"Knead" Methad for the Measurement of Peanuts Lipase

Abstract

A crude lipase was prepared form peanut seed. Some examinations on the nature of crude enzyme and the measurement of lipase activity were carried out. 1. "Knead" method: Cotyledons of germinating or ripening seed were homogenized with water, defatted with acetone and ether, and fine powder was obtained. This powder was put into a test tube and mixed thoroughly with water (in case of hydrolysis) or glycerin (in case of synthesis). Olive oil or oleic acid under test was added at zero time and the mixture was quickly kneaded with the glass rod. After the reaction period, lipid in incubated mixtures was titrated with ethanolic KOH. Lipase activity was expressed by the titer of KOH which was corrected by appropriate blanks. 2. The standard error of hydrolysing activity in the knead method was ±0.01, while, in other methods, the corresponding figures were ±0.086 and ±0.068. Hydrolysing activity was in proportion to the amount of added crude enzyme in reaction mixtures, so lipase activity per 1 unit of crude enzyme did not change. By drying in the desiccator in which silica gel was enclosed, crude enzyme could be stored at room temperature for five days without considerable lessening in hydrolysing activity. The optimum pH of hydrolysing activity of the enzyme prepared from 3-day-old and 6-day-old seedlings was 7.0 and 5.6, respectively. When distilled water was used as a solvent instead of bufer soltution, the enzyme activity was egual to that at the optimum pH in case of buffer solution. In synthesis, when 1 ml. of glycerin was added to reaction mixtures, only one-half as much activity was shown as when 0.5 ml. was added. So it is evident from these results that the proposed method is sufficently applicable to practical purposes.