Fish Physiology and Biochemistry

, Volume 43, Issue 2, pp 373–383 | Cite as

Proteolytic activity in some freshwater animals and associated microflora in a wide pH range

  • V. V. Kuz’mina
  • G. V. Zolotareva
  • V. A. Sheptitskiy


Proteolytic activity in some freshwater animals (crustacean plankton, sandhopper Amphipoda sp., larvae of chironomids Chironomus sp., oligochaetes Oligohaeta sp., dreissena Dreissena polymorpha, roach Rutilus rutilus heckelii, rudd Scardinius erythrophthalmus, ruff Acerina cernua, and monkey goby Neogobius fluviatilis) prevailing within the food of fishes of various ecological groups as well as in their associated microflora in a wide pH range was investigated. It has been shown that the optimum pH of proteases in the animals’ whole organism varies: 6.0 for sandhopper; 8.0 for chironomid larvae, oligochaetes, monkey goby, and ruff; 8.0–9.0 for zooplankton; and 10.0 for roach and rudd. The optimum pH of associated microflora proteases is 6.0 for monkey goby; 7.0 for sandhopper and roach and ruff ; 8.0–9.0 for oligochaetes; 9.0 for zooplankton; and 10.0 for chironomid larvae and rudd. The compensatory role of food items and enteric microbiota proteases in digestive processes in fish of different ecological groups at low pH is discussed.


Digestion Proteases pH Freshwater animals Potential preys Associated microflora 



The authors would like to thank Dr. K. Iliadi, the Hospital for Sick Children Peter Gilgan Centre for Research and Learning, Toronto, for his exceptional linguistic help. This study was partly supported by the Russian Foundation for Basic Research, project 13-04-00248.


