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The characterization of three digestive enzymes from the crayfish Procamabarus clarkii.

ABSTRACT

Crayfish consume a wide variety of organisms and use a diverse array of digestive enzymes to breakdown foods. Very little is known about the enzyme reactions involved in crayfish digestion yet these reactions are crucial to absorption and assimilation. The activities of the digestive enzymes [alpha]-amylase, trypsin, and a nonspecific esterase in a crude homogenate of hepatopancreas were described in relation to changes in substrate concentration, pH, and temperature. The hepatopancreas was removed from three crayfish, homogenized in an enzyme buffer, and crudely purified using centrifugation. The activity of [alpha]-amylase increased with increasing substrate concentration and reached maximum maltose production at 15 mg/ml starch solution. The maximum velocity and apparent Michaelis constant for this reaction was approximately 328.3 units/g wet weight and 2.4 mg/ml starch, respectively. The pH optimum for [alpha]-amylase was 6.5. The activity of [alpha]-amylase increased with increasing temperature from 8 to 44 C with incremental (6 C) [Q.sub.10] values ranging from 1.6-3.5. Trypsin activity increased with increasing substrate concentration to reach a maximum at 1.6 mM [alpha]-tosyl arginine methyl ester (TAME). The maximum velocity and apparent Michaelis constant for this reaction was approximately 342.2 units/g wet weight and 0.308 mM TAME, respectively. The pH optimum for trypsin was 8.0. The activity of trypsin increased with increasing temperature from 8 to 44 C with incremental (6 C) [Q.sub.10] values ranging from 1.5-1.9. Nonspecific esterase activity increased with increasing substrate concentration to reach a maximum activity at 1 mM 4-nitrophenol caproate (4-NPC) solution. The maximum velocity and apparent Michaelis constant for this reaction was approximately 0.804 units/g wet weight and 0.053 mM 4-NPC, respectively. The pH optimum for the nonspecific esterase reaction was 8.5. Nonspecific esterase activity increased with increasing temperatures from 8 to 44 C with incremental (6 C) [Q.sub.10] values ranging from 1.2-3.1. Procambarus clarkii has the capacity to digest carbohydrates, proteins, and lipids, typical of an omnivore. Changes in physiological or environmental conditions such as gut pH and temperature may affect the rate at which these substrates are digested in the hepatopancreas.

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INTRODUCTION

Crayfish are the dominant macrocrustacean in many freshwater ecosystems and are capable of feeding on a variety of plants, animals, and microorganisms, including bacteria (Brown, 1995). Native species of crayfish exist on every continent with the exception of Africa and Antarctica (Hobbs, 1988). In the United States, the crayfish Procambarus clarkii is the most extensively cultured crustacean (Brown, 1995).

The process of digestion in crustaceans has been described by several authors (Brown, 1995; Dall and Moriarty, 1983; Gibson and Barker, 1979; Icely and Nott, 1992; Jones et al., 1997; van Weel, 1970; Vonk, 1960). The process of chemical digestion by digestive enzymes occurs primarily in the hepatopancreas, the site for food absorption, digestive enzyme synthesis, and secretion (reviewed in van Weel, 1970; Dall and Moriarty, 1983; DeVillez and Fyler, 1985; Gibson and Barker, 1979; Icely and Nott, 1992). The analysis of [alpha]-amylase, trypsin, and nonspecific esterase in the hepatopancreas, has been shown previously to be an effective approach to understanding crustacean digestive processes (DeVillez, 1965; Brockerhoff et al., 1970; Loizzi and Peterson, 1971; DeVillez and Fyler, 1985; Biesiot, 1986; Lovett and Felder, 1990; Fang and Lee, 1992; Kamarudin et al., 1994; Jones et al., 1997). In addition, many of these previous studies have used crude digestive enzyme preparations to provide a fast and easy method to observe changes in the digestive physiology (Biesiot, 1986; Lovett and Felder, 1990; Kamarudin et al., 1994; Jones et al., 1997).

Understanding the digestive physiology of Procambarus clarkii may provide insight into nutrition, dietary preferences, and strategies of resource utilization that could lead to improved management strategies for aquaculture practices. Ecologically, this information may also aid in defining the niche that Procambarus clarkii occupies in aquatic systems. The objective of this study was to characterize crude preparations of [alpha]-amylase, trypsin and nonspecific esterase from Procambarus clarkii.

