Temperature and pH characteristics of amylase and proteinase of adult freshwater pearl mussel, Hyriopsis (Hyriopsis) bialatus Simpson 1900

Aquaculture 234 (2004) 575 – 587 www.elsevier.com/locate/aqua-online

Temperature and pH characteristics of amylase and proteinase of adult freshwater pearl mussel, Hyriopsis (Hyriopsis) bialatus Simpson 1900 Mayuva Areekijseree a, Arunee Engkagul b,*, Uthaiwan Kovitvadhi c, Amara Thongpan d, Mingkwan Mingmuang d, Pannee Pakkong e, Krisna Rungruangsak-Torrissen f a

Department of Biology, Faculty of Science, Silpakorn University, Thailand b Department of Biochemistry, Faculty of Science, Kasetsart University, 50 Paholyotin Road Chatuchak, Bangkok 10900, Thailand c Department of Zoology, Faculty of Science, Kasetsart University, Thailand d Department of General Science, Faculty of Science, Kasetsart University, Thailand e Department of Applied Radiation and Isotope, Faculty of Science, Kasetsart University, Thailand f Matre Aquaculture Research Station, Department of Aquaculture, Institute of Marine Research, N-5984 Matredal, Norway Received 4 March 2003; received in revised form 26 September 2003; accepted 4 December 2003

Abstract The enzymatic properties of the main digestive enzymes, amylase and proteinase, from stomach and intestine of adult freshwater pearl mussel, Hyriopsis (Hyriopsis) bialatus Simpson 1900, were studied at various pH’s (1 – 11) and temperatures (20 – 80 jC) as well as their enzymatic stability at various pH’s. The pH and temperature profiles of the enzymatic activities of amylase and proteinase were similar between male and female mussels. At least seven amylase activities were observed in both stomach and intestine. Four acidic amylases showing pH stability at 2 – 11 had temperature optima at 35, 45, 55 and 65 jC. Two neutral amylases with pH stability at 6 – 7 showed temperature optima at 30 and 40 jC, and alkaline amylase(s) with pH stability at 8 – 10 had temperature optima range at 60 – 75 jC. Proteinase, on the other hand, showed its optimal activity at pH 5 (acidic proteinase) and at 60 – 70 jC in the stomach, while at a pH range of 6 – 8 (alkaline proteinase) and at 35 – 40 jC in the intestine. A wide pH stability range was observed for both acidic proteinase (pH 2 – 6) and alkaline proteinase (pH 4 – 7). Six proteinase activities were differentiated, two acidic forms with pH stability at 2 – 3 and 6 having respective optimal temperature at 60 and 70 jC, and four * Corresponding author. Tel.: +66-2-942-8526-8x130; fax: +66-2-561-4627. E-mail address: [email protected] (A. Engkagul). 0044-8486/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.aquaculture.2003.12.008

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alkaline forms with pH stability at 4, 6, 7 and 10 showing respective optimal temperature of 25, 40, 35 and 55 – 80 jC. Digestive enzymes seemed to be more sensitive in females than in males. Females seemed to prefer a more alkaline condition for optimal food digestion compared to male mussels, as their alkaline proteinases were less stable in acidic conditions. At optimal conditions, both amylase and proteinase enzymes showed higher specific activities in the stomach than in the intestine. At habitat temperature (28 – 30 jC), amylase specific activities dominate in the stomach, while both amylase and proteinase specific activities dominate in the intestine. D 2004 Elsevier B.V. All rights reserved. Keywords: Amylase; Freshwater pearl mussels; Hyriopsis bialatus; Proteinase