  1. Aoki H, Ahsan MN, Watabe S (2003) Molecular cloning and characterization of cathepsin B from the hepatopancreas of northern shrimp Pandalus borealis. Comp Biochem Physiol 134B:681–694CrossRefGoogle Scholar
  2. Aranishi F, Hara K, Osatomi K, Ishihara T (1997a) Purification and characterization of cathepsin B from hepatopancreas of carp Cyprinus carpio. Comp Biochem Physiol 117B(4):579–587CrossRefGoogle Scholar
  3. Aranishi F, Hara K, Osatomi K, Ishihara T (1997b) Cathepsin B, H and L in peritoneal macrophages and hepatopancreas of carp Cyprinus carpio. Comp Biochem Physiol 117B(4):601–605Google Scholar
  4. Ashie INA, Simpson BK (1997) Proteolysis in food myosystems—a review. J Food Biochem 21:91–123CrossRefGoogle Scholar
  5. Askarian F, Zhou Z, Olsen RE, Sperstad S, Ringo E (2012) Culturable autochthonous bacteria in Atlantic salmon (Salmo salar L.) fed diets with or without chitin. Characterization by 16S rRNA gene sequencing, ability to produce enzymes and in vitro growth inhibition of four fish pathogens. Aquac Res 326–329:1–8CrossRefGoogle Scholar
  6. Austin B (2006) The bacterial microflora of fish, revised. Sci World J 6:931–945CrossRefGoogle Scholar
  7. Belchior SGE, Vacca G (2006) Fish protein hydrolysis by a psychrotrophic marine bacterium isolated from the gut of hake (Merluccius hubbsi). Can J Microbiol 52:1266–1271CrossRefPubMedGoogle Scholar
  8. Butler AM, Aiton AL, Warner AH (2001) Characterization of a novel heterodimeric cathepsin L-like protease and cDNA encoding the catalytic subunit of the protease in embryos of Artemia franciscana. Biochem Cell Biol 79:43–56CrossRefPubMedGoogle Scholar
  9. Cahill MM (1990) Bacterial flora of fishes: a review. Microb Ecol 19:21–41CrossRefPubMedGoogle Scholar
  10. Capasso C, Lees WE, Capasso A, Scudiero R, Carginale V, Kille P, Kay J, Parisi E (1999) Cathepsin D from the liver of the Antarctic icefish Chionodraco hamatus exhibits unusual activity and stability at high temperatures. Biochim Biophys Acta 1431:64–73CrossRefPubMedGoogle Scholar
  11. Clements KD (1997) Fermentation and gastrointestinal microorganisms in fishes, Ch 6. In: Mackie RI, White BA (eds) Gastrointestinal ecosystems and fermentations. Chapman and Hall, New York, pp 156–198Google Scholar
  12. Dabrowski K (1979) The role of proteolytic enzymes in fish digestion. In: Cultivation of fish fry and its live food, vol 5. Eur Maricult Soc Bradine, Belgium. Special Publ., pp 107–126Google Scholar
  13. Dabrowski K, Glogowski J (1977a) Studies on the proteolytic enzymes of invertebrates constituting fish food. Hydrobiologia (Hagua) 52:171–174CrossRefGoogle Scholar
  14. Dabrowski K, Glogowski J (1977b) The role of exogenic proteolytic enzymes in digestion processes in fish. Hydrobiologia (Hagua) 54:129–134CrossRefGoogle Scholar
  15. Das KM, Tripathi SD (1991) Studies on the digestive enzymes of grass carp, Ctenopharyngodon idella (Val.). Aquaculture 92:21–32CrossRefGoogle Scholar
  16. Deguara S, Jauncey K, Agius C (2003) Enzyme activities and pH variations in the digestive tract of gilthead sea bream. J Fish Biol 62:1033–1043CrossRefGoogle Scholar
  17. Dendinger JE, O’Connor KL (1990) Purification and characterization of a trypsin-like enzyme from the midgut gland of the Atlantic blue crab Callinectes sapidus. Comp Biochem Physiol. 95B:525–530Google Scholar
  18. Diaz-Tenorio LM, Garcia-Carreňo FL, Navarrete Ángeles, del Toro M (2006) Characterization and comparison of digestive proteinases of the Cortez swimming crab, Callinectes bellicosus, and the arched swimming crab Callinectes arcuatus. Invertebr Biol 125(2):125–135CrossRefGoogle Scholar
