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Fish Physiology and Biochemistry

, Volume 36, Issue 4, pp 1041–1060 | Cite as

Purification and properties of digestive lipases from Chinook salmon (Oncorhynchus tshawytscha) and New Zealand hoki (Macruronus novaezelandiae)

  • Ivan Kurtovic
  • Susan N. Marshall
  • Xin Zhao
  • Benjamin K. Simpson
Article

Abstract

Lipases were purified from delipidated pyloric ceca powder of two New Zealand-sourced fish, Chinook salmon (Oncorhynchus tshawytscha) and hoki (Macruronus novaezelandiae), by fractional precipitation with polyethylene glycol 1000, followed by affinity chromatography using cholate-Affi-Gel 102, and gel filtration on Sephacryl S-300 HR. For the first time, in-polyacrylamide gel activity of purified fish lipases against 4-methylumbelliferyl butyrate has been demonstrated. Calcium ions and sodium cholate were absolutely necessary both for lipase stability in the gel and for optimum activity against caprate and palmitate esters of p-nitrophenol. A single protein band was present in native polyacrylamide gels for both salmon and hoki final enzyme preparations. Under denaturing conditions, electrophoretic analysis revealed two bands of 79.6 and 54.9 kDa for salmon lipase. It is proposed that these bands correspond to an uncleaved and a final form of the enzyme. One band of 44.6 kDa was seen for hoki lipase. pI values of 5.8 ± 0.1 and 5.7 ± 0.1 were obtained for the two salmon lipase forms. The hoki lipase had a pI of 5.8 ± 0.1. Both lipases had the highest activity at 35°C, were thermally labile, had a pH optimum of 8–8.5, and were more acid stable compared to other fish lipases studied to date. Both enzymes were inhibited by the organophosphate paraoxon. Chinook salmon and hoki lipases showed good stability in several water-immiscible solvents. The enzymes had very similar amino acid composition to mammalian carboxyl ester lipases and one other fish digestive lipase. The salmon enzyme was an overall better catalyst based on its higher turnover number (3.7 ± 0.3 vs. 0.71 ± 0.05 s−1 for the hoki enzyme) and lower activation energy (2.0 ± 0.4 vs. 7.6 ± 0.8 kcal/mol for the hoki enzyme) for the hydrolysis of p-nitrophenyl caprate. The salmon and hoki enzymes are homologous with mammalian carboxyl ester lipases.

Keywords

Bile salt Calcium Chinook salmon Enzyme inhibition Hoki Lipase Organic solvents Zymography 

Notes

Acknowledgments

This study was supported by funds from the New Zealand Foundation for Research, Science & Technology (FRST) under contracts C02X0301 and C02X0806. We thank The New Zealand King Salmon Co Ltd. for providing salmon tissue samples and Sealord Ltd. for providing hoki tissue samples. This research was facilitated using infrastructure provided by the Australian Government through the National Collaborative Research Infrastructure Strategy (NCRIS), and we thank Australian Proteome Analysis Facility, Macquarie University for amino acid composition and N-terminal analyses.

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Copyright information

© Springer Science+Business Media B.V. 2010

Authors and Affiliations

  • Ivan Kurtovic
    • 1
    • 2
  • Susan N. Marshall
    • 2
  • Xin Zhao
    • 1
  • Benjamin K. Simpson
    • 3
  1. 1.Department of Animal ScienceMcGill University (Macdonald Campus)Ste. Anne de BellevueCanada
  2. 2.The New Zealand Institute for Plant & Food Research LimitedNelsonNew Zealand
  3. 3.Department of Food Science and Agricultural ChemistryMcGill University (Macdonald Campus)Ste. Anne de BellevueCanada

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