粘红酵母ATP:柠檬酸裂解酶基因的克隆表达和酶学性质

Cloning,Expression and Characteristics of ATP: Citrate Lyase from Rhodotorula Glutinis

从粘红酵母(Rhodotorula glutinis)中克隆得到ATP:柠檬酸裂解酶基因两个亚基acl1和acl2,转化毕赤酵母(Pichia pastoris)GS115进行诱导表达,通过Ni柱纯化目的蛋白,以实时波长扫描对重组蛋白的酶学性质进行测定。SDS-PAGE分析表明,两个亚基ACL1和ACL2的分子量分别为66 kDa和55 kDa。重组蛋白的酶学测定表明,经毕赤酵母表达后,单个亚基酶活性不到1.0 U.mg-1,双亚基混合后具有较高的酶活性,当混合质量比为1∶1时全酶的酶比活力达到最大,为6.5 U.mg-1,酶反应的最佳条件为:pH值8.5、37℃反应10 min。为提高油料作物油脂含量提供了新的转基因材料。

The ATP：citrate lyase gene sequence including acl1 and acl2 were first obtained by PCR from Rhodotorula glutinis,and then were transformed into Pichia pastoris GS115 strain through electroporation.The expression proteins were purified to homogeneity to high yield.SDS-PAGE Analysis suggested that the relative molecular weight of the subunit polypeptides ACL1 and ACL2 were about 66 kDa and 55 kDa,respectively.Enzymatic determination of recombinant protein showed that the highest ACL specific activity was 6.5 U·mg-1 when the mass ratio of the two subunit proteins was 1∶1,both gene products were essential for ATP：citrate lyase activity.And it also indicated the recombinant enzyme had optimal activity when it reacted for 10 min at 37 ℃ with pH value of 8.5.This provides a new genetic material that can express in oil crops in order to substantially increase the lipid accumulation.