1. Introduction Hyriopsis (Hyriopsis) bialatus Simpson 1900 is a native species of freshwater mussel of Thailand. They are important economically as multi-utility aquatic animals widely distributed in the bottoms of reservoirs and rivers in central, northern and northeastern Thailand (Brandt, 1974; Kovitvadhi and Kovitvadhi, 2002). Mussels have been an important source of feed for various animals. The nacreous mussel shells have been used for inlaying pearl furniture, ornaments, kitchen utensils and souvenirs. The species also has a potential industrial use for producing freshwater pearl (Yeemin, 1997). Apart from their versatile use as animal feed and other commercial usage with the ability to adapt to the natural habitat throughout most parts of the country, H. bialatus has one other major advantage in that they can reproduce all year round which makes them a good source of low cost high protein diet for the local population. Mussels are filter-feeders and they siphon nutrients from the water column. It is thought that these filtering activities contribute to maintaining river and stream ecosystem. They are also used as bioindicator of the ecological system (Hudson and Isom, 1984; Biggins et al., 1997). Moreover, mussels have antioxidant enzymes and biotransformation enzymes in their digestive gland to detoxify substances in water (Birmelin et al., 1999). The problem arises from the fact that the number of freshwater mussels has been declining drastically due to environmental deterioration, resulting in water quality problems, reduction of fish hosts and increasing commercial demand for mussel use. Attempts have been made to replenish the number of mussels through artificial culture techniques (Isom and Hudson, 1982, 1984; Hudson and Isom, 1984; Keller and Zam, 1990; Uthaiwan et al., 2001; Uthaiwan et al., 2002). Recently, Kovitvadhi and Kovitvadhi (2002, 2003) have successfully cultured Hyriopsis (Limnoscapha) myersiana from glochidia to juvenile stage with 100% transformation rate. However, culture of juvenile H. myersiana resulted in a very low survival rate due to limited information on the relationship between feeding physiology and feed availability (Hawkins et al., 1998; Wong and Cheung, 2001). A previous study in Thailand on feeding of freshwater pearl mussel H. myersiana indicated 99.99% phytoplankton and 0.01% zooplankton in the gastrointestinal tract plankton content (Kovitvadhi et al., 2000). The ultimate goal of this study is to establish a feed formula suitable for culturing of juvenile H. bialatus via in vitro digestibility method (Rungruangsak-Torrissen et al.,

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2002). However, since juvenile H. bialatus are transparent and very small in size (60.6  186.5  216.3 Am), it is impossible to obtain samples from natural habitat. As culturing of juveniles to reach adult stage is the key to complete the life cycle of this species in artificial culture, the information on digestive enzymes and feed digestibility has to be based on available adult enzymes. The aim of this investigation was to study the characteristics of the main digestive enzymes (amylase and proteinase) of adult freshwater mussels in various conditions. Changes in certain digestive enzyme activities from stomach and intestine were examined in adult male and female H. bialatus. Temperature and pH profiles as well as stability at various pH’s of these enzymes were studied. This is the first study on Thai freshwater pearl mussels. The work could provide knowledge on food digestion in mussels, which is prerequisite for future development of artificial feed formulation for mussels at various developmental stages cultured under different rearing conditions.

2. Materials and methods 2.1. Animal and rearing Adult H. bialatus were obtained from the Mun River Basin in the northeastern Thailand. They were cultured in circle nets from January to August 2001 in the pond at the Department of Aquaculture, Faculty of Fisheries, Kasetsart University, Bangkok, Thailand. The mussels were fed freely with natural plankton in the pond. 2.2. Sex determination The experiment was performed to find the differences in amylase and proteinase activities from the pooled extracts of stomach and intestine of mussels of each sex. The mussels were sexually identified by needle fluid suction from gonad to observe the gametes under microscope. Nine males and nine females were used with the average body weight of 49.6 F 2.1 and 57.2 F 9.0 g, respectively. 2.3. Enzyme study 2.3.1. Enzyme extraction The outside shells of mussels were cleaned with dechlorinated tap water and any adhering detritus was removed. The shells were opened by cutting anterior and posterior adductor muscles. The stomach and intestine were dissected and the organs were weighed and pooled (three organs of either stomach or intestine for each sample), then homogenized on ice without the addition of buffer solution. The homogenate was centrifuged at 13,000  g for 15 min at 4 jC and the upper lipid layer was discarded. The supernatant was collected, divided into small portions and kept at 20 jC for later determination of the enzyme specific activities of amylase and proteinase. The protein content of the stomach and the intestinal extracts was determined using the method described in Lowry et al. (1951).

