Construction Of Nattokinase Gene-engineering Strain And Its High-Density Fermentation

Thrombosis takes an increasingly high possession in human morbidity and mortality.Thrombolytic agents are the most effective drugs,but all the current clinical agents have some disadvantages.It is necessary to develope and improve new agents,especially prophylactic food-borne thrombolytic agents.Nattokinase(NK)originated from Japanese traditional food natto,which is a kind of serine protease,produced by the Bacillus subtilis natto(B.subtilis natto)and encoded by the aprN gene.NK is a new type of thrombolytic agents.With the features of having no immunogenicity,significant thrombolytic effect,safe and non-toxic,nattokinase is becoming a functional,preventive and healthy food product owning development and utilization value.Although by traditional solid-state fermentation,the yield of nattokinase is high,the separation and purification steps were complicated andthe loss of its activity during these steps is serious.The liquid fermentation of genetically engineered bacteria may cause some problems,such as producing inclusion body and low enzyme activity.In order to improve the yield of NK,the main contents of this study are as follows:(1)The aprN gene from B.subtilis natto strain was cloned and the codons which encode the initiation 30 amino acids were optimized on the basis of codons preference of B.subtilis.Recombinant vector pHT01-aprN1 was constructed.After being analyzed by restriction enzyme digestion,PCR and sequencing,the exogenous subcloned gene was confirmed to be aprN1.The pHT01-aprN1 was then transformed into B.subtilis 168 by electroporation,and the engineering strains(B.subtilis 168/pHT01-aprN1)were isolated on LB plates containing chloramphenicol.(2)The effects of IPTG,2,6-pyrid inedicarboxylic acid,induction temperature,medium concentration and inoculation amount on enzyme activity were investigated.The highest enzyme activity was 289 U/mL(RSD%=1.1)cultured by flask and detected by tetra peptide substrate.The stability of enzyme activity was good which was 3.9 times of that of B.subtilis natto.Fermentation supernatant was purified by Nickel column and SDS-PAGE and Western-Blot were used to determine the purity of purified nattokinase.(3)In order to achieve industrial production,fed-batch fermentation experiments were carried out.The enzyme activity reached 4521.78 IU/mL(RSD%=1.2),and the optimal feeding schedule was 7778.5 IU/mL(RSD%=1.1).Fermentation supernatant was concentrated by ammonium sulfate with the recovery rate of 89.02%.The protein concentrate was lyophilized and then purified by Nickel column with recovery rate of 93.78%.The purification solution was desalinated and lyophilized into powder,whose enzyme activity is 4.07 times as that of standard powder(20000 FU/mL).