Studies On The Expression Of Nattokinase In Recombinant Lactococcus Lactis

Nattokinase (NK) is a protein kinase extracted from Japanesetraditional food natto. It has shown strong fibrinolytic property. It couldmake up for the shortcomings of traditional thrombolytic drug very well,so it is likely to be used as a new generation of oral agents for thromboticdiseases.To study on the expression of the nattokinase gene, the followingwork has been carried out.Firstly, the pro-nk sequence was cloned into high level expressionvector pNZ8048 for Lactococcus. lactis NZ9000, the result expressionvector pFY002 was constructed and transformed into Lactococcus lactisNZ9000.The recombinant strain Lactococcus lactis NZ9000/ pFY002could express active nattokinase(NK) under the induction of nisin. Theoptimum conditions including nisinA concentration, temperature andinduction time for the expression of nattokinase in the engineering L.lactis were investigated. The optimum concentration of nisinA, theoptimum temperature and induction time for nattokinase were 7.5ng/ml,28℃and 3.5h respectively.The high yield reached 98.55U/L forNZ9000/pFY002.Secondly, an integrative expression vector pFY008 was constructedwith hisH gene as target site for homologous recombination. In order to torealize nattokinase gene (aprN) food-grade expression in Lactococcuslactis, PnisA-aprN gene expression cassette was integrated into thegenome of Lactococcus lactis NZ9000 through double homologousrecombination. The result food-grade expression system recombinantLactococcus lactis with high safe and stable properties was constructed.Itcan produce the active nattokinase. The work mentioned above has laid a solid foundation for the development of a new lactic acid drink withthrombolytic activity.