Directed Evolution Of Douchi Fibrinolytic Enzyme And Expression Of Mutant Enzyme Gene

DFE(Douchi fibrinolytic enzyme) is a new fibrinolytic enzyme which was discovered in Douchi. DFE is worthy developing as either natural thrombolytic agent or health food to prevent thrombotic diseases.The gene encoding pro-DFE from DC-12 was cloned and expressed in E. col.i The catalytic efficiency of DFE was improved by directed evolution. The mutant DFE was expressed in Bacillus subtiilis WB800 by induction with IPTG and the fermentation conditions were optimized. The main results of the thesis are as follows:A functional expression is favorable for high throughput screening(HTS) which is applied to screen libraries consisting of a large number of variants produced by random mutation experiments such as directed evolution. The pro-DFE gene was amplified from DNA of Bacillus subtiilis DC-12 isolated from Douchi-a traditional Chinese fermented-soybean food, The fragment was cloned into PET-32a, and then transformed into E.coli BL21(DE3).The recombinant strain was grown at 37℃. At an OD600 of 0.6,cells were induced by additional IPTG(0.4 mmol/L final concentration). Growth was continued at 24℃. The expressed products were analyzed through SDS-PAGE, the recombinant protein exist in the supernatants and pellets. The strong fibrinolytic activity was detected in the supernatants and reached 200 IU/mL. The recombinant protein with a molecular weight of 28 kDa was purified by Ni2+-NTA column and Sephadex G-75 column.N-terminal sequence of the recombinant protein is in according with the result deduced by DNA sequence.DFE gene was confirmed by the functional expression in E.coli, the molecular weight and N-terminal sequence of the recombinant protein.Mutagenesis on Douchi fibrinolytic enzyme gene was performed by using error-prone PCR strategy. After three cycles of error prone PCR and screening by substrate H-D-Val-Leu-Lys-pNA, the mutant enzyme with improved substrate specificity and catalytic efficiency was obtained. Gene analysis of the mutant enzyme gene showed that the mutant had six nucleotide substitutions(C119G/T236C/A308T/G397A/A633T/T705C)and four of them caused amino acid changes(P40R/ V79A/ Q103L/ A133T).In order to highly express of mDFE3 gene in B.subtilis WB800 and shorten the fermentation period,the full Kanr gene including its promoter seuqence was cloned from pBE3 and inserted into a shuttle vector pHT43 to construct the expression vector pHK11. The mDFE3 encoding sequence was cloned into pHK11 and expressed in B. subtilis WB800. The recombinant strain was grown in LB to the mid-log phase,cells were induced by additional IPTG (1 mmol/L final concentration). Growth was continued at 37℃for up to 5 h. Its maximum fibrinolytic activity of the supernatant in LB medium was 1164 IU/mL. The results of SDS-PAGE analysis showed that there were indeed recombinant proteins with a molecular mass of 28 kDa in supernants. Plasmid stability was also measured. The recombinant plasmid didn't appear obvious gene deletion, showed structural stability after 50 generations. However, the plasmid showed some segregational instability, the plasmid-free cells appeared under no Kanamycin selection pressure.This result indicates that cells carrying certain recombinant pHK-mDFE3 plasmids should be grown in the presence of Kanamycin to avoid loss of the plasmids.In order to further enhance production of recombinant Douchi fibrinolytic enzyme,the optimal fermentation conditions of pHK-mDFE3/WB800 were determined. The compositions of fermentation medium are 2% glucose;2% soybean peptone;0.6% K2HPO4,0.2% KH₂PO₄, 0.04% CaCl2,0.075% MgSO₄;pH7.0; Initial growth of the cells at 37℃to the mid-log phase and subsequent induction with IPTG(1mmol/L final concentration) at 30℃;40 mL of media are in 250 mL flask. Under these conditions the fibrinolytic activity of the supernatant can reach 1948 IU/mL.