Expression optimization and molecular modification of heparin C5 epimerase.

Bingbing Wang; Zhengxiong Zhou; Xuerong Jin; Jianghua LI; Zhongping Shi; Zhen Kang

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Expression optimization and molecular modification of heparin C5 epimerase.

Author: Bingbing WANG ¹ ; Zhengxiong ZHOU ¹ ; Xuerong JIN ¹ ; Jianghua LI ¹ ; Zhongping SHI ¹ ; Zhen KANG ¹
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1. Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi 214122, Jiangsu, China.
Publication Type:Journal Article
Keywords: glucuronic acid-C5-epimerase; heparin; heterologous expression; rational design; substrate binding pocket
MeSH: Animals; Carbohydrate Epimerases; biosynthesis; chemistry; genetics; Escherichia coli; Gene Expression; Heparin; metabolism; Heparitin Sulfate; metabolism; Iduronic Acid; metabolism; Zebrafish Proteins; biosynthesis; chemistry; genetics
From: Chinese Journal of Biotechnology 2020;36(7):1450-1458
CountryChina
Language:Chinese
Abstract: Heparin and heparan sulfate are a class of glycosaminoglycans for clinical anticoagulation. Heparosan N-sulfate-glucuronate 5-epimerase (C5, EC 5.1.3.17) is a critical modifying enzyme in the synthesis of heparin and heparan sulfate, and catalyzes the inversion of carboxyl group at position 5 on D-glucuronic acid (D-GlcA) of N-sulfoheparosan to form L-iduronic acid (L-IdoA). In this study, the heparin C5 epimerase gene Glce from zebrafish was expressed and molecularly modified in Escherichia coli. After comparing three expression vectors of pET-20b (+), pET-28a (+) and pCold Ⅲ, C5 activity reached the highest ((1 873.61±5.42) U/L) with the vector pCold Ⅲ. Then we fused the solution-promoting label SET2 at the N-terminal for increasing the soluble expression of C5. As a result, the soluble protein expression was increased by 50% compared with the control, and the enzyme activity reached (2 409±6.43) U/L. Based on this, site-directed mutations near the substrate binding pocket were performed through rational design, the optimal mutant (V153R) enzyme activity and specific enzyme activity were (5 804±5.63) U/L and (145.1±2.33) U/mg, respectively 2.41-fold and 2.28-fold of the original enzyme. Modification and expression optimization of heparin C5 epimerase has laid the foundation for heparin enzymatic catalytic biosynthesis.