Improving the activity of heparinase I by the directed evolution, its enzymatic properties and optimal conditions for heparin degrading by recombinant cells

Abstract

Heparinase I can degrade heparin and heparin sulfate to produce low molecular weight heparins (LMWHs) that are widely used as an anticoagulant and antithrombotic drug. In this study, directed evolution was used to improve the activity of heparinase I. The clone E. coli-heparinase-I133/P316 with two amino acid substitutions was screened and identified. This clone produced 366U/L of heparinase I, which was 57.8% higher than that from the control clone (232U/L). The optimal temperature of heparinase-I133/P316 was lower than that of the original heparinase I. The mutated enzyme and original one presented almost the same varying trend as an function of pH, and their optimal pH was near 7.5. Ca2+ at 1mM could significantly increase the activity of heparinase-I133/P316. Cu2+, Ni2+, Mg2+, Mn2+ and Zn2+ at 1mM were found to inhibit its activity, and Ba2+ and Fe2+ at 1mM almost had no obvious effect on its activity. The optimal substrate concentration, temperature, pH, shaker speed and cell concentration for heparin degradation by E. coli-heparinase-I133/P316 were 125μg/mL, 40°C, 7.5, 400rpm and 40mg/mL, respectively. After reaction for 6h, degradable products are basically concentrated in a small molecular weight region. This study provides a new insight into the high production of heparinase I and the preparation of LMWHs by E. coli-heparinase-I133/P316 cells.