Design Of Multifunctional Fusion Heparinase And Its Application In The Production Of Low Molecular Weight Heparin

For the side effects of heparin as the anticoagulant and antithrombotic agent, low molecular weight heparin (LMWH) capable of maintaining the anticoagulant activity while showing fewer side effects has been widely used and replaced heparin in clinics. Clean LMWH production by enzymatic depolymerization of heparin is a typical application of enzyme engineering, while the low productivity of heparinase, the complexity of the purification, the high cost, the difficulty in the directed immobilization of the enzyme, and the time-consuming detection of the enzymatic activity. In this thesis, by using the fusion protein technology, mutifuctional heparinase I (HepA), combining the special affinity to maltose of maltose binding protein (MBP) and the easy quantitative detection of the fluorescent proteins (including green flurescent protein, GFP and red flurescent protein, RFP), was designed to realize the process intergration of the enzyme production, separation, purification and applications in LMWH production for reducing the enzyme cost.Double-fusion protein systems by fusion of fluorescent proteins (GFP or RFP) and HepA with different linker lengths were constructed. The results showed that the rapid detection of the HepA’s activity can be achieved by the easy detection of the GFP or RFP fluorescent intensity. Although the inner filter effect and background interference is less in the RFP-HepA fusion system, the GFP-HepA system has stronger fluorescent signal and better thermal stability than RFP-HepA, which wasd presumably related with the easy oligomerization of RFP.Different triple-fusion systems with different linker strengths both in the sequence of GFP-MBP-HepA and MBP-GFP-HepA were constructed and compared. The former had higher specific enzymatic activity, but the MBP fusion partner lost the special affinity to the maltose, and breakage of the fusion porotein was found at the site before the HepA; while the latter was completely expressed without breakage, maintained the special affinity of MBP to the maltose, and had higer special fluorescent intensity, enhanced thermal stability with a half life of 216 min, which was 21.6 times of the half life of MBP-HepA fusion system.Further analysis of the effects of the linker length and strength on the fuctions of the fusion proteins suggested that shorter linker could improve the heparin’s binding to HepA, resulting in the higher catalytic efficiency of HepA and flexibler linker seemed helpful to enhance not only the catalytic efficiency of HepA, but also the thermal stability of the fusion protein and the special affinity of MBP to the maltose.Based on the results obtained above, enzymatic production of LMWH from heparin was successfully realized by the use of MBP-HepA fusion protein, and the operating conditions and the factors affecting the yield of LMWH together with the cost estimation were studied. The effectiveness of using the absorbance at 235 nm of the reaction mixture for the quality control of LMWH production was illustrated.