Amylase and proteolytic activities were determined at different stages of ontogenic development of carp, Catla catla in relation to natural diet. Carp larvae were reared under 4 feeding schemes: continuously fed (CF), continuously starved (CS), initially fed for 4 days and then starved (IFS), and initially starved for 4 days and then fed (ISF). Enzyme activities were detected in day-old larvae. Along with ontogenic development of carp larvae enzyme activities also increased. Maximum amylase and proteolytic enzyme activities were obtained in 12 days and 11 days larvae of CF group. Removal of food after initial 4 days of feeding resulted in sharp decline of enzyme activities. It can be concluded that during initial feeding of catla larvae digestive enzymes are highly regulated by exogenous feeding of zooplankton.

Kumar, S. and Chakrabarti, R. 1998. Ontogenic development of amylase activity in three species of Indian major carps, Catla catla, Labeo rohita and Cirrhinus mrigala in relation to natural diet. Asian Fisheries Science 10: 259-263.

Three species of Indian major carps, catla, rohu and mrigal, were reared from 4th day after hatching to 32 days in a recirculating system. Final average weight was 27.80±2 mg and 31.53±1.24 mg for catla and rohu, respectively, in the first experiment. In a second trial, the final weights for rohu and mrigal were 35.90± 3.44 mg and 23.39±1.27, respectively. Amylase activity was very low during transition period from endogenous to exogenous feeding. Enzyme activity increased with age in all three species. It can be concluded that these three species of cyprinid larvae can easily hydrolyze starch and digest glycogen present in animal tissues a few days after the initial feeding.

Kumar, S., Sharma, J. G. and Chakrabarti, R. 2000. Quantitative estimation of proteolytic enzyme and ultrastructural study of anterior part of intestine of Indian major carp (Catla catla) larvae during ontogenesis. Current Science 79: 1007-1011.

Specific proteolytic enzyme activity and ultrastructure of the anterior part of the intestine of Catla catla larvae were studied during ontogenesis. Specific proteolytic activity of larvae cultured under three feeding schemes of live-food, refrigerated-plankton food and starvation showed that the enzyme activity increased along with age regardless of feeding conditions. There was no significant difference in enzyme activity between larvae fed live-plankton and refrigerated-plankton during the initial 9 days, but it became significantly (P<0.001) higher in the former than the latter from day-10 onwards. Highest activity was observed in the 26-day larvae of live-food treatment (0.987±0.02 mg of tyrosine/mg of protein/h). In the starvation treatment, activity was significantly lower throughout the culture period. Ultrastructure study of the digestive system also supported the results of quantitative estimation. Microvilli, microfilament bundles and secretary granules showed progressive changes along with age. Age-related changes in the diversity and quantity of digestive enzymes appear to represent evolutionary adaptations to the different natural diets and nutritional requirements of distinct life-history stages.

Rathore, R. M., Kumar, S. and Chakrabarti, R. 2005. Digestive enzyme profile of Cyprinus carpio during ontogenic development. World Aquaculture 36 : 37-39.

Digestive enzyme activities of common carp, Cyprinus carpio were studied from 4 to 30 days after hatching (DAH). Total amylase activity in 4 DAH common carp was 133.33±5.88 µg maltose/ml of tissue extract/h and then reduced enzyme activity was found up to day 10. This was followed by a gradual increase in total enzyme activity up to day 30. Highest specific amylase activity was found in 30 DAH fish. Total protease activity was minimum in 4 DAH fish and enzyme activity gradually increased along with increase of age of fish except days 10 and 18. Specific protease activity was 49 µg tyrosine/mg of protein/h in 4 DAH common carp. Enzyme activity gradually increased up to day 12 and then showed a decreasing trend up to day 26. The enzyme activity was 68% higher in 30 DAH common carp than 4 DAH one. Trypsin activity increased from 0.09 mUnit/mg of protein/min in 4 DAH fish to 0.26 mUnit/mg of protein/ min in 10 DAH one. Then trypsin activity decreased up to day 22 of culture. Trypsin activity was maximum on day 28. SDS-PAGE of crude enzyme extract showed one faint band of 52 kDa in 4 to 8 DAH common carp. More bands having molecular weight of 37 and 72 kDa were found in 10 and 12 DAH fish, respectively. 42 and 26.5 kDa bands appeared in 18 DAH fish. From 20 day onwards total 6 bands ranging from 26.5 to 72 kDa were clearly visible. All these bands expected to correspond to serine proteases with some evidence of increasing proteolytic enzyme activity with the age of fish.