MATERIALS AND METHODS

Adult Procambarus clarkii were captured in ponds at the Louisiana State University Aquaculture Center and transported to the University of Alabama at Birmingham (UAB). They were maintained for approximately 3 months in aerated, recirculating raceways (0.6 X 2.5 m) at 27 [+ or -] 3 C with a 12 hr light-dark cycle and fed three to four times weekly (KS diet, UAB Research Foundation). Individuals were anesthetized for several minutes in ice water, blotted dry, weighed, and then dissected. The hepatopancreas was removed from each animal, weighed, and immediately placed into ice-cold general enzyme buffer (20 mM Tris-HCl with 1 mM EDTA and 10 mM calcium chloride at pH 7.4). The hepatopancreas was then homogenized using a hand-held Tissue Tearor (Biospec Products Inc.) and centrifuged (Beckman model TJ-6) at 1,500 X g for 30 min at 4 C. The supernatant was decanted into a 50 ml polypropylene tube, frozen in liquid nitrogen, and stored at -80 C until analysis. During subsequent characterizations, thawed homogenates were kept on ice for the duration of analyses.

The activity of [alpha]-amylase was measured using a kinetic endpoint assay modified from (Bernfeld, 1955). The starch substrate buffer consisted of 0.1 M sodium phosphate dibasic, 6 mM sodium chloride, and 15 mg/ml (wt/vol) starch adjusted to pH 6.9. The absorbance was determined spectrophotometrically using a Shimadzu UV-100 spectrophotometer at 540 nm and 25 C. The optical density of the sample was computed as the average difference of the sample minus the control consisting of substrate and homogenizing buffers. The micromoles of maltose, generated by the enzymatic reaction, were derived from a standard curve generated with known amounts of maltose. The results were recorded as units per g wet weight of hepatopancreas; a unit of [alpha]-amylase activity liberates 1 [micro]mmole of maltose per minute at pH 6.9. The result was converted to units per g wet weight of hepatopancreas. All of the samples for characterization were assayed in duplicate or triplicate with single controls.

To define substrate-saturating conditions, the effects of substrate concentration were assayed. Starch solutions of 0, 1.25, 2.5, 5, 10, and 15 mg/ml were made as serial dilutions (with distilled water) from a 20 mg/ml starch (in distilled water) solution and buffered similarly (6 mM sodium chloride and 0.1 M sodium phosphate dibasic). The source of [alpha]-amylase was a 10 mg/ml (wt/vol) homogenate of hepatopancreas. To determine the optimal pH for [alpha]-amylase, assays were performed from pH 4 to 8 (in increments of 0.5 pH units). A citrate phosphate buffer was used for the pH range of 4 to 6 (0.1 M citrate, 0.1 M sodium phosphate dibasic, 0.05 M sodium chloride, and 15 mg/ml starch) and the standard buffer (0.1 M phosphate dibasic, 0.05 M sodium chloride, and 15 mg/ml starch) was used for the pH range of 6 to 8. The source of [alpha]-amylase for the pH determination was a 2.5 mg/ml (wt/vol distilled water) homogenate of hepatopancreas. The effect of assay temperature on the enzymatic activity of [alpha]-amylase was tested from 8 to 44 C (in increments of 6 C). A temperature-controlled water-circulating system (Neslab Endocal RTE-210) was used to equilibrate the test tubes to temperature prior to and during the reactions. The source of [alpha]-amylase was a 2.5 mg/ml (wt/vol) homogenate of hepatopancreas. The [Q.sub.10] was calculated by using a modified formula to extrapolate the [Q.sub.10] equivalent values (Prosser and DeVillez, 1991).

Trypsin was assayed using a technique described initially by (Hummel, 1959) and later modified by (Bergmeyer et al., 1974); the protocol was provided by Sigma Chemical Company (St. Louis, MO). This spectrophotometric assay uses the substrate [alpha]-tosyl-arginine-methyl-ester (TAME) dissolved in trypsin buffer (100 mM potassium phosphate dibasic and 100 mM potassium phosphate monobasic combined to a pH of 8.0) to a final concentration of 0.8 mM and then placed on ice for the duration of the assay. The absorbance was measured using a temperature-controlled Shimadzu UV-100 spectrophotometer at 247 nm and 25 C. Measurements were recorded when the change in optical density was linear (substrate concentration was saturating). Trypsin activity was measured as the average change in optical density per minute at 247 nm and then converted to units per g wet weight of hepatopancreas (1 unit = 1 micromole of TAME hydrolyzed per minute). The change in absorbance accompanying the hydrolysis of 1 [micro]mmole of TAME per milliliter of assay solution is 0.409 [cm.sup.-1] [mM.sup.-1] (Walsh, 1970). The result was converted to units per g wet weight of hepatopancreas. All of the samples for characterization were assayed in duplicate, and substrate-only blanks were assayed at the beginning of each analysis.

