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Purification and Characterization of Milk Lipase^{1, 2,} II. Characterization of the Purified Enzyme

Department of Dairy Husbandry, University of Nebraska, Lincoln

ABSTRACT

The enzymic characteristics of a pure and homogeneous milk lipase were studied. The enzyme was best stored in a frozen state and was extremely unstable at 20, 30, 37, and 45 C. The enzyme exhibited a single pH optimum of 9.0 to 9.2, and its optimum temperature was about 37 C.

The enzyme hydrolyzed both milk fat and tributyrin. Upon storage at 37 C the loss of the activities toward milk fat and tributyrin was of the same magnitude. Also, sodium para-chloromercuribenzoate, iodoacetic acid, and N-ethyl maleimide inhibited the activity of the enzyme toward the two substrates to the same extent, indicating that the lipolysis of both the substrates may be catalyzed by a single enzyme.

The enzyme showed little or no activity on ethyl acetate, ethyl decanoate, ortho-nitrophenyl acetate, and para-nitrophenyl laurate esters in solution, but hydrolyzed the emulsions of milk fat, olive oil, and tributyrin very actively, thus demonstrating the characteristics of a true lipase. It appears to be an enzyme with a high specificity for glycerol esters.

The presence of milk constituents seemed to exert an inhibitory effect upon the enzymic activity. With the natural emulsion substrates a lag phase was observed in the time-activity curves for a period of 20 min, after which the reaction rate increased with time up to 2 hr.

¹ Published with the approval of the Director as paper no. 1322, Journal Series, Nebraska Agricultural Experiment Station, Lincoln.

² Data taken from a thesis presented by R. C. Chandan in partial fulfillment of the requirements of the Doctor of Philosophy degree, University of Nebraska. February, 1963.