【作者】 苏平；

【导师】 赵开弘；

【作者基本信息】 华中科技大学 ， 环境工程， 2008， 博士

【摘要】 蓝藻是光合作用微生物,在适宜水体中会大量生长繁殖,形成蓝藻水华。藻胆蛋白是藻类光合作用捕光复合物的功能组份,藻胆蛋白是存在于蓝藻、红藻和隐藻中的一类同源蛋白家族。蓝藻体内,藻胆蛋白生物合成的最后一步为藻胆色素与脱辅基藻胆蛋白的连接,藻胆色素的正确连接一般都需要特异的裂合酶来催化完成。目前,已知藻红蓝蛋白裂合酶PecE/F催化藻蓝色素PCB异构化形成PVB并与脱辅基蛋白PecA连接;藻蓝蛋白裂合酶CpcE/F催化藻蓝色素PCB与脱辅基蛋白CpcA的连接。但是对于核膜连接蛋白ApcE、藻蓝蛋白和藻红蓝蛋白中CpcB和PecB、变藻蓝蛋白APC各亚基的生物合成,目前还未见报道。本研究应用体外重组和大肠杆菌体内重组的方法,以鱼腥藻PCC7120为材料,对ApcE、CpcB、PecB以及APC各亚基的生物合成进行了研究。研究了核膜连接蛋白脱辅基蛋白ApcE和ApcE(1-240)与PCB的体外重组。研究表明:ME是重组反应所必需的,4mol/L尿素提高了核膜连接蛋白重组产物的产率,蔗糖的存在有利于催化反应。体外重组最佳的反应体系是:20mmol/L磷酸钾缓冲液(简称:KPB),0.1mol/L NaCl,4mol/L尿素,200mmol/L蔗糖,0.1mmol/L ME, pH 7.2。重组色素蛋白LCM(1-240)经过Ni2+螯合亲和层析提纯后,紫外吸收和荧光光谱具有661nm/672nm的特征峰;其圆二色光谱的极值为662、604、383和347nm;通过光谱计算出重组产物LCM(1-240)的紫外可见吸收比、摩尔消光系数和荧光量子产率分别为4.1、93000 M-1 cm-1和0.43;以上均显示了特征藻胆蛋白的性质。LCM(1-240)的聚集状态分析显示,重组色素蛋白主要以二聚体的形式存在。同时我们研究了ApcE(1-240)与PCB反应的动力学参数,得到其Km=1.3μmol/L、Vmax=9.6nmol/L·s-1、kcat=9.6×10-4 s-1。LCM(1-240)的尿素变性和Zn电泳显示,His6-ApcE(1-240)与PCB的连接是正确的共价连接。研究了藻蓝蛋白和藻红蓝蛋白脱辅基蛋白CpcB(C155I)和PecB(C155I)与PCB的大肠杆菌体内重组和体外重组。本研究利用双克隆载体,将脱辅基蛋白CpcB(C155I)( PecB(C155I)、CpcB、PecB)、裂合酶CpeS、血红素氧化酶HO1、胆绿素还原酶PcyA在大肠杆菌中共同表达,重组后色素蛋白具有特征的紫外可见吸收和荧光光谱。其中,所得到的色素蛋白PCB-CpcB(C155I)和PCB-PecB(C155I)的特征峰分别为619nm/643nm和602nm/630nm。色素蛋白的酸性尿素变性实验表明,变性后紫外吸收峰的位置在662nm左右,证明所连接的色素为PCB。在紫外光的照射下色素蛋白可以产生Zn荧光,证明PCB与脱辅基蛋白是正确的共价连接。根据光谱所得到的一系列的光谱参数(可见紫外吸收比、荧光量子产率、摩尔消光系数)都显示了色素蛋白具有天然藻胆蛋白的性质。同时我们还利用胰蛋白酶进行色素蛋白PCB与CpcB(C155I)和PecB(C155I)的水解实验,水解后的色素肽的高效液相色谱与从天然鱼腥藻PCC7120中所得到的CpcB和PecB水解多肽的HPLC图谱相近,可以证明利用大肠杆菌体内重组所得到的色素蛋白具有天然藻胆蛋白的性质。另外,CpeS催化CpcB(C84A)和PecB(C84S)与PCB的连接时,没有正确的产物生成。以上研究均表明了CpeS可以催化CpcB(C155I)、PecB(C155I)、CpcB和PecB中Cys-84与PCB的偶联。参照CpeS催化CpcB(C155I)和PecB(C155I)与PCB的体外重组的最佳反应体系,将天然的CpcB(PecB)与PCB进行重组,得到的色素蛋白与PCB-CpcB(C155I)和PCB-PecB(C155I)相比,特征峰有2～3nm的红移。研究了变藻蓝蛋白脱辅基蛋白ApcA、ApcB、ApcA2、ApcD、ApcF与PCB的大肠杆菌体内重组。本研究利用双克隆载体,将脱辅基蛋白ApcA(ApcB、ApcA2、ApcD、ApcF)、裂合酶CpeS、血红素氧化酶HO1、胆绿素还原酶PcyA在大肠杆菌中共同表达,重组后色素蛋白PCB-ApcA、PCB-ApcB、PCB-ApcA2、PCB-ApcD和PCB-ApcF具有特征的紫外可见吸收和荧光光谱,分别为618nm/642nm、612nm/640nm、622nm/642nm、650nm/663nm(602nm/635nm)和622nm/644nm。色素蛋白的酸性尿素变性实验表明,变性后峰的位置在660～664nm左右,证明所连接的色素为PCB。在紫外光的照射下色素蛋白可以产生Zn荧光,证明PCB与脱辅基蛋白是正确的共价连接。根据光谱所得到的一系列的光谱参数(可见紫外吸收比、荧光量子产率、摩尔消光系数)都显示了色素蛋白具有天然藻胆蛋白的性质。利用胰蛋白酶进行色素蛋白PCB-ApcA和PCB-ApcB水解实验,水解后的色素肽的高效液相色谱与从天然鱼腥藻PCC7120中所得到的APC水解多肽的HPLC图谱相似,证明利用大肠杆菌体内重组所得到的色素蛋白具有天然藻胆蛋白的性质。色素蛋白Zn电泳在紫外光的照射下可以产生Zn荧光,证明色素PCB与脱辅基蛋白是正确的共价连接。色素蛋白的圆二色光谱具有天然藻胆蛋白的性质,可见区CD显示的是与脱辅基蛋白所连接的色素的构象,分别在350nm和600nm左右有特征的吸收峰。紫外区显示的是色素蛋白多肽链的构象,其中PCB-ApcA、PCB-ApcB、PCB-ApcD和PCB-ApcF和其他藻胆蛋白一样是α螺旋,只有PCB-ApcA2比较特别是β折叠。