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 the 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.

中文翻译：

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硫酸乙酰肝素N-磺基转移酶制备N-硫酸乙酰肝素稳定性和催化效率的提高

硫酸乙酰肝素和肝素的化学酶法和酶法合成被认为是从动物组织中提取肝素的有吸引力的替代方案。脱乙酰氨基葡萄糖2位羟基的硫酸化是后续酶促修饰的先决条件。本研究采用基于B因子值的截断诱变、多重序列比对和结构分析引导的定点诱变等多种策略来提高人N-磺基转移酶的稳定性和活性。最终成功构建了组合变体Mut02（MBP-hNST-NΔ599-602/S637P/S741P/E839P/L842P/K779N/R782V），其37℃半衰期和催化活性分别提高了105倍和1.35倍。分别折叠。使用大肠杆菌表达系统进行有效过表达后，将变体 Mut02 用于化学脱乙酰肝素的 N-硫酸化。N-硫酸化含量达到82.87%左右，比野生型高出近1.88倍。Mut02变体具有高稳定性和催化效率，在肝素生物制造方面具有巨大潜力。