  19. Doke SN, Ninjoor V, Nadkarni GB (1980) Characterization of cathepsin D from the skeletal muscle of fresh water fish, Tilapia mossambica. Agric Biol Chem 44:1521–1528Google Scholar
  20. Erickson MC, Gordon DT, Anglemier AF (1983) Proteolytic activity in the sarcoplasmic fluids of parasitized Pacific whiting (Merluccius productus) and unparasitized True cod (Gadus macrocephalus). J Food Sci 48:315–1319CrossRefGoogle Scholar
  21. Esakkiraj P, Immanuel G, Sowmya SV, Iyapparaj P, Palavesam A (2009) Evaluation of protease-producing ability of fish gut isolate Bacillus cereus. Food Bioprocess Technol 2:383–390CrossRefGoogle Scholar
  22. Fernández Gimenez AV, García-Carreňo FL, Navarrete del Toro MA, Fenucci JL (2001) Digestive proteinases of red shrimp Pleoticus muelleri (Decapoda, Penaeoidea): partial characterization and relationship with molting. Comp Biochem Physiol 130B:331–338CrossRefGoogle Scholar
  23. Ganguly S, Prasad A (2012) Microflora in fish digestive tract plays significant role in digestion and metabolism. Rev Fish Biol Fish 22:11–16CrossRefGoogle Scholar
  24. García-Carreňo FL, Albuquerque-Cavalcanti C, Navarrete del Toro MA, Zaniboni-Filho E (2002) Digestive proteinases of Brycon orbignyanus (Characidae, Teleostei): characteristics and effects of protein quality. Comp Biochem Physiol 132B:343–352CrossRefGoogle Scholar
  25. Ghosh K, Sen SK, Ray AK (2002) Characterization of bacilli isolated from gut of rohu, Labelio rohita, finderlings and its significance in digestion. J Appl Aquac 12:33–42CrossRefGoogle Scholar
  26. Gildberg A (1988) Aspartic proteinases in fishes and aquatic invertebrates. Comp Biochem Physiol 91B:425–435Google Scholar
  27. Glass HJ, MacDonald NL, Moran RM, Stark JR (1989) Digestion of protein in different marine species. Comp Biochem Physiol 94B:607–611Google Scholar
  28. Hau PV, Benjakul S (2006) Purification and characterization of trypsin from pyloric caeca of bigeye snapper (Pricanthus macracanthus). J Food Biochem 30:478–495CrossRefGoogle Scholar
  29. Hidalgo MC, Urea E, Sanz A (1999) Comparative study of digestive enzymes in fish with different nutritional habits. Proteolytic and amylase activities. Aquaculture 170:267–283CrossRefGoogle Scholar
  30. Hiraiwa M (1999) Cathepsin A protective protein: an unusual lysosomal multifunctional protein. Cell Mol Life Sci 56:894–907CrossRefPubMedGoogle Scholar
  31. Hoshino T, Ishizaki K, Sakamoto T, Kumeta H, Yumoto I, Matsuyama H, Ohgiya S (1997) Isolation of a Pseudomonas species from fish intestine that produces a protease active at low temperature. Lett Appl Microbiol 25:70–72CrossRefPubMedGoogle Scholar
  32. Jančarik A (1964) Die Verdauung der Hauptnährstoffe beim Karpfen. Z Fisch Hilfswiss 12:601–684Google Scholar
  33. Kishimura H, Tokuda Y, Klomklao S, Benjakul S, Ando S (2006) Enzymatic characteristics of trypsin from pyloric ceca of spotted mackerel (Scomber australasicus). J Food Biochem 30:466–477CrossRefGoogle Scholar
  34. Kishimura H, Klomklao S, Benjakul S, Chun B-S (2008) Characteristics of trypsin from the pyloric ceca of walleye pollock (Theragra chalcogramma). Food Chem 106:194–199CrossRefGoogle Scholar
  35. Kolkovsky S, Tandler A, Kissil GW, Gertler A (1993) The effect of dietary exogenous digestive enzymes on ingestion, assimilation, growth and survival of gilthead seabream (Sparus aurata, Sparidae, Linnaeus) larvae. Fish Physiol Biochem 12:203–209CrossRefGoogle Scholar
  36. Kottelat M, Freyhof J (2007) Handbook of European freshwater fishes. Publications Kottelat, Cornol, Switzerland 646 ppGoogle Scholar
