【作者】 高柱虎；

【导师】 齐义鹏；

【作者基本信息】 武汉大学 ， 微生物学， 2004， 博士

【摘要】 在朝鲜的清曲酱、中国的豆豉、日本的纳豆等大豆发酵食品中存在着大量的枯草杆菌纤维蛋白溶解酶，这种酶具有安全性高、规模化生产、直接分解纤维蛋白、口服使用、特异性高、预防和治疗血栓疾病等许多优点，因此对这类纤溶酶进行了深入的研究。到目前为止大部分研究者都使用从市售食品中分离的酶来研究，尚无筛选食品中纤溶酶活性最高的菌株的、更无有关这种酶的抗氧化作用、细胞保护作用、靶向性等多种功能的研究。 本文从发酵大豆中筛选并分离了一株可产生高活性纤溶酶的枯草杆菌，并对该纤溶酶进行了多种功能的研究，为开发第三代溶栓剂奠定了基础。 作者利用纤维蛋白平板法从用水稻叶包裹的大豆发酵品中筛选了一株产高活性纤溶酶的枯草杆菌Bacillus subtilis QK02，经中国典型培养物保藏中心鉴定并保存，保存序号为CCTCC No：M203078。这个菌种的纤溶酶活性比一般枯草杆菌的高出5-60倍。从该菌的培养液中纯化出两种纤溶酶(称为Subtilisin,QK-1和QK-2)，分子量分别为42，000和28，000，比活性为1930IU／mg和41，000IU／mg。它们都直接分解纤维蛋白的Aα-，Bβ-，γ-链，对胰蛋白酶的降解具有抗性。其中低分子量的纤溶酶QK一2不受血液中纤溶酶活化抑制剂(PAI一1)的抑制，QK一2的N一末端氨基酸序列为A一Q一S一V一P一Y一G一I一S一Q一I一K一A一P一A一L一H一S一Q一G，和Subtilisin NAT、SubtilisinJ、SubtilisinE、Subtilisin Amylosaeeharitieus、MensentrieoPePtidase的完全一致，故命名QK一2为Subtilisin QK。以Subtilisin QK(以后简称QK)的N一端氨基酸序列和已发表的纳豆激酶C一端序列设计引物，从枯草杆菌B.subtilis QKOZ基因组中扩增了qk基因，该基因由828 bp的DNA组成，编码一个由2 75个氨基酸组成的蛋白质。氨基酸序列中含有纤溶酶活性中心Asp32、His64、ser22‘和底物结合位点ser‘26、Leu‘27、oly，28，与纳豆激酶很相似。从推断的氨基酸序列和限制性酶切图谱分析，该酶和已报道的Subtilisin家族的5个蛋白酶不一样，与此酶同源性最高的是Subtilisin NAT(高于96.8%)。根据subtilisin BPN’的3维结构模拟了QK蛋白酶的3维结构，该酶具有6个a一螺旋和9个p一折叠，由活性中心和底物结合位点的氨基酸组成的裂缝(d eft)结构，便于与纤维蛋白分子的接触和结合。通过常规操作获得了含有qk基因的重组表达质粒，在大肠杆菌中表达的重组纤溶酶r一QK具有与天然蛋白相同的纤溶酶活性。QK在体外拮抗由氧化剂引起的牛血清白蛋白氧化的能力用分光光度计、荧光光谱、ELISA和westhern blot进行了检测，在体内用ELISA检测了QK对亚硝酸钠和过氧化氢引起的老鼠的脑、心、肝、肾和肌肉中的蛋白质的酪氨酸硝酸化的抑制作用。结果表明QK在体外和体内都有明显的抗蛋白质硝酸化作用。QK还能抑制NO对血红素蛋白的氧化，这种作用可能是一种与纤溶活性无关的酶学反应。除此之外，QK还可以保护人脐血管内皮细胞(ECV一304)不受亚硝酸钠和过氧化氢的毒害。 另一方面，作者还用引物(p4，ps)从枯草杆菌B.subtilis QK02基因组扩增出另一个798bp的DNA片段，从DNA序列推导该片段编码一个257个氨基酸组成的蛋白质，推论的纤溶酶活性中心为Asp38、His6“、Ser222。据同源性比较发现，这个蛋白与已发表的枯草杆菌所有纤溶酶无同源性，而与大肠杆菌的一种丝氨酸蛋白酶DegS的同源性高于98，7%，但缺乏DegS的跨膜区(TM domain)，所以我们将此基因命名为△TM一degs。利用据此△TM一degs和degs基因的N一段与degs基因的C一段设计的引物DSI，PKI和PDZ的菌落PCR从3株大肠杆菌菌液扩增出大肠杆菌的degs基因和叮M一degs基因。在大肠杆菌BLZ lpRA一deg表达的重组蛋白(叮M一D egs)具有纤溶酶活性，分子量为33.4kD。用组氨酸抗体亲和色谱法纯化的△TM一DegS来证明该酶的纤溶酶活性是由血纤溶酶激活机制来引起的，而此作用比尿激酶(SO00IU)高1，4倍。 最近Walsh等(Cell，2003)报道DegS能被大肠杆菌外膜孔蛋白的C一末端多肤活化而分解RseA，诱导6E一依赖的转录，Redford等(Infeet.Immun.2003)报道DegS参与了通过大肠杆菌的P菌毛的侵染过程。文献指出大肠杆菌的P菌毛表面有血纤溶酶活化剂受体，许多细菌通过血纤溶酶活化作用来侵染它的宿主，因此我们推侧叮M一DegS的血纤溶酶活化作用可能与大肠杆菌的侵染有关。 为了使QK在溶解血栓时对纤维蛋白具有靶向性，我们从噬菌体展示抗体库中筛选了人的纤维蛋白单链抗体基因，证明它具有对血纤维蛋白的选择性。为了研究QK和△TM一DegS在细胞内的作用机制，利用pAdEasy系统构建了重组腺病毒载体pAdEasy一qk和真核表达载体PIRES一彭p一叭和PIRES一gip一deg。更多还原

