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The Potential of Freshwater Fish Viscus from Silver Carp Hypophthalmichthys molitrix for Trypsin Source

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Abstract

Purpose Silver carp is widely cultured in China (the product reached 3.5 million ton per year), and approximately 20% of the non-edible viscera are discarded. Utilization of the viscera leads to a reduction in waste. Our previous study showed that silver carp produced two types of myosin isoforms, thermostable myosin in summer and unstable one in winter, for the adaptation of the environmental changes. Therefore, in this study, we purified trypsins from silver carp in summer and winter samples to investigate their thermostabilities.

Methods

Trypsins from summer and winter samples were purified by a series of chromatographies. The temperature dependence of trypsins was determined at pH 8.0 in the range of 20–80 °C, and the effect of temperature on the thermostability was determined by measuring the remaining activity after the incubation at pH 8.0 for 15 min in the range of 20–75 °C.

Results

From the summer sample, two trypsins (SSC-T1 and SSC-T2) were purified 63- and 72-fold with the yields of 24 and 21%, respectively, and a trypsin (WSC-T) was purified 81-fold with a yield of 40% from the winter sample. SSC-T1, SSC-T2 and WSC-T showed the same enzymatic characteristics, especially their optimum temperature (65 °C) and thermostability (stable below 63 °C in 15-min incubation) were similar to mammalian trypsins. Additionally, all were stable at 30 °C for 8 h in the presence of calcium-ion.

Conclusion

These data indicated the silver carp viscus has a potential for thermostable trypsin source all the year round.

Graphic Abstract

Thermostable characteristics of trypsins from viscera of freshwater fish including silver carp (Hypophthalmichthys molitrix).

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References

  1. Rypniewski, W., Perrakis, A., Vorgias, C.E., Wilson, K.S.: Evolutionary divergence and conservation of trypsin. Protein Eng. Des. Sel. 7, 57–64 (1994). https://doi.org/10.1093/protein/7.1.57

    Article  Google Scholar 

  2. Walsh, K.A.: Trypsinogens and trypsins of various species. Methods Enzymol. 19, 41–63 (1970). https://doi.org/10.1016/0076-6879(70)19006-9

    Article  Google Scholar 

  3. Kossiakoff, A.A., Chambers, J.L., Kay, L.M., Stroud, R.M.: Structure of bovine trypsinogen at 1.9A resolution. Biochemistry 16, 654–664 (1977). https://doi.org/10.1021/bi00623a016

    Article  Google Scholar 

  4. Gupta, R., Beg, Q., Lorenz, P.: Bacterial alkaline proteases: molecular approaches and industrial applications. Appl. Microbiol. Biotechnol. 59, 15–32 (2002). https://doi.org/10.1007/s00253-002-0975-y

    Article  Google Scholar 

  5. Chakrabarti, S., Guha, S., Majumder, K.: Food-derived bioactive peptides in human health: challenges and opportunities. Nutrients 10, 1738 (2018). https://doi.org/10.3390/nu10111738

    Article  Google Scholar 

  6. Kitade, Y., Miyabe, Y., Yamamoto, Y., Takeda, H., Shimizu, T., Yasui, H., Kishimura, H.: Structural characteristics of phycobiliproteins from red alga Mazzaella japonica. J. Food Biochem. 42, e12436 (2018). https://doi.org/10.1111/jfbc.12436

    Article  Google Scholar 

  7. Furuta, T., Miyabe, Y., Yasui, H., Kinoshita, Y., Kishimura, H.: Angiotensin I converting enzyme inhibitory peptides derived from phycobiliproteins of dulse Palmaria palmata. Mar. Drugs 14, e32 (2016). https://doi.org/10.3390/md14020032

    Article  Google Scholar 

  8. Tavano, O.L., Berenguer-Murcia, A., Secundo, F., Fernandez-Lafuente, R.: Biotechnological applications of proteases in food technology. Compr. Rev. Food Sci. Food Saf. 17, 412–436 (2018). https://doi.org/10.1111/1541-4337.12326

    Article  Google Scholar 

  9. Bougatef, A.: Trypsins from fish processing waste: characteristics and biotechnological applications—comprehensive review. J. Clean. Prod. 57, 257–265 (2013). https://doi.org/10.1016/j.jclepro.2013.06.005

    Article  Google Scholar 

  10. Zamani, A., Benjakul, S.: Trypsin from unicorn leatherjacket (Aluterus monoceros) pyloric caeca: purification and its use for preparation of fish protein hydrolysate with antioxidative activity. J. Sci. Food Agric. 96, 962–969 (2016). https://doi.org/10.1002/jsfa.7172

