were reared as per described by. Krishnaswami (1978). The 5th instar larvae were used for present study. The larvae of 5th day were dissected in ice-cold insect ...
Vol. 39 (2-3), 2012
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COMPARATIVE STUDY OF PROTEASE CHARACTERIZATION IN BIVOLTINE RACES OF BOMBYXMORIL N. T. PAWAR, Y. S. MUNIV, S. J. PATIL, G. P. BHAWANE and A. A. KANASE Department of Zoology, Shivaji University, Kolhapur. 416 004, India •
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(Received September 24, 2011; Re-revised April 17, 2012) Key words : Midgut proteases, Bombyx mori, bivoltine races
ABSTRACT
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The pH optima for digestive protease were 11.6 and 11.4 in CSR2 and CSR4, respectively. The protease activity peaked at 40°C in both races. The estimated 50% inhibition time at 55°C was 40 min in CSR2 and at 60°C 14 min in CSR4. The Km and specific activities in CSR2 were 0.185% and 0.888 ug tyrosin/ug protein/h, while in CSR4 Km was 0.277% and specific activity was 0.626. ug tyrosin/ug protein/h. Digestion period was 50 min in CSR2 and 60 min in CSR4. INTRODUCTION The silkworm, Bombyx mori is monophagous insect.The larvae feeds on sole plant i.e. mulberrys.The 5th instar larvae feed voraciously as compared to other instar. The 5lh instar larvae show enhanced rate of growth and development. The holometabolous insect stores energy in fat body in the form of protein, lipid and glycogen. The 5th instar larvae digest and utilize efficiently the nutrient reserves from the mulberry leaves. The properties of proteolytic enzymes of the digestive fluid and midgut of silkworm larvae were investigated by Horie et al. (1963). Despite the wealth of information on the digestive enzymes including protease (Day & Waterhouse, 1956, Brookes, 1961; Gilmour, 1961, Birk, et al., 1962; Engelmann, 1969). s The protease enzyme in digestive fluid of B. mori larvae and adult was studied by Eguchi et al. (1982). The protease of midgut epithelium has long been recognized to contain peptidases mainly and scarcely any protease activity (Shimoda, 1930; Horie et al.,
1963; Eguchi et al., 1982). The midgut protease of the pharate adult is considered to be utilized source of the cocoon digestive enzymes. The proteolytic enzymes have been demonstrated in the peritrophic membrane of the midgut (Eguichi &lwamoto, 1975, Eguchi et al., 1982).The functional differentiation between digestive fluid and midgut tissue, that is molecular protein are hydrolyzed into peptides in the digestive fluid and into amino acids with peptidases in the midgut tissue. (Horie et al., 1963). In the present study, protease th characteristics of the midgut tissue in 5 instar bivoltine races i.e. CSR-2 and CSR-4 were examined with pH, temperature and themolability and digestion period. »
MATERIALS AND METHODS The CSR-2 and CSR-4 bivoltine races were reared as per described by Krishnaswami (1978). The 5th instar larvae th were used for present study. The larvae of 5 day were dissected in ice-cold insect saline.
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Figs. 1-5. Effect of pH (1). temperature (2), time (3), thermolability (4). and substrate concentration (5) on protease characteristics in B. mori. Lineveaver burk plot (6). , , The midgut was removed from larvae and flushed with ice-cold saline to remove leaf debris. The midguts were homogenized in
saline and centrifuged at 3000 rpm for 15 minutes. The supernatant was used as enzyme source.
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Vol. 39 (2-3), 2012 The activity of protease was determined by Sggmfy method. For this assay 1 % casein into 5ml appropriate buffers and 1ml homogenate was used. The mixture was incubated for 30 min at 40°c. The reaction was terminated by adding 6ml 2% TCA. The precipitate was centrifuged at 3000 rpm for 10 min. The concentration of digested protein in supernatant was determined with 5ml 5% sodium carbonate and Folin reagent. The absorbance was measured at 660nm and expressed in moles of tyrosine formed. The protein concentration was determined by Lowry et al. (1951) using Bovine serum albumin as a standard. RESULTS (a) Effect of pH: The optimum pH of midgut protease was 11.6 in CSR2 and 11.4 in CSR4. The pH activity curves are shown in Fig-1, which indicated that both races show different optimum pH for protease. The specific activity was observed 0.688 ug tyrosine/ug protein/h and 0.626 ug tyrosine/ ug protein/h in CSR2 and CSR4 respectively. b) Effect of temperature: The temperature optima for both races are 40°C. At 10°, 20°, 40° and 60°C temperature protease activity gets declined. The effect of temperature shown in Fig.2. c) Effect of time: The Fig.3 shows time course of protease activity in both races. The reaction product increases almost linearly within 90 min. The digestion period for CSR2 is 50 min, while 60 min in CSR4. d) Effect of thermolabiiity. In the CRS2 half life period for protease was 40 min at 55°C, while in CSR4 14 min at 60°C (Fig.4). e) Effect of substrate Concentration: Fig.5 shows substrate concentration and enzyme activity curves. The low concentration of substrate rapidly hydrolyzed than higher
