In:
Journal of Industrial Microbiology and Biotechnology, Oxford University Press (OUP), Vol. 50, No. 1 ( 2023-02-17)
Abstract:
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.
Type of Medium:
Online Resource
ISSN:
1367-5435
,
1476-5535
DOI:
10.1093/jimb/kuad012
Language:
English
Publisher:
Oxford University Press (OUP)
Publication Date:
2023
detail.hit.zdb_id:
1482484-X
SSG:
12
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