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

Paper ID Volume ID Publish Year Pages File Format Full-Text
2694 126 2016 7 PDF Available
Title
Improving the activity of heparinase I by the directed evolution, its enzymatic properties and optimal conditions for heparin degrading by recombinant cells
Abstract

•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.

Keywords
Directed evolution; Error-prone PCR; Heparinase I; Enzymatic properties; Heparin degradation; Recombinant cells
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Improving the activity of heparinase I by the directed evolution, its enzymatic properties and optimal conditions for heparin degrading by recombinant cells
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Publisher
Database: Elsevier - ScienceDirect
Journal: Biochemical Engineering Journal - Volume 114, 15 October 2016, Pages 237–243
Authors
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Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering
Get Full-Text Now
Don't Miss Today's Special Offer
Price was $35.95
You save - $31
Price after discount Only $4.95
100% Money Back Guarantee
Full-text PDF Download
Online Support
Any Questions? feel free to contact us