Gene Cloning, Expression And Site-directed Mutagenesis Of Nattokinase From Bacillus Subtilis Na-002

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.