Clonging and Expression of Heparinase III Gene from Flavobacterium heparinum and Characterization of the Recombinant Fusion Enzyme

Abstract

Abstract: Heparinase III is an important enzyme for heparin structure analysis and low molecular weight heparin production. To achieve the soluble expression of HepC with high activity in recombinant E. coli, the heparinase III gene(hepC)from Flavobacterium heparinum was amplified by the PCR method, and the fusion expression vector and system of expressing fusion protein of a maltose binding protein(MBP)and HepC were constructed. The results showed that the fusion strategy by using MBP was effective to enhance solubility of the MBP-HepC when induced at low temperature of 15℃. By shake flask cultivation, the specific enzyme activity of MBP-HepC could reach about 46.41 IU/mg, which was the highest among the values reported so far. By one-step affinity purification with amylose resin, the MBP-HepC fusion protein was easily purified to more than 95% purity. The enzyme characteristic study showed that the optimum pH value of MBP-HepC was 7.3, the optimum reaction temperature was 42℃-48℃, and the thermal stability of MBP-HepC is better than Heparinase I(MBP-HepA)at 30℃.

Key words: Heparinase III, Maltose binding protein(MBP), Recombinate expression, Purification, Characterization

Li Ye, Wu Jingjun, Ye Fengchun, Su Nan, Zhang Chong, Xing Xinhui. Clonging and Expression of Heparinase III Gene from Flavobacterium heparinum and Characterization of the Recombinant Fusion Enzyme[J]. Biotechnology Bulletin, 2013, 0(6): 133-139.

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