Purification and Characterization of Lipase in Buckwheat Seed

• Published in: Journal of agricultural and food chemistry 2004-12, Vol.52 (24), p.7407-7411
• Main Authors: Suzuki, Tatsuro, Honda, Yutaka, Mukasa, Yuji
• Format: Article
• Language: English
• Subjects: Enzyme Stability; Fagopyrum - enzymology; Flour - analysis; Hot Temperature; Hydrogen-Ion Concentration; Isoenzymes - isolation & purification; Isoenzymes - metabolism; Lipase - isolation & purification; Lipase - metabolism; Molecular Weight; Seeds - enzymology; Substrate Specificity
• ISSN: 0021-8561; 1520-5118
• DOI: 10.1021/jf049271+

To obtain basic information about enzymatic deterioration of buckwheat flour, triacylglycerol lipase (LIP; EC 3.1.1.3) was purified from buckwheat seed. The LIP consisted of two isozymes, LIP I and LIP II, and they were purified with purification folds of 60 and 143 with final specific activities of 0.108 and 0.727 μmol of fatty acid released per minute per milligram of protein at 30 °C using triolein as a substrate. Molecular weights were estimated to be 150 (LIP I) and 28.4 kDa (LIP I) by gel filtration and 171 (LIP I) and 26.5 kDa (LIP II) by SDS−PAGE. Optimal pHs of LIP activities were 3.0 (LIP I) and 6.0 (Lip II) using triolein as a substrate. Both LIP I and II reacted in the acidic pH range. Optimal temperatures were 30 (LIP I) and 40 °C (LIP II), and both LIP I and II were stable below 30 °C when p-nitrophenyl-laurate was used as a substrate. However, they were inactivated above 60 °C. On the other hand, when triolein was used as a substrate, optimal temperatures were 30 °C for both LIP I and II, and they retained 40% of their activity after a 4 h incubation of enzymes at 70 °C. LIP I and II had higher activity against triolein than monoolein or tri/monopalmitin. Most of the LIP activity was distributed in the embryo. Keywords: Buckwheat; lipase; purification; seed; fatty acid; lipid; organ distribution