    Article  Google Scholar 

  11. Hidalgo, M.C., Urea, E., Sanz, A.: Comparative study of digestive enzymes in fish with different nutritional habits: proteolytic and amylase activities. Aquaculture 170, 267–283 (1999). https://doi.org/10.1016/S0044-8486(98)00413-X

    Article  Google Scholar 

  12. Kishimura, H., Hayashi, K., Miyashita, Y., Nonami, Y.: Characteristics of two trypsin isozymes from the viscera of Japanese anchovy (Engraulis japonica). J. Food Biochem. 29, 459–469 (2005). https://doi.org/10.1111/j.1745-4514.2005.00029.x

    Article  Google Scholar 

  13. Kishimura, H., Hayashi, K., Miyashita, Y., Nonami, Y.: Characteristics of trypsins from the viscera of true sardine (Sardinops melanostictus) and the pyloric ceca of arabesque greenling (Pleuroprammus azonus). Food Chem. 97, 65–70 (2006). https://doi.org/10.1016/j.foodchem.2005.03.008

    Article  Google Scholar 

  14. Kishimura, H., Tokuda, Y., Klomklao, S., Benjakul, S., Ando, S.: Enzymatic characteristics of trypsin from the pyloric ceca of spotted mackerel (Scomber australasicus). J. Food Biochem. 30, 466–477 (2006). https://doi.org/10.1111/j.1745-4514.2006.00076.x

    Article  Google Scholar 

  15. Kishimura, H., Tokuda, Y., Klomklao, S., Benjakul, S., Ando, S.: Comparative study on enzymatic characteristics of trypsins from the pyloric ceca of yellow tail (Seriola quinqueradiata) and brown hakeling (Physiculus japonicus). J. Food Biochem. 30, 521–534 (2006). https://doi.org/10.1111/j.1745-4514.2006.00079.x

    Article  Google Scholar 

  16. Kishimura, H., Tokuda, Y., Yabe, M., Klomklao, S., Benjakul, S., Ando, S.: Trypsins from the pyloric ceca of jacopever (Sebastes schlegeli) and elkhorn sculpin (Alcichthys alcicornis): isolation and characterization. Food Chem. 100, 1490–1495 (2007). https://doi.org/10.1016/j.foodchem.2005.11.040

    Article  Google Scholar 

  17. Kishimura, H., Klomklao, S., Benjakul, S., Chun, B.S.: Characteristics of trypsin from the pyloric ceca of walleye pollock (Theragra chalcogramma). Food Chem. 106, 194–199 (2008). https://doi.org/10.1016/j.foodchem.2007.05.056

    Article  Google Scholar 

  18. Kishimura, H., Klomklao, S., Benjakul, S., Chun, B.S.: Comparative study on thermal stability of trypsin from the pyloric ceca of threadfin hakeling (Laemonema Longipes). J. Food Biochem. 34, 50–65 (2010). https://doi.org/10.1111/j.1745-4514.2009.00263.x

    Article  Google Scholar 

  19. Klomklao, S., Benjakul, S., Visessanguan, W., Kishimura, H., Simpson, B.K., Saeki, H.: Trypsins from yellowfin tuna (Thunnus albacores) spleen: purification and characterization. Comp. Biochem. Physiol. B: Biochem. Mol. Biol. 144B, 47–56 (2006). https://doi.org/10.1016/j.cbpb.2006.01.006

    Article  Google Scholar 

  20. Klomklao, S., Benjakul, S., Visessanguan, W., Kishimura, H., Simpson, B.K.: Purification and characterization of trypsin from spleen of tongol tuna (Thunnus tonggol). J. Agric. Food Chem. 54, 5617–5622 (2006). https://doi.org/10.1021/jf060699d

    Article  Google Scholar 

  21. Klomklao, S., Benjakul, S., Visessanguan, W., Kishimura, H., Simpson, B.K.: Purification and characterization of trypsins from skipjack tuna (Katsuwonus pelamis) spleen. Food Chem. 100, 1580–1589 (2007). https://doi.org/10.1016/j.foodchem.2006.01.001

    Article  Google Scholar 

  22. Klomklao, S., Benjakul, S., Visessanguan, W., Kishimura, H., Simpson, B.K.: Trypsin from the pyloric ceca of bluefish (Pomatomus saltatrix). Comp. Biochem. Physiol. B: Biochem. Mol. Biol. 148B, 382–389 (2007). https://doi.org/10.1016/j.cbpb.2007.07.004

