| Article ID | Journal | Published Year | Pages | File Type |
|---|---|---|---|---|
| 2694 | Biochemical Engineering Journal | 2016 | 7 Pages |
•The activity of heparinase I was improved by the directed evolution.•Enzymatic properties of the mutated and original heparinase I were compared.•Optimal conditions for heparin degradation were determined.•Time-course of heparin degradation was analyzed.
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 366 U/L of heparinase I, which was 57.8% higher than that from the control clone (232 U/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 1 mM could significantly increase the activity of heparinase-I133/P316. Cu2+, Ni2+, Mg2+, Mn2+ and Zn2+ at 1 mM were found to inhibit its activity, and Ba2+ and Fe2+ at 1 mM 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 125 μg/mL, 40 °C, 7.5, 400 rpm and 40 mg/mL, respectively. After reaction for 6 h, 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.