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2.3.2. Determination of amylase specific activity Amylase (EC 3.2.1.1) activity was measured from the increase in reducing sugar (maltose) by the hydrolysis of a-D (1,4) glycosidic bond in polysaccharides, and stained with 3,5-dinitrosalicylic acid (DNS) (Bernfeld, 1951). Starch was used as substrate prepared by boiling 1% soluble starch for 10 min in various 100 mM buffers of different pH’s containing 6 mM NaCl. The buffers used were HCl –KCl buffer for pH 1 and 2, citrate phosphate buffer for the pH range 3 –5, phosphate buffer for the pH range 6– 8 and NaHCO3 – Na2CO3 buffer for the pH range 9 –11. The reaction mixture contained 125 Al of the crude enzyme extract (dilution 1:50 with specific pH buffer) and 125 Al of the starch substrate. For pH profile study, the reaction mixture was performed at room temperature and at various pH’s (1– 11). For pH stability study, the crude enzyme extracts were preincubated with various buffers (pH 1– 11) for 15 min at room temperature before assaying in either acidic (citrate phosphate buffer pH 4) or neutral (phosphate buffer pH 7) condition. For temperature profile study, the reaction mixtures were performed at various temperatures (20 –80 jC) in either the acidic or the neutral condition. All reaction mixtures were incubated for 3 min, and then the reaction was stopped by adding 250 Al of DNS and heated in boiling water bath for 5 min. They were cooled down and added with 2.5 ml of distilled water. The reducing sugar produced was determined by measuring the changes in absorbance at 540 nm. The control (blank) mixtures were prepared by adding the crude enzyme after the DNS reagent. Maltose (0.3 –3.0 Amol) was used for preparation of the calibration curve. The amylase specific activity is defined as Amol of maltose produced per min per mg protein (at the specified reaction condition). 2.3.3. Determination of proteinase specific activity Total proteinase activity was assayed by measuring the increase in cleavage of short chain polypeptide as modified from the method of Vega-Villasante et al. (1995). The total proteinase activity was determined by incubating 150 Al of crude enzyme extract (dilution 1:2 with specific pH buffer) with 250 Al of 2% azocasein substrate dissolved in various pH buffers as described above. For temperature profile study, the reaction was performed at various temperatures (20 – 80 jC) in either the acidic (citrate phosphate buffer pH 5) or the alkaline (phosphate buffer pH 8) condition. Thereby, the optimal temperatures of 60 and 40 jC were chosen for further studies in the acidic (as well as in the pH profile) and in the alkaline condition, respectively. For pH profile study, the reaction was performed at 60 jC and at various pH’s (1– 11). For pH stability study, the crude enzyme extracts were pre-incubated with various buffers (pH 1– 11) for 15 min at 60 jC, before assaying at 60 jC in the acidic condition or at 40 jC in the alkaline condition. The reaction mixture were incubated for 15 min and then the reaction was stopped by adding 1.2 ml of 10% trichloroacetic acid (TCA) to each reaction tube. The controls (blanks) were prepared by mixing the crude enzyme with TCA to denature the enzymes before adding the substrate. The mixtures were mixed thoroughly and left standing for 15 min at room temperature to complete precipitation of the remaining azocasein and fragments of azocasein. Then the mixtures were centrifuged at 8000  g for 15 min, and 1.2 ml of the supernatant fluid was transferred to a test tube containing 1.4 ml of 1.0 M NaOH. The change in the absorbance at 440 nm was determined. Total proteinase specific activity was expressed as the number of

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proteinase unit per mg of protein. One unit (U) of proteinase activity was defined as the amount of enzyme giving an increase of 1.0 absorbance unit at 440 nm at the specified reaction condition.