    Article  Google Scholar 

  23. Klomklao, S., Benjakul, S., Visessanguan, W., Kishimura, H., Simpson, B.K.: A 29 kDa protease from the digestive glands of Atlantic bonito (Sarda sarda): recovery and characterization. J. Agric. Food Chem. 55, 4548–4553 (2007). https://doi.org/10.1021/jf063319x

    Article  Google Scholar 

  24. Klomklao, S., Kishimura, H., Benjakul, S., Simpson, B.K., Visessanguan, W.: Cationic trypsin: a predominant proteinase in Pacific saury (Cololabis saira) pyloric ceca. J. Food Biochem. 34, 1105–1123 (2010). https://doi.org/10.1111/j.1745-4514.2010.00352.x

    Article  Google Scholar 

  25. Fuchise, T., Kishimura, H., Sekizaki, H., Nonami, Y., Kanno, G., Klomklao, S., Benjakul, S., Chun, B.S.: Purification and characteristics of trypsins from cold-zone fish, Pacific cod (Gadus macrocephalus) and saffron cod (Eleginus gracilis). Food Chem. 116, 611–616 (2009). https://doi.org/10.1016/j.foodchem.2009.02.078

    Article  Google Scholar 

  26. Kanno, G., Kishimura, H., Yamamoto, J., Ando, S., Shimizu, T., Benjakul, S., Klomklao, S., Nalinanon, S., Chun, B.S., Saeki, H.: Cold-adapted structural properties of trypsins from walleye pollock (Theragra chalcogramma) and Arctic cod (Boreogadus saida). Eur. Food Res. Technol. 233, 963–972 (2011). https://doi.org/10.1007/s00217-011-1592-8

    Article  Google Scholar 

  27. Kanno, G., Kishimura, H., Ando, S., Klomklao, S., Nalinanon, S., Benjakul, S., Chun, B.S., Saeki, H.: Structural properties of trypsin from cold-adapted fish, arabesque greenling (Pleurogrammus azonus). Eur. Food Res. Technol. 232, 381–399 (2011). https://doi.org/10.1007/s00217-010-1404-6

    Article  Google Scholar 

  28. Kanno, G., Yamaguchi, T., Kishimura, H., Yamaha, E., Saeki, H.: Purification and characteristics of trypsin from masu salmon (Oncorhynchus masou) cultured in fresh-water. Fish Physiol. Biochem. 36, 637–645 (2010). https://doi.org/10.1007/s10695-009-9336-4

    Article  Google Scholar 

  29. Kanno, G., Klomklao, S., Kumagai, Y., Kishimura, H.: A thermostable trypsin from freshwater fish Japanese dace (Tribolodon hakonensis): a comparison of the primary structures among fish trypsins. Fish Physiol. Biochem. in press (2018)

  30. Liu, Z.Y., Wang, Z., Xu, S.Y., Xu, L.N.: Two trypsin isoforms from the intestine of the grass carp (Ctenopharyngodon idellus). J. Comp. Physiol. B. 177, 655–666 (2007). https://doi.org/10.1007/s00360-007-0163-6

    Article  Google Scholar 

  31. Khangembam, B.K., Chakrabarti, R.: Trypsin from the digestive system of carp Cirrhinus mrigala: purification, characterization and its potential application. Food Chem. 175, 386–394 (2015). https://doi.org/10.1016/j.foodchem.2014.11.140

    Article  Google Scholar 

  32. Cao, M.J., Osatomi, K., Suzuki, M., Hara, K., Tachibana, K., Ishihara, T.: Purification and characterization of two anionic trypsins from the hepatopancreas of carp. Fish. Sci. 66, 1172–1179 (2000). https://doi.org/10.1046/j.1444-2906.2000.00185.x

    Article  Google Scholar 

  33. Yuan, C., Kaneniwa, M., Wang, X., Chen, S., Cheng, Y., Qu, Y., Fukuda, Y., Konno, K.: Seasonal expression of 2 types of myosin with different thermostability in silver carp muscle (Hypophthalmichthys molitrix). J. Food Sci. 71, C39–C43 (2006). https://doi.org/10.1111/j.1365-2621.2006.tb12386.x

    Article  Google Scholar 

  34. Kishimura, H., Hayashi, K.: Isolation and characteristics of trypsin from pyloric ceca of the starfish Asterina pectinifera. Comp. Biochem. Physiol. B: Biochem. Mol. Biol. 132, 485–490 (2002). https://doi.org/10.1016/S1096-4959(02)00062-3

    Article  Google Scholar 

  35. Lowry, O.H., Rosebrough, N.J., Farr, A.L., Randall, R.J.: Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 265–273 (1951)