Rathore, R. M., Kumar, S. and R. Chakrabarti, R. 2005. Digestive enzyme patterns and evaluation of protease classes in Catla catla (Family: Cyprinidae) during early developmental stages. Comparative Biochemistry and Physiology B: 142: 98-106.
 

Digestive enzymes of Catla catla were studied during ontogenic development. Specific amylase activity was 0.12 ± 0.01 mg maltose mg protein^− 1 h^− 1 in fish 4 days after hatching (DAH) and reached a maximum on (0.41 ± 0.12 mg maltose mg protein^− 1 h^− 1) 34 DAH. Total protease activity was minimum (123.2 ± 16.5 mU mg protein^− 1 min^− 1) on day-8 and reached its highest level (2713 ± 147.2 mU mg protein^− 1 min^- 1) on day-32. Trypsin activity showed constant increasing trend from day-16 onwards and was maximum on day-34 (118.1 ± 7.09 mU mg protein^− 1 min^− 1). Highest chymotrypsin activity was found on day-32 (1789.0 ± 111.7 mU mg protein^− 1 min^− 1). Lipase activity was detected in 4 DAH catla. Lipase activity increased steadily from day-22 onwards. SDS-PAGE of crude enzyme extracts showed that high molecular mass bands (41.8–127.8 kDa) appeared during the early stages followed by low molecular mass bands (17.8–37.2 kDa). The number of protease activity bands in substrate SDS-PAGE increased with age of fish. During ontogenesis of carp, soybean trypsin inhibitor (SBTI), PMSF and TLCK inhibited 75.5 ± 1.19% to 92.8 ± 0.85%, 53.3 ± 9.47% to 90.5 ± 2.6% and 39.8 ± 3.8% to 84.7 ± 1.54% of total protease activity, respectively. There was only 2.58 ± 0.66% to 10.21 ± 0.09% inhibition of protease activity with EDTA. SBTI and PMSF inhibited 8 and 4 activity bands, respectively. TLCK, a specific trypsin inhibitor, inhibited four trypsin-like enzymes in carp during ontogenesis.

Chakrabarti, R., Rathore, R. M. and Kumar, S. 2006. Study of digestive enzyme activities and partial characterization of digestive proteases in a freshwater teleost, Labeo rohita during early ontogeny.Aquaculture Nutrition 12: 35-43

Digestive enzymes of freshwater fish Labeo rohita (Family: Cyprinidae; Class: Actinopterygii; Infraclass: Teleostei; Order: Cypriniformes) were studied during ontogenic development. Amylase, protease and lipase activities showed a polynomial relationships whereas, trypsin, chymotrypsin and lipase activities exhibited exponential trends with the age of rohu larvae. SDS-PAGE of crude enzyme extract revealed that the proteases of higher MW appeared during early ontogeny, whereas low MW proteases were observed at later stages. Substrate SDS-PAGE supported the quantitative study of protease activities as evidenced with the increasing number and intensity of activity bands with the age of fish. The number of protease activity bands observed in 4, 10, 12, and 24 DAH (days after hatching) larvae were 5, 7, 8 and 9, respectively. Inhibition of protease activities with soybean trypsin inhibitor- SBTI (58.6 - 81.2%), phenyl methyl sulfonyl fluoride- PMSF (55.6 - 70%) revealed the presence of serine proteases. Whereas inhibition with N-α-p-tosyl-_L-lysine chloromethyl ketone-TLCK (41.1 - 52.3%) and N-tosyl-_L-phenylalanine chloromethyl ketone-TPCK (27.9 to 44.5%) indicated the presence of trypsin and chymotrypsin-like enzymes in rohu larvae. Inhibition of SBTI and TPCK showed power, TLCK and EDTA (ethylene diamine tetra acetic acid) showed exponential and PMSF showed polynomial relationships with increasing age of rohu larvae

Chakrabarti, R., Rathore, R. M., Mittal, P. and Kumar, S. 2006. Functional changes in digestive enzymes and characterization of proteases of silver carp (♂) and bighead carp (♀) hybrid, during early ontogeny. Aquaculture 253: 694-702.