通过对色素蛋白的质谱分析,可以证明藻蓝胆素是与脱辅基蛋白在Cys-82位连接。以上实验证明了CpeS可以催化以上五个变藻蓝蛋白亚基和PCB的正确连接。大肠杆菌体内重组产物PCB-ApcD存在两种光谱形式:PCB-ApcD600和PCB-ApcD650,经过初步鉴定推测与蛋白的聚集态有关。研究了CpeS催化变藻蓝蛋白脱辅基蛋白ApcA、ApcB、ApcA2、ApcD、ApcF与PCB的体外重组的辅助因子和酶动力学。研究表明:KPB是比较适合的缓冲液,pH 7.2,NaCl的浓度在100mmol/L～200mmol/L,反应温度37℃。对于ApcA来说,Mg2+可以提高ApcA与PCB重组的效率,而对于ApcF和ApcD,Mg2+影响不大,ME对ApcD与PCB的重组产物的光谱形式有很大的影响,其他金属离子抑制了重组产物的生成,其他辅助因子对重组反应也没有很大的影响。因此,在ApcD和ApcF的体外重组中,不添加其它的辅助因子。通过对CpeS酶的动力学研究表明,酶的催化速度与只有单一的连接作用的藻蓝蛋白裂合酶CpcE/F相当,比具有异构作用的PecE/F略快一些。通过无His-tag的CpeS与脱辅基蛋白ApcA、ApcD、ApcF的体外重组,然后利用镍离子鳌合亲和层析提纯,得到较纯的PCB-ApcA、PCB-ApcD、PCB-ApcF,光谱以及通过光谱所计算出的参数显示与大肠杆菌体内重组所得到的产物性质相同,证明CpeS在一定的条件下也可以催化PCB与ApcA、ApcD、ApcF的连接。但是,在体外重组中,CpeS不能催化ApcB和ApcA2与PCB成功偶联。更多还原

【Abstract】 Cyanobacteria is the photosynthesis microorganism. Under the fitting water environment, a great deal of algae can grown which can induced water bloom. Phycobiliproteins, a homologous family of light-harvesting proteins present in cyanobacteria, red algae, and cryptophytes, are the function composition of light-harvesting complexes in the algae photosynthesis. The last step in phycobiliprotein biosynthesis is the phycobilin addition to the apophycobiliproteins. In vivo, the correct attachment of most chromophores is catalyzed by lyases, of which only few have been characterized so far. As we know now, PecE and PecF catalyzed both the attachment and isomerization of phycocyanobilin to PecA, while the covalent attachment of phycocyanobilin to theα-subunit of C-phycocyanin, CpcA, is catalyzed by CpcE and CpcF. But heretofore nothing is reported about the biosynthesis of the core-membrane linker protein (LCM), theβ-subunits of C-phycocyanin and C-phycoerythrocyanin (CpcB and PecB), and the subunits of allophycocyanin (APC).In this paper, the biosynthesis of LCM, CpcB, PecB and the five subumits of APC from Anabaena sp. PCC7120 was studied by the in vitro and in vivo reconstitution.ApcE and C-terminally truncated ApcE(1-240) bind covalently phycocyanobilin (PCB) in an autocatalytic reaction were firstly studied on its cofactor in the reconstitution system in vitro. Mercaptoethanol (ME) is necessary to the reconstitution, 4mol/L urea increased the reconstitution yield and the addition of sucrose is useful for this reaction. The optimal reaction system is 20mmol/L potassium phosphate buffer (KPB, pH 7.2) containing 100mmol/L NaCl, 4mol/L urea, 200mmol/L sucrose, 0.1mmol/L ME. After purified by the Ni2+-affinity