    Google Scholar 

  36. Laemmli, U.K.: Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680–685 (1970). https://doi.org/10.1038/227680a0

    Article  Google Scholar 

  37. Hummel, B.C.W.: A modified spectrophotometric determination of chymotrypsin, trypsin, and thrombin. Can. J. Biochem. Physiol. 37, 1393–1399 (1959). https://doi.org/10.1139/o59-157

    Article  Google Scholar 

  38. Klomklao, S., Benjakul, S., Visessanguan, W.: Comparative studies on proteolytic activity of spleen extracts from three tuna species commonly used in Thailand. J. Food Biochem. 28, 355–372 (2004). https://doi.org/10.1111/j.1745-4514.2004.05203.x

    Article  Google Scholar 

  39. Jones, D.A., Kamarudin, M.S., Vay, L.L.: The potential for replacement of live feeds in larval culture. J. World Aquacult. Soc. 24, 199–210 (1993). https://doi.org/10.1111/j.1749-7345.1993.tb00009.x

    Article  Google Scholar 

  40. Ruan, G.L., Li, Y., Gao, Z.X., Wang, H.L., Wang, W.M.: Molecular characterization of trypsinogens and development of trypsinogen gene expression and tryptic activities in grass carp (Ctenopharyngodon idellus) and topmouth culter (Culter alburnus). Comp. Biochem. Physiol. B: Biochem. Mol. Biol. 155, 77–85 (2010). https://doi.org/10.1016/j.cbpb.2009.10.005

    Article  Google Scholar 

  41. Klomklao, S., Benjakul, S., Kishimura, H., Chaijan, M.: 24 kDa Trypsin: a predominant protease purified from the viscera of hybrid catfish (Clarias macrocephalus × Clarias gariepinus). Food Chem. 129, 739–746 (2011). https://doi.org/10.1016/j.foodchem.2011.05.014

    Article  Google Scholar 

  42. Bkhairia, I., Khaled, H.B., Ktari, N., Miled, N., Nasri, M., Ghorbel, S.: Biochemical and molecular characterisation of a new alkaline trypsin from Liza aurata: structural features explaining thermal stability. Food Chem. 196, 1346–1354 (2016). https://doi.org/10.1016/j.foodchem.2015.10.058

    Article  Google Scholar 

  43. Ahsan, M.N., Funabara, D., Watabe, S.: Molecular cloning and characterization of two isoforms of trypsinogen from anchovy pyloric ceca. Mar. Biotechnol. 3, 80–90 (2001). https://doi.org/10.1007/s101260000055

    Article  Google Scholar 

  44. Klomklao, S., Benjakul, S.: Two trypsin isoforms from albacore tuna (Thunnus alalunga) liver: purification and physicochemical and biochemical characterization. Int. J. Biol. Macromol. 107, 1864–1870 (2018). https://doi.org/10.1016/j.ijbiomac.2017.10.059

    Article  Google Scholar 

  45. Walsch, K.A.: Trypsinogens and trypsins of various species. Method Enzymol. 19, 41–46 (1970). https://doi.org/10.1016/0076-6879(70)19006-9

    Article  Google Scholar 

  46. Hermodson, M.A., Ericsson, L.H., Neurath, H., Walsh, K.A.: Determination of the amino sequence of porcine trypsin by sequenator analysis. Biochemistry 12, 3146–3153 (1973). https://doi.org/10.1021/bi00741a002

    Article  Google Scholar 

  47. Pinsky, K.S.: Differential regulation of trypsinogen: mRNA translation full-length mRNA sequences encoding two oppositely charged trypsinogen isoenzymes in the dog pancreas. Mol. Cell. Biol. 5, 2669–2676 (1985). https://doi.org/10.1128/MCB.5.10.2669

    Article  Google Scholar 

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Authors and Affiliations

  1. Laboratory of Marine Chemical Resource Development, Graduate School of Fisheries Sciences, Hokkaido University, Hakodate, Hokkaido, 041-8611, Japan

    Kenichiro Abe

  2. Department of Food Production and Environmental Management, Faculty of Agriculture, Iwate University, Morioka, 020-8550, Japan

    Chunhong Yuan

  3. Laboratory of Marine Chemical Resource Development, Faculty of Fisheries Sciences, Hokkaido University, Hakodate, Hokkaido, 041-8611, Japan

    Yuya Kumagai & Hideki Kishimura

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  1. Kenichiro Abe
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  4. Hideki Kishimura
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Correspondence to Hideki Kishimura.

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Abe, K., Yuan, C., Kumagai, Y. et al. The Potential of Freshwater Fish Viscus from Silver Carp Hypophthalmichthys molitrix for Trypsin Source. Waste Biomass Valor 11, 3971–3978 (2020). https://doi.org/10.1007/s12649-019-00717-7

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