Study of digestive enzyme and partial characterization of proteases of silver carp (♂) and bighead carp (♀) hybrid during early ontogeny was performed. Specific amylase activity was observed in 4 DAH (0.07 ± 0.01 mg maltose mg protein-1 min-1) carp hybrid.  Specific amylase activity showed polynomial relationship with the age of fish. Total protease, trypsin and chymotrypsin activities were 14.37 ± 2.21, 11.38 ± 1.67 and 2.83 ± 0.50 mUnits mg protein-1 min-1 in 4 DAH fish, respectively. Total protease activity showed exponential trend, whereas trypsin and chymotrypsin activities showed polynomial relationships with the increasing age of the fish. Lipase activity was 2.33 ± 0.18 mUnits in 4 DAH carp hybrid. Lipase activity showed polynomial trend with the increasing age of fish. In inhibition study, SBTI, PMSF, TLCK and TPCK inhibited the protease activity 83.0 to 92.0, 71.0 to 81.0, 45.1 to 55.5 and 35.8 to 48.2%, respectively. SDS-PAGE showed the presence of various protein bands (20.9-69.4 kDa) in carp hybrid during ontogenic development. Substrate SDS-PAGE revealed the presence of several protease activity bands (19.1-73.7 kDa) in digestive tissue extract of fish during ontogenesis. Inhibition of protease activity bands in substrate SDS-PAGE with SBTI and PMSF revealed the abundance of serine proteases and inhibition of activity bands with TLCK and TPCK evidenced the presence of more than one isoform of trypsin and chymotrypsin in the digestive tissue extract of hybrid carp.

Kumar, S., Fernando, L. G., Chakrabarti, R., del Toro, M. A. N. and Cordova-Murueta, J. H. 2007. Characterization of Characterization of digestive proteases of three carps Catla catla, Labeo rohita and Hypophthalmicthys molitrix and in vitro digestibility study. Aquaculture Nutrition 13: 1-9.

Characteristics and functional efficacy of digestive proteases of Catla catla, catla, Labeo rohita, rohu and silver carp were studied. Total protease activity was significantly (P<0.05) higher in rohu (1.219±0.059 U/mg protein/min) followed by silver carp (1.084±0.061 U/ mg protein/min), and catla (0.193±0.006 U/ mg protein/min). Trypsin activity of silver carp and rohu was 89-91% higher than catla. Chymotrypsin activity was significantly (P<0.05) higher in silver carp compared with rohu and catla. The protease activity of rohu and silver carp displayed bell-shaped curves with maximum activity at pH 9; whereas in catla, maximum activity was found between pH 8 and 11. Inhibition of protease activity with soybean trypsin inhibitor (SBTI), phenylmethylsulfonyl fluoride (PMSF) revealed the presence of serine proteases and inhibition of activity with N-a-p-tosyl-L-lysine-chloromethyl ketone (TLCK) and N-tosyl-L-phenylalanychloromethane (TPCK) indicated the presence of trypsin-like and chymotrypsin-like enzymes in all these three carps. SDS-PAGE showed the presence of several protein bands ranging from 15.3 to 121.9 kDa in enzyme extracts of catla, rohu and silver carp. The substrate SDS-PAGE evidenced the presence of various protease activity bands ranging from 21.6-93.7, 21.6-63.8 and 26.7-98.5 kDa for catla, rohu and silver carp, respectively. In pH-stat hydrolysis of Chilean fishmeal showed significantly (P<0.05) higher degree of hydrolysis compared with soybean meal, silver cup (a commercial fish feed of Mexico) and wheat flour, with enzyme preparations of three fishes. The rate of hydrolysis was significantly (P<0.05) higher in silver carp compared with others.

Digestive enzymes of Cirrhinus mrigala (Ham.) were studied during ontogenic development. Specific amylase activity was detected in first feeding fish. The enzyme activity decreased up to day-18 and then it increased with the age of fish to reach the highest level on day-34. Protease activity was 28.61 ± 8.90 mU mg protein^-1 min^-1 on day-4 and increased with the age throughout the study period. Trypsin activity was 31.86 ± 1.12 mU mg protein^-1 min^-1 on day-4. The activity was decreased up to day-10 and from day-12 onwards increased up to day-26. Chymotrypsin activity was 14.56 ± 2.74 mU mg protein^-1 min^-1on day-4 and constantly increased up to day-26. A significant increase in lipase activity was observed between days-24 and 34. SDS-PAGE and Substrate SDS-PAGE showed the diversity of protein (17.4 -127.8 kDa) and protease activity bands (16.6-88.8 kDa) during ontogenesis. SBTI, PMSF, TLCK and TPCK inhibited the protease activity up to 79.72 to 97.21, 65.55 to 94.83, 45.41 to 75.31 and 40.78 to 64.72%, respectively. Inhibition study in substrate SDS-PAGE revealed the abundance of serine proteases and the presence of isoforms of trypsin and chymotrypsin. EDTA showed 5.56 to 22.78% inhibition of metal ion specific enzyme activity.

