The Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, Jiangnan University, Wuxi 214122, China.
Science Center for Future Foods, Jiangnan University, Wuxi 214122, China.
J Ind Microbiol Biotechnol. 2023 Feb 17;50(1). doi: 10.1093/jimb/kuad012.
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.
肝素和硫酸乙酰肝素的化学-酶法和酶法合成被认为是一种有吸引力的替代方法，可替代从动物组织中提取肝素。脱乙酰葡萄糖胺 2 位羟基的硫酸化是随后进行酶修饰的前提条件。在这项研究中，采用了多种策略，包括基于 B 因子值的截断突变、多重序列比对指导的定点突变以及结构分析，以提高人 N-磺基转移酶的稳定性和活性。最终，成功构建了一个组合变体 Mut02（MBP-hNST-NΔ599-602/S637P/S741P/E839P/L842P/K779N/R782V），其半衰期在 37°C 时提高了 105 倍，催化活性提高了 1.35 倍。在大肠杆菌表达系统中进行有效的过表达后，该变体 Mut02 被应用于化学脱乙酰肝素的 N-磺化。N-磺化含量达到约 82.87%，比野生型高近 1.88 倍。该变体 Mut02 具有高稳定性和催化效率，在肝素生物制造方面具有很大的潜力。