Fig. 1. The amylase specific activity (Amol min 1 mg protein 1) in the stomach and the intestinal extracts of adult male and female H. bialatus. The enzymatic reaction was performed at room temperature showing pH profiles (A), and the pH stability within 15 min at various pH’s before assaying at pH 4 (B) and pH 7 (C).

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2.4. Statistics Mean and standard error of the mean of each enzyme specific activity were calculated. Statistical analysis at 95% significance level was determined using analysis of variance (ANOVA), and multiple comparisons were analyzed by least-significant difference (LSD).

3. Results 3.1. Characteristics of amylase Different profiles of amylase activity were observed among various pH and temperature conditions; however, the profiles were similar in both stomach and intestine, regardless of

Fig. 2. The amylase specific activity (Amol min 1 mg protein 1) in the stomach and the intestinal extracts of adult male and female H. bialatus. The enzymatic reaction was performed at pH 4 (A) and pH 7 (B) at various temperatures.

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sex (Figs. 1 and 2). The amylase showed two pH optima for the hydrolysis of its substrate, at 4 – 5 and 6 –8 in both stomach and intestine (Fig. 1A), and it was stable over a broad pH range from 2 to 11 in pH 4 (Fig. 1B) and 6 to 10 in pH 7 (Fig. 1C) assay conditions, in both sexes and organs. By varying temperature at pH 4 assay condition, four isoforms of acidic amylase were observed in the stomach showing optimal temperatures at 35, 45, 55 and 65 jC, whilst three to four isoforms were found in the intestine, in both sexes (Fig. 2A). In the stomach, the acidic amylase specific activities were significantly higher in males than in females ( P < 0.05). From pH stability profiles (Fig. 1C) and by varying temperature at pH 7 assay condition (Fig. 2B), two isoforms of neutral amylase with temperature optima at 30 and 40 jC and at least one alkaline amylase with optimal temperature range at 60 – 75 jC were differentiated. Neutral amylase had higher specific activities than alkaline amylase ( P < 0.05). At optimal conditions, the amylase specific activity was higher in the stomach than in the intestine (Figs. 1A,C and 2B). Females showed a significantly lower amylase specific activity (Fig. 2B), with alkaline stability

Fig. 3. The total proteinase specific activity (mU min 1 mg protein 1) in the stomach and the intestinal extracts of adult male and female H. bialatus. The enzymatic reaction was performed at various temperatures in an acidic condition of pH 5 (A) and in an alkaline condition of pH 8 (B).

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Fig. 4. The total proteinase specific activity (mU min 1 mg protein 1) in the stomach and the intestinal extracts of adult male and female H. bialatus. The enzymatic reactions were performed showing pH profiles at 60 jC (A), and the pH stability within 15 min at various pH’s at 60 jC before assaying (B) in an acidic condition of pH 5 at 60 jC and (C) in an alkaline condition of pH 8 at 40 jC.

