摘要
纳豆激酶是从日本传统的大豆发酵食品——纳豆中发现的新型纤溶酶。这种酶具有较高的纤溶活性，无毒副作用，不引起内出血，在体内半衰期长。纳豆激酶无论作为抗血栓药物还是预防血栓栓塞性疾病的保健食品，都具有重要的开发价值。天然的纤溶酶活性往往较低，通过利用体外DNA分子改造等基因操作技术，构建高比活力的工程菌是实现产业化的重要途径。
本研究主要进行了以下工作：
（1）通过酪蛋白平板初筛和血纤维蛋白平板复筛结合的方法，从豆豉、纳豆样品中筛选到了21株纤溶酶产生菌，获得了一株高产纤溶酶菌株Na-002，纤溶酶活性可达10875U/g，比相关报道的出发菌株活性都要高。
（2）通过形态鉴定、生理生化鉴定和16SrDNA序列分析等多项分类法，对Na-002进行了鉴定，确定其属于枯草芽孢杆菌。
（3）通过PCR扩增技术，克隆获得了纤溶酶基因，与公布的11株纳豆激酶的核苷酸序列相似性达到99.8%~99.2%，氨基酸序列相似性达到98.6%~99.7%，说明该纤溶酶基因是纳豆激酶系列的一种，该基因的序列保守性强。
（4）通过PCR技术克隆得到pro-NK基因，并分别构建pET-32a(+)表达载体、pWB980表达载体与pPIC9K表达载体，实现了在大肠杆菌BL21（DE3）、枯草芽孢杆菌WB800和毕赤酵母GS115中进行表达。实验表明，大肠杆菌表达的酶蛋白表达量高，易于纯化，但是多以包涵体形式存在，需要通过变性复性的方法才能获得有活性的酶；枯草芽孢杆菌表达活性最好，但是表达量少，纯化难度大；毕赤酵母表达系统的纯度高，但存在活性低，表达量相对较小的缺点。
（5）通过定点突变的方法，从pH稳定性的角度对该酶基因进行了定点突变研究，获得了S182L、S182W、S182K、L203E、L203K等5个突变体，并实现了毕赤酵母表达。通过对该酶最适pH的测定发现，该酶最适pH的变化与碱基替换呈现出相关性：酸性氨基酸有利于酶在pH值为酸性范围内稳定，而碱性氨基酸有利于酶在pH值为碱性范围内稳定。
（6）通过同源建模，推测该酶具有两个结构域：结合结构域（CBM）和催化结构域（CD）。CBM是结合在底物表面的，位于N端，用1scjB（同源性97.183%）作为模板，构建CBM的三维结构，它是由第7个氨基酸到第77个氨基酸共71个氨基酸构成，形成了两个α螺旋和3个β折叠；CD的三维结构用1scjA (同源性98.545%)作为模板来构建,由第78个氨基酸到第352个氨基酸共275个氨基酸构成，形成了一个(β/α)7桶状结构。
本研究丰富了纳豆激酶的酶学基础理论知识，并为进一步的基因水平酶分子进化理论的研究以及口服溶栓药物的开发和应用奠定了基础。
Nattokinase(NK) is a novel fibrinolysin which is first found in Japanese traditionalfermentation food,natto.Nattokinase owns the characteristics such as the high activity offibre dissolution,no poisonous and other side effects,no internal hemorrhage and longhalf life in vivo. Nattokinase has the important value whether as an anti-thrombus drugor as a healthy food preventing the thromboembolism. While the activity of naturalfibrinolysin is usually very low, therefore, it is a major approach to industrializenattokinase by DNA operation technique to construct engineering bacterium with highspecific activity. This paper mainly carried out the work as follows:
(1) 21 bacteria producing fibrinolysin were obtained from the samples of natto andfermented soya beans by the way of preliminary screening with casein culture mediumand second screening with blood fibrin culture medium. Among them there was a strainNa-002, which had a high activity of fibrinolysin, achieving to 10875U/g, higher thanany reported strains.
(2) Strain Na-002 was identified as a Bacillus subtilis by morphological indexes,physiological and biochemical indexes and 16SrDNA sequence analyses.
(3) The fibrinolysin gene was cloned by PCR amplification, which had the99.8%~99.2% similarities with the 11 published nucleotide sequence of nattokinase. Thesimilarities of amino acid reached 98.6%~99.7%. It revealed that the cloned fibrinolysingene was one kind of nattokinase and the gene had good sequence conservation.
(4) The pro-NK gene was cloned by PCR technique. Meanwhile, the expressionvector of pET-32α(+), pWB980 and pPIC9K were constructed, respectively. Thecloned gene was expressed in E. coli BL21 (DE3), Bacillus subtilis WB800 and pichiapastoris GS115. It disclosed that the expression amount of E. coli was high and theproduction was purified easier, while the protein existed in the form of inclusion body,which meant more work on obtaining the active enzyme. The expression amount ofBacillus subtilis was in a low level and it was difficult to purify the enzyme, though the production owned a better activity. The expression amount of pichia pastoris was lowand the production carried the poor activity, though it had a high purity.
(5) The 5 mutants, S182L, S182W, S182K, L203E and L203K were obtained bysite-directed mutagenesis based on the pH stability, and they were all expressed in pichiapastoris. There was a close relationship between the optimum pH and the base displace.The acidic amino acid kept the stability of the enzyme in the acidity condition, while thealkaline amino acid kept the stability of the enzyme in the alkalescence condition.
(6) It was presumed by homology modeling that there were two structural domains,combining domain (CBM) and catalyzing domain (CD). CBM combined the substratesurface and located in the N-terminal. The three dimensional structure of CBM wasconstructed with the model of 1scjB (97.183% homology). It was consisted of 71 aminoacid from amino acid 7 to amino acid 77, which formed 2α-helix and 3β-pleatedsheet structure. The three dimensional structure of CD was constructed with the model of1scjA (98.545% homology). It was consisted of 275 amino acid from amino acid 78 toamino acid 352, which formed a (β/α)7 tubbiness structure.
This research enriched the basic theory of nattokinase and laid a foundation not onlyfor further study on the enzyme molecular evolution in gene level but also for theapplication of oral taking drugs of thrombolysis.
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