115 concentration of substrate. At higher concentration protease activity decreases and becomes stable at one point. DISCUSSION Although it has been said that the proteolytic enzymes in the alimentary canal in Bombyx mori are predominantly localized in the gut contents and properties of tissue protease have not been known well (Shimoda, 1930, Horie et al.,1963). In midgut pH optima showed at 11.6 in CSR2 and 11.4 in CSR4. Eguchi et al. (1972) studied the optimum pH of larval protease as 11.2, while in adult about 9. The protease activity changed according to age, sex and feeding in midgut of 5th instar. Jadhav and Kallapur (1987) recorded the protease activity at pH 8.5. In races, C105 and C124 optimum pH in larvae was 11.3 (Eguchi, Iwamoto 1975). In honeybee, pH optimum was 8.5 (Berta moritz et al., 1987), in house cricket pH optimum was 7.6 (Teo and woodring, 1987). The lepidopteran larvae, black cutworm show midgut fluid pH as 8.3, while in corn earworm it was 9.7 and in tobacco budworm 8.9 (Purcell, et al., 1992). *
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There was an increase in activity of protease throughout 20°-40°C (Fig.2). The optimum temperature was 40°C in both races. In house cricket, temperature optima at 35°C, in honey bee 37°C, in B. mori 40°C consistant results with Horie (1963). At high temperature protease is denatured. The digestion period in CSR2 was 50 min in CSR4 in 60 min. In Bombyx mori studied by Eguchi et al. (1972) was 150 min, in tissue protease and 60 min in digestive fluid. The heat stability/thermolability of midgut tissue was 10 min at 50°C for 40% loss of tissue protease activity as studied by Eguchi & Iwamoto (1975) in Bombyx mori In CSR2 loss of 50% activity at 55°C for 40 min and in
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CSR4 at 60°C for 14 min. CSR4 is more thermostable than CSR2 at 60°C. The Km value for protease in CSR2 was 0.185%, while in CSR4 it was 0.277%. The midgut protease is more efficient in CSR2 than CSR4. AKNOWLEDGEMENTS •
The authors are thankful to Department of Zoology, Shivaji University, Kolhapur for providing laboratory facilities. REFERENCES Berta, Moritz and Karl, Crailsheim. Physiology of protein digestion in the midgut of the honeybee (Apis meliifera L ) . J. Insect Physiol., 1987, 33, 923-931. Birk, Y., Harpaz, I., Ishaaya, I., and Bondi, A. Studies on the proteolytic activity of the beetles Tenebrio and Tribolium. J. Insect Physiol., 1962, 8,417-429. Brookes, V. J . , Partial purification of a proteolytic enzyme from an insect, Phormia regin. Biochem. Biophys. Acta, 1961, 46, 13-21. Day, M. and Waterhouse, D.F. The mechanism of digestion. Insect Physiology, Wiley, New York; Chapman and Hall, London, 1956, pp. 311-330. Eguchi, M. and Iwamoto, A. Changes in proteases, esterase and phosphatases in alimentary canal of the silkworm during metamorphosis. Insect Biochem., 1975, 5, 495-507. Eguchi, M., Furukawa, S. and Iwamoto, A. Proteolytic enzyme in the midgut of the pharate adult of the silkworm, Bombyx mori. J.Insect Physiol., 1972, 18, 24572467. Eguchi, M., Iwamoto A. and Yamauchi K. Interrelation of proteases from the midgut lumen; epithelia and peritrophic
membrane of the silkworm, Bombyx mori L J. Comp. Biochem. Physiol., 19&2, 72, 359-363. Engelmann, F. Food stimulated synthesis of intestinal proteolytic enzymes in the cockroach, Leucophaea maderae, J. Insect Physiol., 1969, 15, 217-235. Gilmour, D. The Biochemistry of Insect. Academic Press, New York, 1961, pp.53-58. Horie, Y., Tanaka, M. and Ito, T. Proteolytic enzymes of digestive juice and midgut of the silkworm. Bombyx mori,L. J. Sericulture Science Japan. 1963, 32, 815 (in Japanese). Jadhav, G. and Kallapur, V.L Influence of sex and feeding on the protease activity of certain tissues of fifth instar silkworm, Bombyx mori. Entomon, 1987, 13, 289293 . Krishnaswami, S. New technology of silkworm rearing. CSR and Tl Bulletin, 1978, 2, 123. Lowry, O.H., Rosenbrough, N.J., Farra, L and Randall, R. J. Protein measurement with the folin phenol reagent. J. Biol. Chem., 1951, 193, 265-275. Purcell, J. P., Greenplate, J. T. and Sammons, R. D. Examination of midgut luminal proteanase activities in six economically important insects. Insect Biochem. Molec.Biol., 1992, 22, 41-47. Shinoda, O. Contributions to the knowledge of intestinal secretion in insect-Ill. On the digestive enzymes of the silkworm, Bombyx mori. J. Biochem., 1930,11,345367. Teo, L. H. and Woodring, J. P. The digestive protease and lipase in the house cricket Acheta domesticus. J. insect Bioche., 1987, 18, 363-367.