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higher for stomach amylase and lower for intestinal amylase (Fig. 1C), compared to the male mussels ( P < 0.05). 3.2. Characteristics of proteinase Temperature profiles of the proteinases were similar in both stomach and intestine, regardless of sex (Fig. 3). Both acidic proteinases showed a distinctive active temperature range of 60 –70 jC in the stomach (Fig. 3A). On the other hand, the alkaline proteinase group showed optimal temperature range of 35– 40 jC with a slight activity peak at 25 jC in both organs, and the activities were still observed from 55 jC to as high as 80 jC (Fig. 3B). Similar groups of proteinase showing pH optima at pH 5 and 6 – 8 were observed in both stomach and intestine, regardless of sex (Fig. 4A). The proteinase group showing high specific activities at pH 5 was mainly found in the stomach having a wide pH stability range of 2– 6, while their specific activities were low in the intestine (Fig. 4B). On the other hand, when the assay was performed at the alkaline condition of pH 8, the proteinase group showing pH stability at 6 – 7 was mainly observed in both stomach and intestine with slight activities of proteinases with pH stability at 4 and 10 (Fig. 4C). Six different proteinases were differentiated, two acidic proteinases with pH stability at 2 –3 and 6 (Fig. 4B), and four alkaline proteinases having pH stability at 4, 6, 7 and 10 (Fig. 4C). Based on stomach acidic proteinases having different specific activities at 60 jC (Fig. 4B) and based on both organs in Fig. 3A, the acidic proteinase with higher specific activity had lower optimal temperature (60 jC) than the lower specific activity proteinase (70 jC). Based on different peak levels of proteinase specific activity in Fig. 4C, the four alkaline proteinases having pH stability at 4, 6, 7 and 10 probably had optimal enzymatic activities at 25, 40, 35 and 55 –80 jC, respectively (Fig. 3B). Interestingly, females showed a lower proteinase specific activity ( P < 0.05) than the male mussels at the alkaline assay condition, especially for the alkaline proteinase having pH stability at 4 (Fig. 4C) with optimal activity at 25 jC (Fig. 3B). Besides, female intestinal alkaline proteinases were less stable in acidic condition than male proteinases (Fig. 4C). Similar to amylase activity, total proteinase activity was higher in the stomach than in the intestine at optimal conditions, regardless of the assay pH condition.

4. Discussion and conclusion In H. bialatus, the stomach and the intestine comprise 2.9% and 3.5% of body weight, respectively. It is difficult to measure the pH in these organs and, by measuring the pH of the crude enzyme without adding buffer solution, it was found to be neutral (pH 7) from both stomach and intestine. The optimal pH for amylase activity in H. bialatus was in the range of 6– 8 in the stomach and the intestine, which is similar to that found in the marine mussel Mytilus chilensis (Fernandes-Reiriz et al., 2001). In the current experiment, the optimal activity for amylase was in the range of 30– 45 jC at pH 7 (Fig. 2B), which is also

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close to the habitat temperature (28 – 30 jC) of the freshwater mussel resulting in a high level of activity for carbohydrate digestion in this animal. The neutral and alkaline forms of amylase seemed to be more important than the acidic forms, as they showed higher specific activities at habitat temperature with two times higher activity in the stomach than in the intestine (Fig. 2). Similar to what Sabapathy and Teo (1992) suggested, the most suitable condition for amylase activity would be neutral pH. Further work to identify these isoforms of amylase is needed. Similar to amylase, there are many types of proteinase enzymes, which show specific activity at different pH’s (Mortensen et al., 1994; Powers and Kam, 1995) and temperatures (Rungruangsak-Torrissen and Male, 2000). However, for quantification of total proteinase activity, azocasein is generally used as substrate (Garcia-Carreno and Harrd, 1993). The acidic proteinases showed a distinctive optimal activity at 60 –70 jC in the stomach, while the alkaline proteinases showed somewhat similar optimal activity at 35– 40 jC in both stomach and intestine (Fig. 3). Total proteinase activity in the stomach of both sexes is higher than in the intestine even at the habitat temperature of 28 – 30 jC. This could be related to intracellular protein digestion, which would occur in the stomach more than in the intestine (Ibarrola et al., 1996). At least seven isoforms of amylase and six isoforms of proteinase were found in H. bialatus under the conditions studied, suggesting functional diversity of each digestive enzyme in the mussel so that their secretions would be properly adjusted/regulated according to variations in environmental conditions and food supply. Different isoforms of amylase (Alemany and Rosell-Perex, 1973; Mayzaud et al., 1987; Le Moine et al., 1997) and proteinases (Blow, 1976; Torrissen, 1987; Mayzaud et al., 1987; Harel et al., 1991; Asgiersson and Bjarnasson, 1991; Rungruangsak-Torrissen and Male, 2000) have been reported. The results suggest that the acidic proteinases having pH stability at 2– 3 and 6, very active at 60 –70 jC with acidic condition preference, could be two isoforms of pepsin-like proteinase. The main alkaline proteinases found in both stomach and intestine having optimal activity at 25– 40 jC and functioning best in alkaline condition could probably be characterized as serine proteinase group (such as trypsin-like and chymotrypsin-like enzymes). Further identification of these main enzymes with the use of different specific synthetic substrates (Table 1) is needed for confirmation. Interestingly,

