Preparation Of The Genetic Engineering Of Nattokinase

Arterial thrombosis is the underlying cause of a wide variety of cerebro-cardiovascular diseases. Most of the third-generation thromblytic agents are variants of tissue type plasmino-gen activator, such as reteplase, monteplase, tenecteplase and lanoteplase. Natural thromblytic agents are extracted from micro-organisms, plants and animals.Natto, a Japanese traditional food, is a kind of fermented soybean with Bacillus subtills. Nattokinase is a serine proteinase of natto produced in the process of soybean fermentement, which was found to have high fibrinolytic activity in 1987. It is competently stable in the normal pH and temperature. In recent years, much attention has been paid on it for its advantages of super ability in dissolving thrombus with safety, long half-time period and effectivity by oral taking as medicine. Therefore, there is potential for nattokinase to be developed as a natural thrombolytic agent. Technology of genetic engineering would be the important base for the industrialization of nattokinase.At present, most of the studies have been done on strain improvement and fermentation condition optimization as well as the purification of Nattokinase, in order to improve Nattokinase productivity. However, high-yielded strains of nattokinase Bacillus subtilis still need to be screened from nattoes produced in Japan. There is an urgent need to develop the technology of genetic engineering for industrial production of Nattokinase.In our precent study, with primary screening from three kinds of nattoes from Japan for the nattokinase producing strains of bacteria and subsequently secondary screening on casein plate, 10 bacteria strains with nattokinase producing activity were isolated. Among them, the strain N07 showed the highest capability in nattokinase producing and enzymatic satability, through fermentation and nattokinase activity assay of the fermentation products. The activity of nattokinase produced from N07 is equivalent to 1384.73 IU/mL urokinase. Routine tests of N07, including morphology, physiological and biochemical characteristics, suggest that strain N07 is Bacillus subtilis. Then, genome DNA of Bacillus subtilis N07 was extracted, and two PCR segments, 1143 bp and 825 bp long, respectively, were obtained and sequenced with nattokinase-specific primers. Sequence analysis with Blast on NCBI website showed that these two fragments are 99.8% similar to the reported nattokinase gene. Bioinformatic analysis showed that the nattokinase is composed of 275 amino acids, and its relative molecular weight is 27,728. The pre-pro-nattokinase is composed of 381 amino acid residues, the signal peptide 106 amino acid residues and mature peptide 275 amino acid residues. The isoelectric point of nattokinase is 8.6±0.3.The two gene segments were separately cloned into the vector pMDl8-T to construct two nattokinase expression vectors, pET30a-NK1 and pET30a-NK2, which were confirmed for correct gene recombination with PCR and sequencing analysis. The vectors were then transferred into E.coli BL21, which was followed by the multiplying in E.coli BL21 culture induced by IPTG, the isolation and purification of nattokinase and the detection of nattokinase activity on fibrin plates. The results showed that the vector pET30a-NK1 carrying fragment 1 (for pre-pro-NK gene) successfully expressed the secretion protein of nattokinase and produced high activity of nattokinase. However, the vector pET30a-NK2 for the fragment 2 (NK gene) could not produce active nattokinase. We speculate that the leading peptide sequence of nattokinase can help correctly folding of mature peptide of nattokinase, thereby enhancing the solubility and activity of nattokinase. The probability of correct folding of mature peptide is very low without help of the leading peptide, and so .they form inclusion bodies and loss the activity. The signal peptide of nattokinase can help mature peptide transfered to extracellular. It achieved nattokinase extracellular expression in E.coli and maked separation and purification simpler.The further works for the improvement of the gene enginnering technology include the optimization of fermentation conditions, and the technology of nattokinase purification and activity determination.