Cloning Of Nattokinase Gene And Expression In Pichia Pastoris

Natto is the Japanese traditional fermented food and has a very long history of at least 2,000 years. Nattokinase was first found and named by Hiroyuki Sumi in 1987. It is a serine proteinase of natto produced in the process of soybean fermentement, which was found to have high fibrinolytic activity.At present cardiovascular disease has become one of the biggest threats of human health. Streptokinase is an effective method for the treatment of cardiovascular disease..Now nattokinase becomes a hot study due to its advantages in safety, super ability of dissolving thrombus, long half time of degradation, easiness to be absorbed by body and cost low. Recently,a series of researches to explore the biochemical characteristics have been done.The results showed that the isoelectric point of Nattokinase is 8.6±0.3, and nattokinase is composed of 275 amino acids, its relative molecular weight is calculated to be 27 Kda ,according to the sequence of its'amino acids.Now nattokinase is mainly be developed as a new potent fibrinolytic medicine and health products.The obtains of nattokinase mainly has two ways. First, enhancing the fibrinolytic activity of NK by improve on the strain. Secondly, cloning the NK gene and expressing it in a host to increase nattokinase productivity.In this research, genome DNA of Bacillus subtilis N07 was used as the PCR template. Pro-NK was amplified by PCR . Then the 825bp gene segment was cloned into the vector of pMD19-T, transferred into the host strain, multiplied, purified, and analyzed using electrophoresis and sequencing. Sequence analysis with Blast on NCBI website showed that the gene fragment is identical to the reported nattokinase gene. This NK gene was cloned in expression vector pPICZaA. After being analyzed by restriction enzyme EcoRI and XbaI, PCR and sequencing, it was confirmed that heterogeneous gene spliced into vector pPICZaA was NK gene.The recombinant vector pPICZaA-NK was linearized and transformed into host cell Picchia pastoris x-33. Recombinant Picchia pastorix-33 was isolated on YPDS plates containing Zeocin and it was induced expression by 1% concentration of methanol in BMMY. Fibrinogenolysis test showed that fermentation liquor is thrombolytic. Through purifying the enzyme by the ammonium sulfate, dialyze, SephadexG-50 Chromatography and so on, the purified enzyme showed a single protein band in the SDS-PAGE and it is 27 KDa. The purified enzyme has Fibrinogenolysis amount to urokinase 195 U/ml. And the temperature and pH optimum for nattokinase were 45℃and pH8.0, respectively.This research has cloned and expressed the NK gene in Picchia pastoris x-33 successfully, and it would be helpful in producing nattokinase by engineering.