Isolation, properties and the use of a chymosin-like enzyme from harp seal (Pagophilus groenlandicus)

Shamsuzzaman, Kazi M. (1983) Isolation, properties and the use of a chymosin-like enzyme from harp seal (Pagophilus groenlandicus). Doctoral (PhD) thesis, Memorial University of Newfoundland.

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Abstract

Four zymogens of acidic proteases A, B, C, and C’ were isolated from the gastric mucosa of harp seal (Pagophilus groenlandicus) by ion exchange chromatography on a DEAE Sephadex A-50 column. Zymogens A and C were further purified by affinity chromatography using carbobenzoxy-D-phynylalanine-triethylene-tetramine and gel filtration on a Sephadex G-100 column. Certain physical and catalytic properties of proteases A and C were compared with those of calf chymosin (E.C.3.4.23.4) and porcine pepsin (E.C.3.4.23.1). -- Zymogen C and the corresponding enzyme were homogeneous on analytical polyacrylamide gel electrophoresis. Zymogen A was homogeneous as judged by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate and high performance liquid chromatography but was heterogenous as judged by polyacrylamide gel electrophoresis in the absence of SDS. Zymogens A and C had molecular weights of 26,300 ± 1540 and 37,100 ± 1075 daltons respectively as estimated by gel filtration but 33,800 + 1800 and 44,000 + 2100 daltons respectively as estimated by SDS polyacrylamide gel electrophoresis. Protease A had an isoelectric point of 4.90. The energies of activation of protease A and calf chymosin were 15.6 and 13.1 Kcal/more calculated from the milk clotting activity of the enzymes. -- Protease A was similar to calf chymosin with respect to several criteria. It had a higher ratio of milk clotting to proteolytic activity than those of seal protease C and porcine pepsin, clotted milk up to pH 7.0, and had a pH optimum of 2.2-3.5 for hemoglobin hydrolysis. It did not inactivate ribonuclease, had very low activity on APDT and lost activity in 6M urea. These results indicate that protease A is chymosin-like rather than pepsin-like. -- Experimental Cheddar cheese made with protease A, calf rennet, crude seal gastric protease (SGP) and Fromase (Musor miehei protease) had comparable yields and proximate compositions. Sensory analysis of experimental cheeses after 30 weeks of aging showed that the product made with crude SGP had higher sensory scores than those made with the other coagulants. Chemical analyses showed more extensive degradation of protein in cheeses made with calf rennet and Fromase than in the cheeses made with the seal proteases. The electrophoretic patterns of the proteins from cheeses made with seal proteases were qualitatively similar to those of calf rennet cheese. It was concluded that protease A and seal gastric proteases are promising as rennet subtitutes.

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