【作者】 张玲；

【导师】 赵开弘；

【作者基本信息】 华中科技大学 ， 环境科学， 2006， 硕士

【摘要】 目前在藻胆蛋白裂合酶研究方面,虽然已知CpcE/F只是催化藻蓝色素PCB与脱辅基蛋白CpcA的连接,但对该酶的催化机理的研究还是空白。本研究从氨基酸序列入手,通过构建缺失突变、定点突变的方法对其功能区域及其关键性的氨基酸残基进行分析。鉴于,α-APC中Cys-81和β-APC中Cys-82与PCB的连接的催化酶还未见报道,本课题拟通过同源性分析获得与已知裂合酶同源的基因,用分子克隆的方法克隆基因并表达相应的蛋白,用于体外重组实验。本文通过比较CpcE/F和PecE/F的氨基酸序列,对CpcE/F进行缺失和定点突变,共构建七个突变体:CpcF(1-160),CpcF(10-213),CpcF(I9K),CpcE(1-274),CpcE(L276D),CpcE(1-272),CpcE(L275D)。CpcF(1-160)主要以包涵体形式存在而不能与CpcE结合,CpcF(I9K)对酶活性基本无影响,CpcF(10-213)缺失的片段可能是高级结构上比较关键的区域,CpcE(1-272)活性仅17~28%,缺失的片段可能参与活性中心的构建,CpcE(L275D)和CpcE(L276D)两个定点突变体表明L276非常重要。对鱼腥藻PCC7120的两个亚基α-APC,β-APC进行体外重组实验,结果表明EDTA与去污剂Triton X-100抑制酶的活性。找出ApcA最佳反应条件为70mmol/L左右的Mg2+、0.2mmol/L左右的ME,终浓度为800~1000mmol/L的磷酸钾缓冲液(pH 8.0),最佳温度为30℃~37℃。ApcB体外重组不能成功,与PCB-pET-Alr0617进行重组实验,能部分成功,可能是由于ApcB重组对PCB构象要求很严,游离的PCB构象不适合。更多还原

【Abstract】 As we know now, CpcE/F is the lyase catalyzing the attachment of phycocyanobilin to phycobiliprotein, but the mechanism is still unknown. In this work, we aimed to validate this proposal and to map in more detail the functions to the protein sequence, using the ways of deletion mutation, site-directed mutations . What’s more, Since there was no report about the lyase for Cys-81 ofα-APC and Cys-82 ofβ-APC, our work mainly deals with the finding and characterization of this (or these) lyase. Some gene fragments was found by homology analyses, these gene fragments were cloned and expressed in E.coli for further research.Through analyzing CpcE/F and PecE/F, seven deletion mutants: CpcF(1-160), CpcF(10-213), CpcF(I9K), CpcE(1-274), CpcE(L276D), CpcE(1-272), CpcE(L275D) of the CpcE/F of the cyanobacterium, Mastigocladus laminosus PCC7603, were constructed to probe the functional domains. A 53 aa truncation in CpcF C-terminus caused a loss of the ability to form a complex with CpcE, CpcF(I9K) had not influence to activity of the lyase, CpcE(1-272) had only 17~22% activity of the wild type,possibly due to misfolding. the deletion interferes with the refolding of CpcE. According to the activity of CpcE(L275D) and CpcE(L276D),L276 is a critical residue.Through the experimences ofα-APC andβ-APC in the reconstitution system in vitro, it can be proved that the reaction was mostly quenched by the detergent Triton X-100 and EDTA. The best reaction conditions were 0.2mM mercaptoethanol,70mM Mg2+,800~1000mM Phosphate buffer(pH8.0). The optimum reaction temperature was 30~37°C. It can’t succeed in the reconstitution system ofβ-APC in vitro and only partly succeeded when reconstitution with PCB-lyase-84. Maybe it was because of the construction of PCB.更多还原