藻红蓝蛋白裂合异构酶E基因突变体的构建、表达与酶活性检测

CLONING,EXPRESSING AND ACTIVITIES STUDY OF MASTIGOCLADUS LAMINOSUS PECE MUTATED GENES

通过诱变分别得到PecE的N端 ,C端和中间位置缺失突变的一系列突变基因的编码产物 ,测定了这 5种缺失蛋白质的酶活性的大小。实验结果显示 :来自层理鞭枝藻 (M laminosus,UTEX 1931)的PecE[简称PecE(UTEX1931) ]与来自层理鞭枝藻 (M laminosus, PCC 76 0 3)的PecE[简称PecE(PCC 76 0 3) ]有 81%的同源性。PecE(UTEX1931) PecF(PCC 76 0 3)的酶催化活性与PecE(PCC 76 0 3) PecF(PCC 76 0 3)的酶催化活性相当。PecE(UTEX 1931)的N端缺失 2 2个氨基酸的蛋白质显示出了约 2 0 %的催化活性 ;而N端缺失 36个氨基酸、5 6个氨基酸的蛋白质 ,C端缺失 2 3个氨基酸的蛋白质和PecE(PCC 76 0 3)中间缺失 2 7个氨基酸的蛋白质均丧失活性 。

Phycoerythrocyanin(PEC)is an intergral compent of the Phycobilisomes(PBS). Unlike other phycobiliproteins, isolated PEC shows a remarkable photochemistry. It is photoreversibly photochromic, and the reactivity has been related to the unusual chromophore-PVB bound to Cys 84 on the subunit via a thioether linkage. For PVB, the problem has recently been clarified by the identificatin of a new enzymatic activity of the two lyase subunits (PecE, PecF), which genes are located in the pec operon downstream from the structural( pecB, A )and linker genes( pecC ). PecE and PecF, the product of two phycoerythrocyanin lyase genes( pecE and pecF )of Mastigocladus laminosus ( fischerella ), catalyze two reactions:(1) the regiospecific addition of phycocyanobilin (PCB) to Cys 84 of the phycoerythrocyanin subunit(PecA), and (2) the isomerization of the PCB to the phycoviolobilin (PVB) chromophore. In the pnesent study, we have used deletion and mutagenesis to produce a seires of mutated forms of PecE. The mutated PecE (PecEn22, PecEn36, PecEn56;PecEc23, PecEm27)which means losing 22, 36, 56 amino acid in N end;23, amino acid in C end; and 27 amino acid in middle, were got with molecular design. First, the PCR products were purified and digested with Smal and Xhol, then recombined with pBluescript vector and transformed into the E. coli TG1 strain. The mutated fragments were inserted into pBluescript vector and were confirmed with sequencing. Meanwhile the mutated fragments were subcloned into another expression vector pET30. After induction of IPTG, the mutated recombinants of PecE and the PecF are highly expressed in E. Coli BL21. In vitro, when we combined the mutated PecE and PecF with PCB and PecA together, we got a series of spectra characterizing the enzymatic reconstitutions. Compared to the highly reversible photochemistry of the natural α PEC, the variations in relative reconstitution activity were confirmed. Incubation of PecE, PecF with PCB resulted in an absorption increase at 570 nm which has the characteristic absorption properties, the same as the native α PEC. The absorption blue shiffed to 500nm after irradiation with 570nm light, and the remarkable different absorption were measured and used to denote the native abilities. Compared to PecE between UTEX1931 and PCC7603, we can find that they displayed high homology (81%)in level of amino acid. Based on this reason, the enzymatic activities were measured and PecE(UTEX1931)exhibited 80% activity compared to PecE(PCC7603)which exhibited 100% activity. The mutated PecE (UTEX 1931)which lost 36, 56 amino acid in N end , 23 amino acid in C end lost the enzymatic activities, which means that there must exist the important residue in these lost amino acid. The mutated PecE(PCC 7603)which lost 27 amino acid in the middle lost the enzymatic activity, which means that the lost of these 27 amino acid had destroyed the original structure of PecE inevitablely. However, the mutated PecE (UTEX 1931)which lost 22 amino acid in N end had enzymatic activity of 20%. We can conclude that maybe the lost of 1-22 amino acid had not destroyed the structure as a whole, but induced the change on local structure. The results provide the important information on active center and space structure of enzyme PecE, and the relationship between the structure and enzymatic activity of this enzyme was arisen from the results.