Arunee Engkagul

Digestive enzymes and in-vitro digestibility of different species of phytoplankton for culture of the freshwater pearl mussel, Hyriopsis (Hyriopsis) bialatus

Hyriopsis (Hyriopsis) bialatus has been cultured during the mussel life cycle from glochidia to the adult stage with a low total survival of 6% up to 130-day-old juveniles. The main digestive enzymes (amylase and proteinases) were not detectable in one-day-old juveniles, and increased during development. The stomach, including digestive glands, was the major digestive organ for both carbohydrate and protein. The optimum conditions for amylase activity were 40°C and pH 7; for acidic proteinases they were 60°C and pH 5. Two main alkaline proteinases were found in the intestine, with optimum conditions of 30°C and pH 8 and 60°C and pH 8. To improve mussel survival by finding suitable phytoplankton species and age as food for juveniles and adults, an in-vitro digestibility test was performed on ten algal species three and seven days old using amylase and proteinases in crude enzyme extracts from different mussel life stages. Among the phytoplankton selected, the three most efficiently digested by juveniles were seven-day-old Chlorella sp.2, seven-day-old Chlorococcum sp. and seven-day-old Kirchneriella incurvata, in the ratio 1:1:3 for 30-day-old juveniles and 3:1:1 for 130-day-old juveniles. For the adult mussel, three-day-old Chlorella sp.2, seven-day-old Coccomyxa sp., and seven-day-old Monoraphidium sp., in the ratio 3:1:1, were the most digestible phytoplankton. Levels of in-vitro digestibility were related to the quality (not the concentrations) of carbohydrate and protein in the phytoplankton mixtures, and protein digestibility seemed to be the key factor determining food quality for the mussel.

Development of digestive enzymes and in vitro digestibility of different species of phytoplankton for culture of early juveniles of the freshwater pearl mussel, Hyriopsis (Hyriopsis) bialatus Simpson, 1900

Summary In vitro culture of the glochidia of the freshwater pearl mussel, Hyriopsis (Hyriopsis) bialatus Simpson, 1900, in M199 medium supplemented with common carp plasma resulted in 95 ± 2% survival, of which 97 ± 2% were transformed to juveniles. Transformation occurred within 10 days. After transformation, early juvenile mussels were reared and fed for 15 days with a mixture of four species of phytoplankton (Chlorella sp.2, Kirchneriella incurvata, Navicula sp. and Coccomyxa sp.). Digestive enzyme activities, not detected in glochidia, were induced after feeding in transformed juveniles. Analysis of juvenile crude enzyme extracts after 15 days of feeding revealed the presence of amylase, proteinases, lipase and cellulase with specific activities 7.29 ± 0.12 U mg protein−1, 0.003 ± 0.0001 U mg protein−1, 1.85 ± 0.24 U mg protein−1 and 0.03 ± 0.01 U mg protein−1, respectively, which corresponded to 39%, 25%, 42% and 6%, respectively, of those found in the gastrointestinal tract of adult mussels. In vitro digestibility of the four species of phytoplankton using juvenile crude enzyme extract resulted in digestion of carbohydrate, protein and lipid content of Chlorella sp.2, K. incurvata, Navicula sp. and Coccomyxa sp., respectively, at the following levels: carbohydrate, 414 ± 46, 234 ± 38, 98 ± 19 and 188 ± 31 μg of reducing sugar per mg phytoplankton; protein, 75 ± 13, 164 ± 19, 63 ± 7 and 120 ± 11 μg of cleaved peptides per mg phytoplankton; lipid, 369 ± 24, 18 ± 3, 56 ± 7 and 80 ± 11 μg of fatty acid per mg phytoplankton. Among different nutrient digestibilities, that of protein is apparently the key factor determining food quality, followed by carbohydrate as the secondary factor in herbivorous species like mussels. The data indicate that a combination of Chlorella sp.2 and K. incurvata is a suitable food formula for juvenile culture.

Biochemical Characterization of the α-Amylase Inhibitor in Mungbeans and Its Application in Inhibiting the Growth of Callosobruchus maculatus

The insect Callosobruchus maculatus causes considerable damage to harvested mungbean seeds every year, which leads to commercial losses. However, recent studies have revealed that mungbean seeds contain alpha-amylase inhibitors that can inhibit the protein C. maculatus, preventing growth and development of the insect larvae in the seed, thus preventing further damage. For this reason, the use of alpha-amylase inhibitors to interfere with the pests digestion process has become an interesting alternative biocontrolling agent. In this study, we have isolated and purified the alpha-amylase inhibitor from mungbean seeds (KPS1) using ammonium sulfate precipitation, gel filtration chromatography and reversed phase HPLC. We found that the alpha-amylase inhibitor, isolated as a monomer, had a molecular weight of 27 kDa. The alpha-amylase inhibitor was purified 750-fold with a final yield of 0.4 mg of protein per 30 g of mungbean seeds. Its specific activity was determined at 14.5 U (mg of protein)(-1). Interestingly, we found that the isolated alpha-amylase inhibitor inhibits C. maculatus alpha-amylase but not human salivary alpha-amylase. After preincubation of the enzyme with the inhibitor, the mungbean alpha-amylase inhibitor inhibited C. maculatus alpha-amylase activity by decreasing V(max) while increasing the K(m) constant, indicating that the mungbean alpha-amylase is a mix noncompetitive inhibitor. The in vivo effect of alpha-amylase inhibitor on the mortality of C. maculatus shows that the alpha-amylase inhibitor acts on C. maculatus during the development stage, by reducing carbohydrate digestion necessary for growth and development, rather than during the end laying/hatching stage. Our results suggest that mungbean alpha-amylase inhibitor could be a useful future biocontrolling agent.

Characterisation of thermostable trypsin and determination of trypsin isozymes from intestine of Nile tilapia (Oreochromis niloticus L.)

Trypsin from intestinal extracts of Nile tilapia (Oreochromis niloticus L.) was characterised. Three-step purification - by ammonium sulphate precipitation, Sephadex G-100, and Q Sepharose - was applied to isolate trypsin, and resulted in 3.77% recovery with a 5.34-fold increase in specific activity. At least 6 isoforms of trypsin were found in different ages. Only one major trypsin isozyme was isolated with high purity, as assessed by SDS-PAGE and native-PAGE zymogram, appearing as a single band of approximately 22.39 kDa protein. The purified trypsin was stable, with activity over a wide pH range of 6.0-11.0 and an optimal temperature of approximately 55-60 °C. The relative activity of the purified enzyme was dramatically increased in the presence of commercially used detergents, alkylbenzene sulphonate or alcohol ethoxylate, at 1% (v/v). The observed Michaelis-Menten constant (Km) and catalytic constant (Kcat) of the purified trypsin for BAPNA were 0.16 mM and 23.8 s(-1), respectively. The catalytic efficiency (Kcat/Km) was 238 s(-1) mM(-1).