To define saturating-substrate conditions, TAME solutions of 0.016, 0.069, 0.08, 0.16, 0.27, 0.4, 0.69, 0.8, and 1.6 mM were made as dilutions (with trypsin buffer) from a 1.6 mM stock solution. The enzyme source was a 20 mg/ml (wt/vol) homogenate of hepatopancreas. To determine the optimal pH for trypsin, assays were performed from pH 4.5 to 9.5 (in 0.5 unit intervals). Trypsin buffer (100 mM potassium phosphate) was adjusted to provide buffers for the pH range of 4.5 to 8.5, and a 100 mM glycine-phosphate-sodium hydroxide buffer (pH adjusted with sodium hydroxide) was used for the range of pH 8.5 to 9.5. A 45 mg/ml (wt/vol) homogenate of hepatopancreas was used as the enzyme source for pH determinations. The effect of temperature on the enzymatic activity of trypsin was tested from 8 to 44 C (in 6 C increments). A 45 mg/ml (wt/vol) homogenate of hepatopancreas was used as the enzyme source for temperature determinations. The [Q.sub.10] was calculated as described previously.

The activity of lipolytic enzymes (nonspecific esterases or lipases) was determined by a method modified from Bishop (1997) and Gjellesvik et al. (1992). A 100 mM stock solution of 4-nitrophenol caproate (4-NPC) was made by dissolving 4-NPC in ice-cold 100% ethanol; this solution was mixed thoroughly and aliquoted into separate borosilicate test tubes that were sealed air-tight and stored at -80 C. Aliquots of the 4-NPC stock solutions were removed from the 100 mM stock tube at the time of assay and added to ice cold buffer solution (0.5 M Tris-HCl and 0.1 M sodium chloride) at pH 8.5 (pH determined at 25 C) to give a final concentration of 0.4 mM 4-NPC. This substrate buffer solution is very sensitive to temperature, time, and pH. Substrate buffer was kept ice cold for the duration of the assay and used immediately. The absorbance was recorded from a temperature-controlled Shimadzu UV-100 spectrophotometer at 400 nm and 25 C. Measurements were recorded when the change in optical density was linear (substrate concentration was saturating). Nonspecific esterase activity was measured as the average change in optical density per minute at 400 nm and then converted to units (micromoles of 4-NPC hydrolyzed per minute) using the extinction coefficient for nitrophenol of 19,800 [M.sup.-1] [cm.sup.-1] at pH 8.5 and 405 nm (Gjellesvik et al., 1992). This result was converted to units per g wet weight of hepatopancreas. All of the samples for characterization were assayed in duplicate with single substrate-only controls with each assay.

To define substrate-saturating conditions, solutions of 4-NPC were made to 0, 0.025, 0.05, 0.1, 0.4, 0.6, and 1 mM. The source of nonspecific esterase was a 50 mg/ml (wt/vol) homogenate of hepatopancreas. To determine the optimal pH of nonspecific esterase activity, assays were performed from pH 6 to 11 (in increments of 0.5 pH units). The standard buffer (0.5 M Tris-HCl and 0.1 M sodium chloride) was used for the entire pH range and was adjusted with sodium hydroxide or hydrochloric acid solutions when appropriate. The source of nonspecific esterase was a 50 mg/ml (wt/vol) homogenate of hepatopancreas. The 4-NPC substrate was not pH stable and rapidly degraded at pH levels below 6 and above 11. The effect of temperature on the enzymatic activity of nonspecific esterase was tested from 8 to 44 C (in increments of 6 C). The source of nonspecific esterase was a 50 mg/ml (wt/vol) homogenate of hepatopancreas. The [Q.sub.10] was calculated as described previously.

RESULTS

Kinetic analysis of [alpha]-amylase activity indicated that the enzyme was saturated at approximately 10 mg/ml starch and remained saturated with increasing starch concentration (Figure 1A). The maximum velocity and apparent Michaelis constant was approximately 328.3 units/g wet weight and 2.4 mg/ml starch, respectively, as determined by Lineweaver-Burk plot (Figure 2A). The enzymatic activity of [alpha]-amylase increased with increasing pH from 4 to 5.5, remained relatively constant from pH 5.5 to 6.5, and then decreased to pH 8 (Figure 1B). The enzymatic activity of [alpha]-amylase increased with increasing temperature (Figure 1C). The [Q.sub.10] values for [alpha]-amylase ranged from 1.56 to 3.50 (Table 1).