【Abstract】 The fibrinolytic enzymes in the Chongguk- Jiang of Korea, Dou-Shi of China, Natto of Japan have some advantages such as the high safety, mass production, oral administration, high specify to fibrin and direct dissolving fibrin, so many researches on this enzyme had been done. Till now, all researchers have investigated by using the enzymes purified from commodity food, but no one has researched the characteristics of this fibrinolytic enzyme such as anti-oxidation, cell protection and targeting by using purified from the culture of the strain which have screened from these foods by measuring fibrinolytic activity.In this treatise, it was subscribed the multi-functional properties of the fibrinolytic enzymes purified from the culture of the strain with the high fibrinolytic activity which was screened from natural source by fibrin plate method.We have isolated the strain with high fibrinolytic activity from the soybean food fermented by packing with rice leaves and identified it as Bacillus subtilis QK02 to be deserved in China Center for Type Culture Collection (CCTCC) with accession numberNo. M203078. The fibrinolytic activity of B. subtilis QK02 was higher 5-60 times than those of the other strains isolated from the fermented soybean food. Two fibrinolytic enzymes (called as Subtilisin, QK-1 and QK-2) purified from the culture medium of B. subtilis QK02 were exhibited 42,000 and 28,000 kD of molecular weight, 1930 IU/mg and 41,000 IU/mg of specific activity, respectively. They all directly dissolved the Act-, B|3-,y- fragment of fibrin and have activity on the fibrin plate after being digested by trypsin. The fibrinolytic activity of QK-2 which is smaller molecular weight had not inhibited by any substances in blood such as plasminogen activator inhibitor-1. The N-terminal amino acid sequence of QK-2 wasA-Q-S-V-P-Y-G-I-S-Q-I-K-A-P-A-L-H-S-Q-G, which is identical with those ofSubtilisin NAT Subtilisin J Subtilisin E Subtilisin Amylosacchariticus Mensentricopeptidase and from the biochemical characteristics it was named as subtilisin QK.In reference of the N-terminal amino acid sequence of subtilisin QK (QK) and the C-terminal amino acid sequence of subtilisin NAT (NK), the primers was designed and synthesized and the DNA fragment, qk gene, encoded subtilisin QK was amplified from B. subtilis QK02 genome DNA by PCR. The qk gene was composed with 828bpnucleotides encoded a protein consisted by 275 amino acids which had Asp32, His64Ser221 as catalytic center, Ser126 Leu127 Gly128 as the substrate binding site that are aliketo NK. A comparison of the deduced amino acid sequence of QK and restriction enzyme sites of qk gene with those of above five enzymes showed that QK was different from those and the high homolog enzyme with it was subtilisin NAT (96.8%). Through the 3D structure modeling of QK protein by using those of subtilisin BPN’, it was indicated that there were six regions of a-helical and nine P-strand structure and one cleft structure formed with catalytic center and substrate binding site in order to have fibrin easily contact and combine. Through the general molecular cloning method, qk gene was inserted into the expression vector to be expressed in E. coli and the expressionproduct, r-QK, had the fibrinolytic activity like to natural protein.The anti-nitrotyrosine formation activity of QK in BSA was analyzed by using spectrophotometer, ELISA, Westhern blot and fluorescence emission spectra assay, in vitro. The protein protection action of QK against protein nitration, in vivo, was investigated with the homogenates of brain, heart, lung, liver, kidney, spleen, and skeletal muscle between the ribs from the mice injected with oxidants. As results, QK had significantly exhibited the anti-protein nitration activity in vitro and in vivo. And QK had be able to inhibit the oxidation of oxy-hemoglobin by nitrite, which was not concerned with its fibrinolytic activity, and also protect the human更多还原