Table 1 Specific synthetic substrates used for specific proteolytic enzyme assays Enzyme

Substrate

Pepsin (EC 3.4.23.1) Trypsin (EC 3.4.21.4) Chymotrypsin (EC 3.4.21.1)

H-Pro-Thr-Glu-Phe-Phe (NO2)-Arg-Leu-OHa Benzoyl-L-arginine-p-nitroanilideb N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilideb Benzoyl-L-tyrosine ethyl esterc Hippuryl-L-phenylalanined Hippuryl-arginined

Carboxypeptidase A (EC 3.4.12.2) Carboxypeptidase B (EC 3.4.12.3) a

Dunn et al. (1986). Rungruangsak-Torrissen et al. (2002). c Rick (1974). d Appel (1974). b

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intestinal alkaline proteinase from females is less stable in acidic condition than those from male mussels (Fig. 4C), suggesting that females prefer a more alkaline condition than males for optimal food digestion. In addition, the alkaline proteinases showing stability at pH 4 (Fig. 4C) and optimal temperature close to habitat (Fig. 3B), as well as the neutral and alkaline amylases (Fig. 2B), had a significantly lower specific activity in females than in males in both organs ( P < 0.05). Digestive enzymes of female mussels seemed to be more sensitive than those of males. The lower digestive activities of the enzymes found in females may indicate that female mussels were having a lower consumption rate than the male mussels, as digestive enzyme activity usually relates to rate of food consumption. The plankton content in the gut of mussels in Thailand was found to consist of 99.99% phytoplankton and 0.01% zooplankton (Kovitvadhi et al., 2000), which may support the importance of carbohydrate as a food source in mollusk (Emerson, 1967; Teo and Sabapathy, 1990; Sabapathy and Teo, 1992). In this study, although the amylase specific activity was found to be considerably higher than the total proteinase specific activity at habitat temperature of 28– 30 jC, this may not be directly comparable to in vivo condition as azocasein resulted in lower proteinase activity compared to other specific synthetic substrates (Lemos et al., 1999) and the phytoplankton consumed contained 40 –60% protein (IFRPD, 1973). The existence of proteinases in the digestive system is vital for the hydrolysis of peptide bonds to maintain the cellular metabolism of the animal itself, and freshwater mussels usually have body protein compositions as high as 40%. Enzymes are proteins; hence, pH and the temperature could play a vital role on their activities and stability. Moreover, each enzyme has its own specific structure upon which a slight change of these factors could drastically affect its catalytic performance. Therefore, the range of pH and temperature for optimal food digestion as well as the pH range of enzyme stability are good indicators of in vivo digestion in the animal, and would be useful for formulating artificial diets that would be suitable for optimal digestion under different rearing conditions of the freshwater mussel at various developmental stages. This work is the first undertaken to provide an insight into different characteristics of the main digestive enzymes (amylase and proteinase) of the Thai freshwater pearl mussels. This study provides some practical knowledge of the properties of the mussel digestive enzymes. Some information is also gained with a possibility to improve diet formulation for numerous developmental stages, not only for cultured H. bialatus but probably also for other mussel species.

Acknowledgements This work was supported by the grant from the Graduate School, Kasetsart University, and the Reverse Brain Drain Project, Ministry of University Affairs, through Dr. Pichan Sawangwong (Office of International Relations, Burapha University, Thailand). We would like to thank the Department of Aquaculture, Faculty of Fisheries, Kasetsart University for providing a pond for culturing mussels, and Asst. Prof. Satit Kovitvadhi (Department of Agriculture, Rajabhat Institute Bansomdejchaopraya, Thailand) for statistical analysis and providing the mussels used in this experiment.

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