. It was therefore of interest to examine the effect of centrifugation of homogenates on the amylase activity. In the present study with Dover sole, the level of c~-amylase was halved when a centrifuged homogenate was used in place of a whole homogenate containing particulate material. It is therefore evident that in Dover sole the intestinal membrane either binds some of the ~-amylase, as is believed to occur in land animals (Dalqvist, 1978), or contributes in some other way to amylase action. Other activities
As can be seen from Table 1, several substrates were not attacked by Dover sole intestinal homogenares. These included the polysaccharides laminarin, cellulose, xylan and pullulan. In addition, the disaccharides sucrose, lactose and cellobiose were only hydrolysed very slightly as judged by paper chromatography. A study of the rate of hydrolysis of these disaccharides compared with that of maltose was carried out using a glucose oxidase method (Fig. 5). Whole homogenates were used in these experiments and it is evident that whilst maltose undergoes rapid and steady hydrolysis, the other disaccharides are hydrolysed only to a very limited extent even after prolonged incubation. Hydrolysis o f chitin
Chitinase occurs in the digestive system of certain fishes and other vertebrates (Fange and Grove, 1979) and has been examined in a number of different species of fishes (Yoshida and Sara, 1970). In the present study chitinase and chitobiase activities were detected in the digestive system of Dover sole. Chitin itself is a hard and insoluble substrate which is difficult to use in enzyme assays and chitobiose is expensive. Hence, other than for the initial survey, the artificial substrates chitin azure and p-nitrophenyl N-acetyl-/3-glucosaminide were used. Chitinase was optimal at 9.5 whilst the chitobiase showed maximum activity at pH 5.0. (Fig. 7). Further studies will be necessary to ascertain whether these enzymes are produced by intestinal bacteria or by the digestive system of the Dover sole itself. However the present studies indicated that chitobiase existed as isoenzymes which appeared in the non-particulate fraction of homogenates. In conclusion the present studies indicate that
homogenates of Dover sole intestine have the ability to hydrolyse starch-type polysaccharides and chitin to their respective monosaccharide units. The amylase/c~-glucosidase system will convert starch, (which may be present as such in artificial diets) to glucose; but in the natural environment glycogen, which is a constituent of crustaceans (Kjolberg et al,, 1963) forms part of the diet. Whilst the chitinase-chitobiase system can, at least qualitatively, hydrolyse the shells of crustaceans to N-acetylglucosamine it is not known to what extent this occurs on a quantitative basis. Further work is in progress to study the factors that are of significance in the synthesis of these enzymes and in particular to examine the role of digestive enzymes during the earlier periods of the life cycle of the Dover sole. Acknowledgements--This work was supported by an SERC Marine Technology Grant and forms part of the research programme of the Aquaculture Enginering Group, HeriotWatt University. The authors also wish to thank Mr S. T. Hull and his staff (Seafresh Farms Ltd) for their help.
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