A robust protocol for directed aryl sulfotransferase evolution towards GlcNAc
Congratulations to Shohana Islam on her recent Publication!Copyright: © Bio VI
Sulfurylation is of biomolecules is well-spread in the nature, which plays an important role in biological events such as signal transduction, detoxification, molecular recognition, and hormone regulation. Chemical sulfurylation is performed in the industries that often requires several steps, hazardous chemicals and usually lacks regio- and chemoselectivity. On the other hand, the enzyme class, sulfotransferase catalyzes the selective transfer of a sulfuryl group from a donor to an acceptor molecule. Among the sulfotransferases, the class of bacterial aryl sulfotransferases are of growing interest because of their broad acceptor spectrum and cost-effective sulfuryl donor. As an alternative to chemical methods enzymatic sulfurylation is proposed, in which directed evolution can play an important role to improve the catalytic efficiency and substrate specificity of industrially relevant aryl sulfotransferases.
A directed aryl sulfotransferase evolution protocol was successfully validated to improve the specific activity towards a monosaccharide, N-acetylglucosamine – GlcNAc. A random mutagenesis library of aryl sulfotransferase B from Desulfitobacterium hafniense was generated using sequence saturation mutagenesis - SeSaM. The screening of 1760 clones was performed via advanced and optimized para-nitrophenylsulfate based screening system in 96-well format. The identified best variant, ASTB-V1 with a substitution Val579Asp showed an up to 3.4-fold increased specific activity and 2.4-fold higher monosulfurylated N-acetylglucosamine production determined via high-performance liquid chromatography and mass spectrometry.
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Islam S. S., Mate M. D., Martínez, R., Jakob, F., Schwaneberg, U.; A robust protocol for directed aryl sulfotransferase evolution towards the carbohydrate building block GlcNAc; Biotechnology & Bioengineering, Accepted manuscript online: 30 December 2017, DOI: 10.1002/bit.26535