Trypsin was purified from the digestive system of carp Catla catla (Hamilton) by ammonium sulfate fractionation, diethylaminoethyl-cellulose column chromatography, and Benzamidine Sepharose 4 fast flow column affinity chromatography. Trypsin was purified 26.2-fold with an 11.1% yield. The purified enzyme was active between pH 7.0 and 9.8, and maximal activity of the enzyme was observed at pH 7.0. Highest activity was found at 40°C. The activity was reduced to 52.84% at 60°C and was completely lost at 70°C. An addition of 2 mM CaCl2 enhanced trypsin activity during the 8-h incubation. The Km, Kcat, and catalytic efficiency values of purified enzyme were 0.062 mM and 19.23/s, and 310.16/s/mM, respectively. The enzyme activity was inhibited by soybean trypsin inhibitor, phenylmethylsulfonylflouride, and N-a-p-tosyl-L-lysine chloromethyl ketone. The molecular mass of the purified enzyme was 20.2 kDa by sodium dodecyl sulphate polyacrylamide gel electrophoresis. Mass spectrometry study of purified enzyme gave the peptide sequences LGEHNIAVNEGTEQFIDSVK (MW = 2,027.9568) and HPSYNSRNLDNDIM (MW = 1,692.6952) showing identical sequence with trypsin from various fishes.

Lukram, M. I., Ninawe, A. S. and Chakrabarti, R. 2014. Influence of Feeding Regimes on the Digestive Enzyme Profile and Ultrastructure of Digestive Tract of First Feeding Catla catla (Hamilton) Larvae. Aquaculture Research and Development (OMICS Publishing Group, http://dx.doi.org/10.4172/2155-9546.1000243).

The present investigation aimed to evaluate the effect of feeding regimes composed of various combinations of live food (LF), artificial food (AF) and mixed food (MF: LF and AF 50% each) on the digestive physiology of first feeding Catla catla (catla) larvae. Larvae (0.92 ± 0.21 mg) were cultured for 30 days under six different feeding regimes of LF, AF, MF, two groups were shifted from LF to AF on day-13 (LF-AF-13) and day-18 (LF-AF-18) and one group from LF to MF on day-12 (LF-MF-13). Significantly (p<0.05) higher survival and average weight of catla were found in MF, LF, and LF-MF-13, respectively compared to others. Chitinase and chitinobiase activities were significantly (p<0.05) higher in LF compared to others. This group was followed by LF-MF-13. Amylase, carboxypeptidase A and B activities were significantly (p<0.05) higher in LF-AF-18, MF and LF-AF-13, respectively compared to others. Total protease, trypsin, chymotrypsin and lipase activities were significantly (p<0.05) higher in LF-MF-13 compared to the other feeding regimes. SDS-PAGE and substrate SDS-PAGE showed the presence of 14 (14.34-97.57 kDa) and 8 (15.26-72.88 kDa) bands, respectively in the digestive tissue extract. The ultrastructure of the digestive tract viz. the height of microvilli, number and size of mitochondria, lipid droplet and goblet cell were influenced by the feeding regime of larvae. LF-MF-13 group was conspicuous by the abundance of mitochondria, nucleus and goblet cell and the absence of lipid droplet. Considering the digestive enzyme profile, survival and growth of larvae, it seems LFMF-13 feeding regime is suitable for catla larvae and co-feeding should be started after 12 days of hatching.