chromatography, LCM(1-240) had absorption at 661 nm and fluorescence at 672 nm. The CD extrema (662, 604, 383 and 347 nm) match the absorption maxima. This and the absorption ration (QAvis/uv =4.1), the fluorescence quantum yield (Φf=0.43), the extinction coefficient (ε=93000 M-1 cm-1) suggest that PCB in the reconstituted products is in a near native chromophore conformation of LCM . The kinetic analyses of the reaction of PCB with ApcE(1-240) have also studied, and giving the following parameters: Km=1.3μmol/L、Vmax=9.6 nmol/L·s-1、kcat=9.6×10-4 s-1. It’s also verified by means of acid-urea denaturation and UV-induced fluorescence by Zn2+-SDS gels tests.CpcB(C155I) and PecB(C155I) bind covalently PCB in vitro and in vivo in E. coli catalyzed by CpeS were firstly studied. The apo-proteins CpcB(C155I) (PecB(C155I), CpcB, PecB), the lyase CpeS, heme oxygenase 1 (HO1) and PCB:ferredoxin oxidoreductase (PcyA) were co-expressed in E. coli by a dual vector system. Reconstitution chromoproteins, purified by Ni2+-affinity chromatography had absorption maxima at 619 and 602 nm and fluorescence emission maxima at 643 and 629nm respectively. After denaturation in acidic urea solution (8mol/L, pH 2.0), reconstituted chromoproteins gave maximal absorption at 662 nm. Reconstituted and purified PCB-CpcB(C155I) and PCB-PecB(C155I) were analyzed via SDS-PAGE: in the presence of Zn2+ they showed the fluorescence that is characteristic for bilins covalently bound to the proteins. We have got the the absorption ration, the fluorescence quantum yield and the extinction coefficient on the base of the experimental spectra. Purified PCB-CpcB(C155I) and PCB-PecB(C155I) biosynthesized in E. coli were finally digested with pepsin under acidic conditions. They have the same maximal absorption and HPLC analysis as the nativeβ-PEC andβ-CPC. PCB also can attach to CpcB and PecB in vitro reconstitution, and the maximal absorption and fluorescence has a 2-3nm red shift compared to the chromoproteins biosynthesized in E. coli. All these declared that CpeS catalyzes the site-selective attachment of PCB to cysteine-β84 in both CpcB and PecB.The five subunits (ApcA, ApcB, ApcA2, ApcD, ApcF) of allophycocyanin bind covalently PCB in vivo in E. coli catalyzed by CpeS were firstly studied. The apo-protein ApcA (ApcB, ApcA2, ApcD, ApcF), the lyase CpeS, heme oxygenase 1 (HO1) and PCB:ferredoxin oxidoreductase (PcyA) were co-expressed in E. coli by a dual vector system. Reconstitution