Purification and properties of digestive lipases from Chinook salmon (Oncorhynchus tshawytscha) and New Zealand hoki (Macruronus novaezelandiae).
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.
Kurtovic
Ivan
I
Department of Animal Science, McGill University (Macdonald Campus), 21,111 Lakeshore Road, Ste. Anne de Bellevue, QC, H9X 3V9, Canada, Ivan.Kurtovic@plantandfood.co.nz.
Marshall
Susan N
SN
Zhao
Xin
X
Simpson
Benjamin K
BK
D000595:Amino Acid Sequence; D000818:Animals; D002118:Calcium; D002384:Catalysis; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D004064:Digestive System / Q000201:enzymology*; D005590:Fractional Precipitation; D048388:Gadiformes / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D006923:Hymecromone / Q000031:analogs & derivatives / Q000378:metabolism; D007700:Kinetics; D008049:Lipase / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism*; D008969:Molecular Sequence Data; D010261:Paraoxon / Q000494:pharmacology; D012474:Salmon / Q000378:metabolism*; D020539:Sequence Analysis, Protein; D020358:Sodium Cholate; D013696:Temperature
C056366:4-methylumbelliferyl butyrate; D006923:Hymecromone; D008049:Lipase; D020358:Sodium Cholate; D010261:Paraoxon; D002118:Calcium