Cloning Of Gene Encoding The Fibrinolytic Enzyme And Expression In E.coli And Pichia Pastoris

In our study, a fibrinolytic enzyme from BacillusZLW-2 screened from slaughtering plant in baoding can strongly dissolved blood clots, which should be developed as a new drug for cardiovascular and cerebrovascular diseases. The gene of the enzyme was cloned and sequenced, whose GeneBank accession number was EU734749. The sequence contained one open reading frame of 1146 bp encoding 381 animo acids, whichhad 99%homology with Nattokinase YF308 and NAT. The deduced animo acid sequence included a signal peptide with 29-residue, a propeptide with 77-residue and a mature enzyme with 275-residue by analysis with SignalP3.0. This mature enzymehad 8 Lys residues without Cys and the same activity center of Asp32,his64 and Ser221 and binding center of Ser125, Leu126 and Gly127 as the reported nattokinase. Due to the similarity of DNA sequence and deduced amino acid sequence between it and the other fibrinolytic enzymes, this newly cloned enzyme was identified as nattokinase.The pre-pro-fibrinolytic enzymeNK1 fragment(encoding signal peptide, propeptide and mature peptide) pro-fibrinolytic enzyme NK2 and fibrinolytic enzyme NK3(encoding mature peptide) were isolated and ligased with pET28a vector respectively, then transformed into BL21(DE3). The result revealed that the expression product was no fibrinolytic enzyme activity, which should be inclusion body. The expression product from NK1 induced with 0.1mmol/L IPTG at 30℃for 20h showed enzyme activity of 183U/mL after modified freeze-thawing.In order to improve the stability and expression yields of exogenous gene, the genes of pro-nk and nk were cloned into Pichia pastoris for further research. According to the gene EU734749 and eukaryotic expression vector pPIC9K, we redesigned the primers to construct the recombinant plasmid pPIC9K-pro-nk, pPIC9K-nk.. Recombinant plasmids were transformed to Pichia pastoris GS115 after being linearized. After MD MM and activity transparent circles screening a recombinant yeast named PK53 withhigher enzyme activity was elected. By the study on the growth and fermentation conditions of the PK53, it was found that the genetic stability of the gene was so well that the rate of gene losing is still zero after 10 generation of breeding, even in the absence of selection pressure. The optimization of fermentation conditions was discribed as follows: inoculation amount, 1%; liquid volume, 40 mL / 300 mL; amount of Methanol, 0.7% ; 30℃, 250 rpm. After fermentation optimization, the enzyme activity of recombinant yeast was up to 675.53 U/mL, 4.99 times of the original strain. The optimal temperature of the enzyme was 50℃, the pH was 8.0, which were consistent with the original strain.