Khangembam, B. K. and Chakrabarti, R. 2015. Trypsin from the digestive system of carp Cirrhinus mrigala: Purification, Characterization and its potential application. Food Chemistry (Elsevier B.V., DOI: 10.1016/j.foodchem.2014.11.140)

Trypsin was purified 35.64-fold with 4.97% recovery from the viscera of carp Cirrhinus mrigala (mrigal) by ammonium sulfate precipitation, ion exchange and affinity chromatography. The purified enzyme was active at a wide range of pH (7.0–9.2) and temperature (10–50°C). The purified enzyme exhibited high thermal stability up to 50°C for 1 h. The enzyme activity was stabilized by Ca⁺² (2 mM) up to 7 h at 40°C. The Km and kcat values of purified enzyme were 0.0672 mM and 92.09/s/mM, respectively. Soybean trypsin inhibitor and phenylmethylsulphonylflouride completely inhibited the enzyme activity. The specific inhibitor of trypsin, N-a-p-tosyl-L-lysine chloromethyl ketone inhibited 99.67% activity. Na⁺, K⁺ and Li⁺ inhibited 20.99 ± 5.25%, 16.53 ± 4.80% and 18.99 ± 1.42% of enzyme activity, respectively. Divalent ions Mg⁺², Zn⁺², Co⁺², Hg⁺² and Cd⁺² inhibited 21.61 ± 2.22%, 31.62 ± 1.78%, 31.62 ± 1.96%, 85.68 ± 1.51% and 47.95 ± 2.13% enzyme activity, respectively. SDS–PAGE showed that the molecular mass of purified enzyme was 21.7 kDa. MALDI-TOF study showed a peptide sequence of AFCGGSLVNENKMHSAGHCYKSRIQV at the N-Terminal. This sequence recorded 76–84% identity with trypsin from Thunnus thynnus and other fish species. This confirmed that the purified protein was trypsin. The purified enzyme has potential applications in detergent and food industry because of its thermal stability and alkaline nature.

Sharma, J. G., Kumar, A., Saini, D., Targay, N. L., Khangembam, B. K. and Chakrabarti, R. 2015. In vitro digestibility study of some plant protein sources as aquafeed for carps Labeo rohita and Cyprinus carpio using pH-Stat method. Indian Journal of Experimental Biology (accepted, NISCAR, CSIR).

The protein content of twenty plant ingredients, including aquatic weeds, cereals, pulses and oil-cakes was determined using micro-Kjeldahl method. In vitro digestibility of these ingredients was evaluated for rohu Labeo rohita and common carp Cyprinus carpio using pH-Stat method. The protein contents of water fern, duckweed, almond oil-cake and soybean product were 20.81, 39.75, 47.78 and 57.48%, respectively. Species-specific digestibility was found for the same plant ingredient. The degree of hydrolysis for water fern, duck weed, almond oil-cake and soybean product were 14.17, 4.80, 17.30 and 3.57%, respectively for rohu and 4.58, 6.03, 12.17 and 3.35%, respectively for common carp. This study showed that incorporation of water fern and almond oil-cake in the diet of rohu, and duck weed and almond oil-cake in the diet of common carp are beneficial considering their protein content and digestibility. These are cost-effective, protein-rich feed ingredients for aquafeed.

Nilssen, K. J., Ngasainao, M. R., Sharma, J. G., Srivastava, S. K., Chandra, S., Moiranthem, K. S., Khangembam, B. K., Kumar, S. and Chakrabarti. 2015. Activities of digestive enzymes in relation to ingested natural food in three carp species of the Himalayan River Ladhiya, northern India. Aquaculture Nutrition (Blackwell Science Ltd., accepted).

The present investigation was carried out to study gut content and accompanying digestive enzyme profiles of the three carp species, Golden mahseer Tor putitora, Snow-trout Schizothorax richardsonii and Hill-trout Barilius barila. The fishes were captured from the River Ladhiya, Champawat district, Uttarakhand, northern India. The digestive tract length was on average 1.8 and 2.3 times longer than the body, while members of Bacillariophyceae, Chlorophyceae, Cyanophyceae and Zygnematophyceae made up 95.15 and 99.63% of gut content in the Golden mahseer and Snow trout, respectively. In Hill-trout, the digestive tract being only half the body length, gut content was insects (72.73%), eggs of aquatic organisms (22.73%), and nematodes (4.55%). Estimated gut enzyme activities demonstrated that amylase activity was significantly (P < 0.05) higher in Golden mahseer and Snow trout compared to Hill-trout, while total protease, trypsin and chymotrypsin activities were all significantly (P < 0.05) higher in the Hill-trout compared to other species. A strong co-relation was found among relative gut length, food composition and digestive enzyme activities of each species. This may guide the future formulation of feed to these important species of carps kept in farm.