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A robust protocol for directed aryl sulfotransferase evolution toward the carbohydrate building block GlcNAc
Author(s) -
Islam Shohana,
Mate Diana M.,
Martínez Ronny,
Jakob Felix,
Schwaneberg Ulrich
Publication year - 2018
Publication title -
biotechnology and bioengineering
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.136
H-Index - 189
eISSN - 1097-0290
pISSN - 0006-3592
DOI - 10.1002/bit.26535
Subject(s) - chemistry , carbohydrate , biochemistry , aryl , mutagenesis , sulfotransferase , stereochemistry , sulfation , organic chemistry , mutation , gene , alkyl
Bacterial aryl sulfotransferases (AST) utilize p ‐nitrophenylsulfate ( p NPS) as a phenolic donor to sulfurylate typically a phenolic acceptor. Interest in aryl sulfotransferases is growing because of their broad variety of acceptors and cost‐effective sulfuryl‐donors. For instance, aryl sulfotransferase A (ASTA) from Desulfitobacterium hafniense was recently reported to sulfurylate d ‐glucose. In this study, a directed evolution protocol was developed and validated for aryl sulfotransferase B (ASTB). Thereby the well‐known p NPS quantification system was advanced to operate efficiently as a continuous screening system in 96‐well MTP format with a true coefficient of variation of 14.3%. A random mutagenesis library (SeSaM library) of ASTB was screened (1,760 clones) to improve sulfurylation of the carbohydrate building block N‐acetylglucosamine (GlcNAc). The beneficial variant ASTB‐V1 (Val579Asp) showed an up to 3.4‐fold increased specific activity toward GlcNAc when compared to ASTB‐WT. HPLC‐ and MS‐analysis confirmed ASTB‐V1's increased GlcNAc monosulfurylation (2.4‐fold increased product formation) representing the validation of the first successful directed evolution round of an AST for a saccharide substrate.