Cloning, Expression, and Characterization of a Highly Stable Heparinase I from IBacteroides xylanisolvens/I

Heparinase I (Hep I), which specifically degrades heparin to oligosaccharide or unsaturated disaccharide, has an important role in the production of low molecular weight heparin (LMWH). However, low productivity and stability of heparinase I hinders its applications. Here, a novel heparinase I (BxHe...

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Veröffentlicht in:Polymers 2023-04, Vol.15 (7)
Hauptverfasser: Pei, Jia-Lu, Wei, Wei, Wang, Ding-Ran, Liu, Cai-Yun, Zhou, Hua-Ping, Xu, Chen-Lu, Zhang, Ye-Wang
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container_issue 7
container_start_page
container_title Polymers
container_volume 15
creator Pei, Jia-Lu
Wei, Wei
Wang, Ding-Ran
Liu, Cai-Yun
Zhou, Hua-Ping
Xu, Chen-Lu
Zhang, Ye-Wang
description Heparinase I (Hep I), which specifically degrades heparin to oligosaccharide or unsaturated disaccharide, has an important role in the production of low molecular weight heparin (LMWH). However, low productivity and stability of heparinase I hinders its applications. Here, a novel heparinase I (BxHep-I) was cloned from Bacteroides xylanisolvens and overexpressed in soluble form in Escherichia coli. The expression conditions of BxHep-I were optimized for an activity of 7144 U/L. BxHep-I had a specific activity of 57.6 U/mg at the optimal temperature and pH of 30 °C and pH 7.5, with the Km and Vmax of 0.79 mg/mL and 124.58 U/mg, respectively. BxHep-I catalytic activity could be enhanced by Ca[sup.2+] and Mg[sup.2+], while strongly inhibited by Zn[sup.2+] and Co[sup.2+]. Purified BxHep-I displayed an outstanding thermostability with half-lives of 597 and 158 min at 30 and 37 °C, respectively, which are the highest half-lives ever reported for heparinases I. After storage at 4 °C for one week, BxHep-I retained 73% of its initial activity. Molecular docking revealed that the amino acids Asn25, Gln27, Arg88, Lys116, His156, Arg161, Gln228, Tyr356, Lys358, and Tyr362 form 13 hydrogen bonds with the substrate heparin disaccharides in the substrate binding domain and are mainly involved in the substrate binding of BxHep-I. These results suggest that the BxHep-I with high stability could be a candidate catalyst for the industrial production of LMWH.
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However, low productivity and stability of heparinase I hinders its applications. Here, a novel heparinase I (BxHep-I) was cloned from Bacteroides xylanisolvens and overexpressed in soluble form in Escherichia coli. The expression conditions of BxHep-I were optimized for an activity of 7144 U/L. BxHep-I had a specific activity of 57.6 U/mg at the optimal temperature and pH of 30 °C and pH 7.5, with the Km and Vmax of 0.79 mg/mL and 124.58 U/mg, respectively. BxHep-I catalytic activity could be enhanced by Ca[sup.2+] and Mg[sup.2+], while strongly inhibited by Zn[sup.2+] and Co[sup.2+]. Purified BxHep-I displayed an outstanding thermostability with half-lives of 597 and 158 min at 30 and 37 °C, respectively, which are the highest half-lives ever reported for heparinases I. After storage at 4 °C for one week, BxHep-I retained 73% of its initial activity. 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subjects Cloning
Ethylenediaminetetraacetic acid
Sugars
title Cloning, Expression, and Characterization of a Highly Stable Heparinase I from IBacteroides xylanisolvens/I
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