Kinetic analysis of trypsin activity indicated that the enzyme was saturated at approximately 0.7 mM TAME and remained saturated with increasing TAME concentrations (Figure 3A). The maximum velocity and apparent Michaelis constant for this reaction were approximately 342.2 units/g wet weight and 0.308 mM TAME, respectively, as determined by Lineweaver-Burk plot (Figure 2B). The enzymatic activity of trypsin increased with increasing pH from 4.5, peaked at pH 8.0, and decreased to pH 9.5 (Figure 3B). The enzymatic activity of trypsin increased with increasing temperature (Figure 3C). The [Q.sub.10] values for trypsin activity ranged from 1.53 to 1.88 (Table 1).

Kinetic analysis of nonspecific esterase activity indicated that the enzyme was saturated at approximately 0.8 mM 4-NPC are remained saturated with increasing substrate concentration (Figure 4A). The maximum velocity and apparent Michaelis constant for this reaction were approximately 0.804 units/g wet weight and 0.053 mM 4-NPC, respectively, as determined by Lineweaver-Burk plot (Figure 2C). The enzymatic activity of nonspecific esterase increased with increasing pH from pH 6, peaked at pH 8.5, and decreased to pH 11 (Figure 4B). The enzymatic activity of nonspecific esterase increased with increasing temperature (Figure 4C). The [Q.sub.10] values for nonspecific esterase activity ranged from 1.21 to 3.06.

DISCUSSION

The digestive enzyme [alpha]-amylase exhibited substrate saturation kinetics. Similar trends in [alpha]-amylase activity were observed in larvae of the American lobster Homarus americanus (Biesiot, 1986). Though reports are limited in the literature for crustacea, the apparent Michaelis constant reported here (2.4-mg/ml starch) is similar to that (4.5 mg/ml) reported by Mayzaud (1985) for the copepod Acartia clausi.

The optimal pH range determined for [alpha]-amylase in the crayfish Procambarus clarkii was pH 5.5 to 6.5. This pH range is consistent with the pH optima described previously for this species (pH 5.18 to 6.05; van Weel, 1970), the crayfish Astacus fluviatilis (pH 5 to 6; Kooiman, 1964) and other crustaceans (Biesiot, 1986; Blandamer and Beechey, 1964; Mayzaud, 1985; Wojtowicz and Brokerhoff, 1972).

The direct effect of temperature on the specific activity of [alpha]-amylase was similar to that described for other crustaceans. Biesiot (1986) found that [alpha]-amylase activity increased from 25 to 50 C in Homarus americanus. The enzymatic activity of [alpha]-amylase increased from 10 to 40 C then decreased in the crab Carcinus maenus (Blandamer and Beechey, 1964) and the copepod Acartia clausi (Mayzaud, 1985). The relatively high [Q.sub.10] values for [alpha]-amylase in the low temperature intervals suggest that low temperatures have a greater effect on [alpha]-amylase activity than high temperatures. However, the amount of [alpha]-amylase activity present is very much greater than either trypsin or nonspecific esterase, and this reduction in specific activity probably has little physiological significance.

Trypsin also demonstrated saturation kinetics and an apparent Michaelis constant of 0.31 mM TAME. This value is consistent with the Michaelis constants of 0.33, 0.27, 0.42, and 0.39 mM TAME reported for trypsins A, B, C, and D, respectively, from Procambarus clarkii (Kim et al., 1994).

The optimum pH for trypsin determined in this study is 8.0. This pH is consistent with the reported optima of 8.0, 8.0, 7.5, and 7.5 for trypsins A, B, C, and D, respectively, from Procambarus clarkii (Kim et al., 1994), identical to that of Orconectes virilis (Devillez, 1965,) and slightly more acidic than the optimum pH of 8.5 reported for Astacus astacus (Kleine, 1967). Similar pH optima were reported for several other crustacean species, including the lobster Homarus americanus (Brockerhoff et al., 1970), the white shrimp Penaeus setiferus (Gates and Travis, 1969), the tiger prawn Penaeus monodon (Pei-Jung, Hsien-Ching, and Inn-Ho, 1990), and Peneaus japonicus (Galgani et al., 1985).

