硫酸化
催化作用
硫转移酶
硫酸乙酰肝素
硫酸盐
化学
生物化学
糖胺聚糖
有机化学
作者
Xintong Xi,Litao Hu,Hao Huang,Yang Wang,Ruirui Xu,Guocheng Du,Jian Chen,Zhen Kang
出处
期刊:Journal of Industrial Microbiology & Biotechnology
[Oxford University Press]
日期:2023-01-01
卷期号:50 (1)
被引量:3
摘要
The chemo-enzymatic and enzymatic synthesis of heparan sulfate and heparin are considered as an attractive alternative to the extraction of heparin from animal tissues. Sulfation of the hydroxyl group at position 2 of the deacetylated glucosamine is a prerequisite for subsequent enzymatic modifications. In this study, multiple strategies, including truncation mutagenesis based on B-factor values, site-directed mutagenesis guided by multiple sequence alignment, and structural analysis were performed to improve the stability and activity of human N-sulfotransferase. Eventually, a combined variant Mut02 (MBP-hNST-NΔ599-602/S637P/S741P/E839P/L842P/K779N/R782V) was successfully constructed, whose half-life at 37°C and catalytic activity were increased by 105-fold and 1.35-fold, respectively. After efficient overexpression using the Escherichia coli expression system, the variant Mut02 was applied to N-sulfation of the chemically deacetylated heparosan. The N-sulfation content reached around 82.87% which was nearly 1.88-fold higher than that of the wild-type. The variant Mut02 with high stability and catalytic efficiency has great potential for heparin biomanufacturing.
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