【作者】 王锋；

【导师】 赵开弘；

【作者基本信息】 华中科技大学 ， 生态学， 2006， 硕士

【摘要】 藻胆蛋白是蓝藻中的捕光蛋白,其生物合成的重要一步是藻胆色素与脱辅基蛋白的连接。大多数藻胆色素的正确连接都需要结合位点专一和对色素的构象有选择性的裂合酶来催化完成,但是这方面的报道不是很多。本实验室通过同源性分析获得的由编号为alr0617基因编码的蛋白为β-CPC、β-PEC中的Cys-84,α-APC和β-APC中的Cys-82与PCB的连接的催化酶,命名为藻蓝蛋白裂合酶CpeS。设计引物通过PCR技术从藻总DNA中扩增藻蓝蛋白β亚基基因cpcB,其编码的蛋白CpcB在CpeS催化下与PCB进行体外重组,找到CpcB的最佳表达条件:温度35°C,时间6h,LB培养基。CpeS存在的情况下,通过体内重组方式得到色素蛋白PCB-β-CPC和PCB-β-PEC,但是却得不到色素蛋白PCB-β-PEC(C84S);没有CpeS存在的情况下,只有11.8%的PCB-β-CPC和6%的PCB-β-PEC生成。这些结果表明:藻蓝蛋白裂合酶起到了催化PCB与Cys-84连接的作用。构建CpeS(H22V)突变体,通过体内重组的方式研究了组氨酸残基突变对CpeS酶活性的影响,结果表明该残基对于CpeS的酶活性具有一定的影响,其活性为野生型CpeS酶活性的14.5%。使用胃蛋白酶对天然色素蛋白β-CPC、β-PEC和APC以及重组色素蛋白PCB-CpcB(C155I)、PCB-PecB(C155I)、PCB-α-APC和PCB-β-APC进行相同条件的水解并得到各自的色素肽。酸性尿素变性实验证明得到的色素肽中的PCB没有被破坏。色素肽的高效液相色谱分析结果表明:藻蓝蛋白裂合酶CpeS催化PCB与β-CPC和β-PEC中Cys-84的连接;APC由α-APC和β-APC中构成,CpeS催化PCB与α-APC和β-APC中Cys-82的连接。更多还原

【Abstract】 Phycobiliproteins are light-harvesting proteins present in cyanobacteria. The most important step in phycobilin biosynthesis is the phycobilin addition to the apophycobiliproteins. In vivo, the correct attachment of most chromophores is catalyzed by binding-site and chromophore-specific lyases, of which only few have hitherto been characterized.By homology analyses, the protein encoded by gene alr0617 was proved to be the lyase of Cys-84 ofβ-CPC andβ-PEC, Cys-82 ofα-APC andβ-APC. Name is CpeS.The gene cpcB from Anabaena PCC 7120 were cloned with vector and verified by sequencing. The overexpressed encoded protein by cpcB were direct used to reconstitution with PCB in vitro. As a result, the better expressive condition is: temperature is 35°C, time is 6 hours and LB culture medium.With CpeS, the holophycobiliproteins PCB-β-CPC and PCB-β-PEC were synthesized in vivo reconstitution. But PCB-β-PEC(C84S) were not obtained. Without CpeS, the productivity of PCB-β-CPC is 11.8% and PCB-β-PEC is 6%.It was shown that CpeS is the lyase of Cys-84 ofβ-CPC andβ-PEC.To study the structure-function relationships and the effect of the histidine residues to the CpeS of phycocyanin, CpeS(H22V) mutants were constructed. The lyase activity of these mutants were tested by reconstitution in vivo to study the effects of histidine to CpeS. Comparing with the wild CpeS, the lyase activity of CpeS(H22V) is 14.5%.The phycocyanobilin peptides were obtained from the naturalβ-CPC,β-PEC, APC and reconstituted PCB-CpcB(C155I),PCB-PecB ( C155I ) ,PCB-α-APC,PCB-β-APC hydrolyzed by pepsin, respectively. After denaturation in the dark with urea in the presence of hydrochloric acid (pH=2), the PCB was not destroyed.With HPLC, we make a comparison of the retention time between the natural and the reconstituted peptides. The results showed that CpeS catalyzes PCB attachment sites at Cys-84 inβ-CPC andβ-PEC, Cys-82 inα-APC andβ-APC. APC were consist ofα-APC andβ-APC.更多还原