  37. Kumar S, Garcia-Carreno FL, Chakrabarti R, Toro MAN, Co´rdova-Murueta JH (2007) Digestive proteases of three carps Catla catla, Labeo rohita and Hypophthalmichthys molitrix: partial characterization and protein hydrolysis efficiency. Aquac Nutr 13:381–388CrossRefGoogle Scholar
  38. Kurokawa T, Shiraishi M, Suzuki T (1998) Qualification of exogenous protease derived from zooplankton in the intestine of Japanese sardine Sardinops melanoticus larvae. Aquaculture 161:491–499CrossRefGoogle Scholar
  39. Kuz’mina VV (2005) Physiological and biochemical principles of exotrothy processes in fish. Nauka Publisher, Moscow (in Russian) Google Scholar
  40. Kuz’mina VV (2008) Classical and modern conceptions of fish digestion, Ch. 4. In: Cyrino JEP, Bureau D, Kapoor BG (eds) Feeding and digestive functions in fishes. Science Publishers, Enfield, NH, pp 85–154CrossRefGoogle Scholar
  41. Kuz’mina VV, Golovanova IL (2004) Contribution of prey proteinases and carbohydrases in fish digestion. Aquaculture 234:347–360CrossRefGoogle Scholar
  42. Kuz’mina VV, Skvortsova EG (2003) Contribution of prey proteolytic enzymes in digestive processes in carnivorous fish. J Ichthyol 43(2):209–214Google Scholar
  43. Kuz’mina VV, Zolotareva GV, Sheptitskiy VA (2014) Effect of pH on the activity of proteinases in intestinal mucosa, chyme, and microbiota of fish from the Cuciurgan Reservoir. J Ichthyol 54(8):591–597CrossRefGoogle Scholar
  44. Kuz’mina VV, Zolotareva GV, Sheptitskiy VA (2016) Effect of pH on the proteinase activities in the intestine mucosa, chyme, and enteral microbiota in the piscivorous fish, differing in their ecological traits. J Ichthyol 56(1):147–153CrossRefGoogle Scholar
  45. Kuz’mina VV, Ushakova NV (2013) The influence of temperature and pH on the effects of zinc and copper on proteolytic activities of intestinal mucosa in planktivorous and benthophagous fishes and their potential preys. Toxicol Environ Chem 95(1):150–162CrossRefGoogle Scholar
  46. Kuz’mina VV, Skvortsova EG, Zolotareva GV, Sheptitskiy VA (2011) Influence of pH upon the activity of glycosidases and proteinases of intestinal mucosa, chyme and microbiota in fish. Fish Physiol Biochem 37(3):345–357CrossRefPubMedGoogle Scholar
  47. Lauff M, Hofer R (1984) Proteolytic enzymes in fish development and the importance of dietary enzymes. Aquaculture 37(4):335–346CrossRefGoogle Scholar
  48. Le Boulay C, Van Wormhoudt A, Sellos D (1996) Cloning and expression of cathepsin L-like proteinases in the hepatopancreas of the shrimp Penaeus vannamei during the intermolt cycle. J Comp Physiol 166B(5):310–318CrossRefGoogle Scholar
  49. Le Chevalier P, Sellos D, Van Wormhoudt A (1995) Purification and partial characterization of chymotrypsin-like proteases from the digestive gland of the scallop Pecten maximus. Comp Biochem Physiol 110B:777–784CrossRefGoogle Scholar
  50. Li J, Ni J, Li J, Wang C, Li X, Wu S, Zhang T, Yu Y, Yan Q (2014) Comparative study on gastrointestinal microbiota of eight fish species with different feeding habits. J Appl Microbiol 117:1750–1760CrossRefPubMedGoogle Scholar
  51. Lubianskienė V, Jastiuginienė R (1996) Antibiotic and fermentative activity of bacteria found in water and digestive tract of fish from lake Druksiai at Ignalina nuclear power plant. Ekologija (Vilnius) 2:3–7Google Scholar
  52. Marquez L, Robles R, Morales GA, Moyano FJ (2012) Gut pH as a limiting factor for digestive proteolysis in cultured juveniles of gilthead sea bream (Sparus aurata). Fish Physiol Biochem 38:859–869CrossRefPubMedGoogle Scholar
  53. Munilla-Moran R, Stark JR, Babour A (1990) The role of exogenous enzymes in digestion in cultured turbot larvae (Scophthalmus maximus L.). Aquaculture 88:337–350CrossRefGoogle Scholar