Temperature-dependent enzyme activity in the present study is comparable with the values reported for Procambarus clarkii (Kim et al., 1994), the crayfish Orconectes virilis (Devillez, 1965), and the white shrimp Penaeus setiferus (Gates and Travis, 1969). The low [Q.sub.10] values for trypsin that are reported in the present study suggest that the enzymatic activity of trypsin is not substantially influenced by changes in temperature.

Similar to that of [alpha]-amylase and trypsin, nonspecific esterase activity was substrate-dependent and saturable. Though this assay has not been used (to our knowledge) for characterizations of crustacean nonspecific esterases, it has been used for larval studies with teleost fish (Bishop and Watts, 1997; Gjellesvik et al., 1992). The apparent Michaelis constant for nonspecific esterase reported in the present study is 0.053-mM 4-NPC. This is within the range reported by Gjellesvik et al. (1992) for cod (0.14 mM) and human (0.044 mM) bile salt-dependent lipase.

Nonspecific esterase exhibited an optimum pH at 8.5. This report supports the earlier study of Loizzi and Peterson (1971), who found two different Tween-60 esterases in Procambarus clarkii; one had an optimum at pH of 7.1 to 7.3 and the other at 8.1 to 8.4. Berner and Hammond (1969) detected two pH optima for lipolytic activity in the crayfish Cambarus virilis, one at pH 4.0 (a pH too acidic to be measured with 4-NPC) and another at pH 9.0 that was described as a true lipase. Kleine (1967) separated four hepatopancreatic fractions with esterase activity from the crayfish Astacus astacus; the pH optima of these enzymes were between 8 and 9. Mansour-Bek (1954) described an esterase from Astacus sp. that had pH optimum of 5.2 to 5.6. A true lipase has also been reported for Homarus americanus, with a pH optimum of 7.0 for adults (Brockerhoff et al., 1970) and 5.5 for early life history stages (Biesiot, 1986). These data suggest that crayfish, as well as other crustaceans, have several esterases that are active in the gut and hepatopancreas.

[FIGURE 1 OMITTED]

[FIGURE 2 OMITTED]

[FIGURE 3 OMITTED]

[FIGURE 4 OMITTED]

The effects of temperature on nonspecific esterase activity are consistent with those reported for lipase in Homarus americanus (Biesiot, 1986). The [Q.sub.10] values for nonspecific esterase suggest that enzymatic activity is affected more by low temperature than by higher temperatures.

As suggested above, a number of factors can potentially influence the digestive capacity of the crayfish hepatopancreas, including pH and temperature. The reported pH for the crayfish gut was 4.7 to 6.6 (Brown, 1995; Gibson and Barker, 1979; van Weel, 1970), however, trypsin and nonspecific esterase have alkaline pH optima. Dall and Moriarty (1983) suggested that these enzymes may function within microenvironments (such as the diverticula of the hepatopancreas) that would allow them to function closer to the pH optima. For example, Kleine (1966) described the gastric juice of the crayfish Astacus astacus as being devoid of lipolytic activity but reported high lipolytic activity in an extract of hepatopancreas. Loizzi and Peterson (1971) described two Tween-60 esterases that were found at the cell-striated borders of absorptive cells and within the vacuoles of enzyme secretory and enzyme synthesizing cells in the hepatopancreas of Procambarus clarkii. Alternatively, Dall and Moriarty (1983) indicated that digestion may occur as fluctuations around neutral pH that would allow sufficient, but not necessarily optimum, digestion by all enzymes. These data suggest that it is possible to change the rate at which various substrates can be digested by changing the gut pH.

Procambarus clarkii maintains a capacity to digest carbohydrates, proteins, and lipids over a wide range of temperatures. At low temperatures enzyme activity is minimal. Reduced consumption rate at low temperatures (Croll, 2002) coupled with reduced enzymatic capacity, would limit the activity and growth of individuals during periods of low water temperature.
Table 1. [Q.sub.10] Values for [alpha]-Amylase, Trypsin, and Nonspecific
Esterase

Temperature [alpha]-Amylase Trypsin Nonspecific Esterase
[degrees]C [Q.sub.10] [Q.sub.10] [Q.sub.10]

 8 to 14[degrees] 3.50 1.82 2.57
14 to 20[degrees] 2.24 1.88 3.06
20 to 26[degrees] 2.10 1.60 1.40
26 to 32[degrees] 2.48 1.58 1.83
32 to 38[degrees] 1.73 1.54 1.32
38 to 44[degrees] 1.56 1.53 1.21


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Hugh S. Hammer, Charles D. Bishop and Stephen A. Watts

Department of Biology

University of Alabama at Birmingham

Birmingham, AL 35294-1170
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