Sidebottom C M, Charton E, Dunn P P, Mycock G, Davies C, Sutton J L, Macrae A R, Slabas A R
Unilever Research, Colworth Laboratory, Sharnbrook, England.
Eur J Biochem. 1991 Dec 5;202(2):485-91. doi: 10.1111/j.1432-1033.1991.tb16398.x.
We have purified and examined the substrate specificity of four lipases from two strains of the mould Geotrichum candidum, ATCC 34614 and CMICC 335426. We have designated the lipases I and II (ATCC 34614), and A and B (CMICC 335426). The enzymes are monomeric and have similar molecular masses and pI. Thus, lipases I and II have native molecular masses of 50.1 kDa and 55.5 kDa, and pI of 4.61 and 4.47, respectively. Lipases A and B are very similar to lipases I and II with native molecular masses of 53.7 kDa and 48.9 kDa, and pI of 4.71 and 4.50, respectively. Treatment with endo-beta-N-acetylglucosaminidase caused a reduction in molecular mass of approximately 4.5 kDa for all four lipases, indicating that these enzymes are glycosylated. Western blotting shows that the lipases are related. However, lipase B from CMICC 335426 shows a remarkable specificity for unsaturated substrates with a double bond at position 9 (cis configuration), and this specificity is not exhibited by the other three lipases. No lipase of this unique specificity has previously been purified to homogeneity. Structural studies using these four lipases should allow insight into the molecular basis of this remarkable specificity.
我们已经从白地霉的两株菌株（ATCC 34614和CMICC 335426）中纯化并检测了四种脂肪酶的底物特异性。我们将脂肪酶分别命名为I和II（ATCC 34614），以及A和B（CMICC 335426）。这些酶是单体，具有相似的分子量和等电点。因此，脂肪酶I和II的天然分子量分别为50.1 kDa和55.5 kDa，等电点分别为4.61和4.47。脂肪酶A和B与脂肪酶I和II非常相似，天然分子量分别为53.7 kDa和48.9 kDa，等电点分别为4.71和4.50。用内切β-N-乙酰氨基葡萄糖苷酶处理后，所有四种脂肪酶的分子量均降低了约4.5 kDa，表明这些酶是糖基化的。蛋白质印迹分析表明这些脂肪酶具有相关性。然而，CMICC 335426的脂肪酶B对9位具有双键（顺式构型）的不饱和底物表现出显著的特异性，而其他三种脂肪酶则没有这种特异性。此前尚未将具有这种独特特异性的脂肪酶纯化至同质。使用这四种脂肪酶进行结构研究应有助于深入了解这种显著特异性的分子基础。