  54. Natalia Y, Hashim R, Ali A, Chong A (2004) Characterization of digestive enzymes in a carnivorous ornamental fish, the Asian bony tongue Scleropages formosus (Osteoglossidae). Aquaculture 233:305–320CrossRefGoogle Scholar
  55. Navarrete del Toro MA, García-Carreño FL, Díaz LM, Celis-Guerrero L, Saborowski R (2006) Aspartic proteinases in the digestive tract of marine decapod crustaceans. J Exp Zool 305A:645–654CrossRefGoogle Scholar
  56. Oh E-S, Kim D-S, Kim JH, Kim H-R (2000) Enzymatic properties of a protease from the hepatopancreas of shrimp, Penaeus oriantalis. J Food Biochem 24:251–264CrossRefGoogle Scholar
  57. Oozeki Y, Bailey KM (1995) Ontogenetic development of digestive enzyme activities in larval walleye pollock, Theragra chalcogramma. Mar Biol 122(2):177–186Google Scholar
  58. Rawlings ND, Tolle DP, Barrett AJ (2004) MEROPS: the peptidase database. Nucleic Acids Res 32(suppl 1):D160–D164CrossRefPubMedPubMedCentralGoogle Scholar
  59. Ray AK, Ghosh K, Ringø E (2012a) Enzyme-producing bacteria isolated from fish gut: a review. Aquac Nutr 18(5):465–492CrossRefGoogle Scholar
  60. Ray AK, Mondal S, Roy T (2012b) Optimization of culture conditions for production of protease by two bacterial strains, Bacillus licheniformis BF2 and Bacillus subtilis BH4 isolated from the digestive tract of Bata, Labeo bata (Hamilton). Proc Zool Soc 65(1):33–39CrossRefGoogle Scholar
  61. Reid RGB, Rauchert K (1976) Catheptic endopeptidases and protein digestion in the horse clam Tresus capax (Gould). Comp Biochem Physiol 54B:467–472Google Scholar
  62. Richter-Otto W, Fehrmann M (1956) Zur methodik von darmflora untersuchungen. Ernährungsforsch 1:584–586Google Scholar
  63. Ringo E, Birkbeck TH (1999) Intestinal microflora of fish and fry: a review. Aquac Res 30(2):73–93CrossRefGoogle Scholar
  64. Teschke M, Saborowski R (2005) Cysteine proteinases substitute for serine proteinases in the midgut glands of Crangon crangon and Crangon allmani (Decapoda: Caridea). J Exp Mar Biol Ecol 316(2):213–229CrossRefGoogle Scholar
  65. Ugolev AM (1985) Evolution of digestion and principles of evolution of functions. Nauka, Leningrad (in Russian) Google Scholar
  66. Ugolev AM, Kuzmina VV (1993) Digestive processes and adaptations in fish. Gidrometeoizdat, Sanct-Petersburg (in Russian) Google Scholar
  67. Vega-Villasante F, Nolasco H, Civera R (1995) The digestive enzymes of the Pacific brown shrimp Penaeus californiensis—II. Properties of protease activity in whole digestive tract. Comp Biochem Physiol 112B:123–129CrossRefGoogle Scholar
  68. Visessanguan W, Menino AR, Kim SM, An H (2001) Cathepsin L: a predominant heat activated proteinase in arrowtooth flounder muscle. J Agric Food Chem 49(5):2633–2640CrossRefPubMedGoogle Scholar
  69. Visessanguan W, Benjakul S, An H (2003) Purification and characterization of cathepsin L in arrowtooth flounder (Atheresthes stomias) muscle. Compar Physiol Biochem 134B(3):474–487Google Scholar
  70. Wang B, Wang C, Mims SD, Xiong YL (2000) Characterization of the proteases involved in hydrolyzing paddlefish (Polyodon spathula) myosin. J Food Biochem 24:503–515CrossRefGoogle Scholar
  71. Warner AH, Pullumbi E, Amons R, Liu L (2004) Characterization of a cathepsin L-associated protein in Artemia and its relationship to the FAS-I family of cell adhesion proteins. Eur J Biochem 271:4014–4025CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media Dordrecht 2016

Authors and Affiliations

  • V. V. Kuz’mina
    • 1
  • G. V. Zolotareva
    • 2
  • V. A. Sheptitskiy
    • 2
  1. 1.I.D.Papanin Institute for Biology of Inland Waters RASBorok, YaroslavlRussia
  2. 2.T.G. Shevchenko State UniversityTiraspolMoldova

Personalised recommendations