chromoproteins, purified by Ni2+-affinity chromatography had absorption maxima and fluorescence emission maxima at 618 nm/642 nm, 612 nm/640 nm, 622 nm/642 nm, 650 nm/663 nm (602 nm/635 nm) and 624 nm/644 nm respectively. After denaturation in acidic urea solution (8mol/L, pH 2.0), reconstituted chromoproteins gave maximal absorption at 662-664 nm. Reconstituted and purified PCB-ApcA , PCB-ApcB, PCB-ApcA2, PCB-ApcD, PCB-ApcF were analyzed via SDS-PAGE: in the presence of Zn2+ they showed the fluorescence that is characteristic for bilins covalently bound to the proteins. We have got the the absorption ration, the fluorescence quantum yield and the extinction coefficient on the base of the experimental spectra. Purified PCB-ApcA and PCB-ApcB biosynthesized in E. coli were finally digested with pepsin under acidic conditions. They have the analogical maximal absorption and HPLC analysis as the native APC. All these and chromoproteins mass spectrum analysis declared that CpeS catalyzes the attachment of PCB to Cys-82 in these five APC subunits.We also have done researchs on the reconstitution of the up five APC subunits of allophycocyanin bind covalently PCB in vitro catalyzed by CpeS. CpeS only can catalyze PCB bind to ApcA, ApcD and ApcF. The cofactors in the reconstitution system and the kinetic analysis have been studied. Potassium phosphate buffer (KPB, pH 7.2) is the opatimal buffer for the reconstitution. The suitable concentration of NaCl is between 100-200mmol/L. 37℃is the proper reconstitution reaction temperature. Mg2+ can enhance the efficiency of PCB-ApcA reconstituted in vitro, but it have not influence on the PCB-ApcD and PCB-ApcF reconstituted in vitro. Mercaptoethanol (ME) has great influence on the spectra form of PCB-ApcD. Other metal ions restrain the reconstitution efficiency. And the other cofactors have not any influence. So we did not add any cofactors in the reconstitution of ApcD and ApcF. The kinetic analysis indicated that these values agree well with the range obtained for PCB attachment to cysteine-84 of CPC and PEC by the E/F-type lyases and CpeS. Purified PCB-ApcA, PCB-ApcD and PCB-ApcF, produced in vitro from the respective apoproteins and PCB under the action of CpeS without His-tag, had the same spectra and spectral parameters as biosynthesized chromoproteins in E. coli. These also show that only a single PCB had been covalently bound in vitro to Cys-82 of ApcA, ApcD and ApcF, respectively.更多还原