Food-grade Expressing And Analysis Of Nattokinase In Lactobacillus Delbrueckii Subsp. Bulgaricus

Nowaday, cardiovascular diseases have become one of the greatest threats to human health, meanwhile, researching on the development and application of thrombotic disease treatment and new thrombolytic drugs has become a studying hot spot. The development of Nattokinase used in functional food and food additives has been very rapid. Nattokinase is able to reduce blood pressure, blood fat, anti-thrombotic, anti-viral, anti-oxidation and prevent cancer and so on, therefore Nattokinase has become a novel foodborne thrombolytic drugs. However, the main source of Nattokinase is a Japanese natto and the unique taste is unacceptable, which used in food weakly. Therefore, it is necessary to use Nattokinase as a functional food. Lactic acid bacteria as safe food-grade microorganisms has been an inevitable trend of the food industry development in the whole world as well. With the rapid development of biology technology and genetic engineering, the researching of lactic acid bacteria does not only need the traditional physiological functions but also the study of the genetic level. Besides, the study about making lactic acid bacteria as receptor strain of recombinant vector has been achieved successfully, which leads researchers to add lactic acid bacteria with exogenous genes and to show expression of the exogenous genes through DNA recombination technology, making such lactic acid bacteria have a new probiotic properties.In this study, Nattokinase achieves food-grade expression in lactic acid bacteria, through the plasmid was electroporated into thy A-null host Lactobacillus delbrueckii subsp. bulgaricus. By measuring plasmid stability, the same expression capacity of the 15 generation is the same as original one and combined with flat dash serially passaged 30 generations can be stable. Experimental results show that the highest expression of Nattokinase is during the decline of Lactobacillus delbrueckii subsp. bulgaricus through semi-quantitative RT-PCR analysis for expression of Nattokinase in Lactobacillus delbrueckii subsp. bulgaricus. The molecular size of expression product is estimated with method of SDS-PAGE. Then Nattokinase is 28 KD. Characterization of heterogeneous expressing food-grade Nattokinase in Lactobacillus delbrueckii subsp. Bulgaricus have been researched which prove that the optimum reaction temperature of heterogeneous expressing Nattokinase in Lactobacillus delbrueckii subsp. bulgaricus was 40℃, optimum p H value was 7.0, it was more stable when its temperature belowed 40℃ and p H 6.0-8.0. The promoting effects of Ca^{2 +}, Mn^{2 +}, Fe^{2 +} on enzyme activity changed with different concentrations, Cu²⁺、Hg²⁺ inhibited the enzyme activity, and the inhibitive effect was more significant with the higher concentration of Ag ⁺. Meanwhile, the inhibitor PSMF had significant inhibitory effect on nattokinase, which indicated that nattokinase was a serine protease. Through the optimization of culture conditions to improve the expression of nattokinase, according to the experimental results of single factor orthogonal experiment to determine the optimal culture conditions for culture time 18 h, inoculum 2%, vaccination age for 16 h, activity reached 3.498 U/m L.The experiments show that Nattokinase is almost no activity in simulation artificial gastric juice and the effect of pepsin to Nattokinase is irreversible. But Nattokinase can stably exist in the intestinal fluid without being degraded by trypsin and the relative activity can reach 60% in intestinal 4 h. In this study, nattokinase was transformed into Lactobacillus delbrueckii subsp. bulgaricus, using lactic acid portable nattokinase successfully reach the intestine through the gastric juice. The results proved that the enzyme activity can reach the highest activity at 3 h, the extracellular enzyme activity of about 32% which due to the disintegration of the cell. Experiments show that trace metal ions can promote self-dissolving bacteria to increase the release of nattokinase. The largest solubility reaches respectively 25% and 24%, the relative activity of about 129% and 123% when Ca^{2 +} and Mg^{2 +} concentration was 10^-4 mol/L; the maximum solubility can reach 20%, the relative activity of around 114% when Mn2 + at a concentration 10-5 mol/L that compared with the blank are improved. It showed the way of the enzyme dissolving fibrin was dissolution directly by inactivating the enzyme plasminogen, and the enzyme showed good thrombolytic activity with experiments in vitro.This study provides us the basis of the food-grade expression vector and heterologous expression in lactic bacteria. Meanwhile we also apply heterologous expression of food-grade nattokinase in lactic acid to actual production, and this can provide theoretical basis for the the development of new functional dairy products.