Directed aryl sulfotransferase evolution toward improved sulfation stoichiometry on the example of catechols.

Sulfation is an important way for detoxifying xenobiotics and endobiotics including catechols. Enzymatic sulfation occurs usually with high chemo- and/or regioselectivity under mild reaction conditions. In this study, a two-step p-NPS-4-AAP screening system for laboratory evolution of aryl sulfotransferase B (ASTB) was developed in 96-well microtiter plates to improve the sulfate transfer efficiency toward catechols. Increased transfer efficiency and improved sulfation stoichiometry are achieved through the two-step screening procedure in a one-pot reaction. In the first step, the p-NPS assay is used (detection of the colorimetric by-product, p-nitrophenol) to determine the apparent ASTB activity. The sulfated product, 3-chlorocatechol-1-monosulfate, is quantified by the 4-aminoantipyrine (4-AAP) assay in the second step. Comparison of product formation to p-NPS consumption ensures successful directed evolution campaigns of ASTB. Optimization yielded a coefficient of variation below 15% for the two-step screening system (p-NPS-4-AAP). In total, 1760 clones from an ASTB-SeSaM library were screened toward the improved sulfation activity of 3-chlorocatechol. The turnover number (kcat = 41 ± 2 s-1) and catalytic efficiency (kcat/KM = 0.41 µM-1 s-1) of the final variant ASTB-M5 were improved 2.4- and 2.3-fold compared with ASTB-WT. HPLC analysis confirmed the improved sulfate stoichiometry of ASTB-M5 with a conversion of 58% (ASTB-WT 29%; two-fold improvement). Mass spectrometry (MS) and nuclear magnetic resonance spectroscopy (NMR) confirmed the chemo- and regioselectivity, which yielded exclusively 3-chlorocatechol-1-monosulfate. For all five additionally investigated catechols, the variant ASTB-M5 achieved an improved kcat value of up to 4.5-fold and sulfate transfer efficiency was also increased (up to 2.3-fold).

Targeting microplastic particles in the void of diluted suspensions.

Accumulation of microplastic in the environment and food chain will be a grand challenge for our society. Polyurethanes are widely used synthetic polymers in medical (e.g. catheters) and industrial products (especially as foams). Polyurethane is not abundant in nature and only a few microbial strains (fungi and bacteria) and enzymes (polyurethaneases and cutinases) have been reported to efficiently degrade polyurethane. Notably, in nature a long period of time (from 50 to >100 years depending on the literature) is required for degradation of plastics. Material binding peptides (e.g. anchor peptides) bind strongly to polymers such as polypropylene, polyethylene terephthalate, and polyurethane and can target specifically polymers. In this study we report the fusion of the anchor peptide Tachystatin A2 to the bacterial cutinase Tcur1278 which accelerated the degradation of polyester-polyurethane nanoparticles by a factor of 6.6 in comparison to wild-type Tcur1278. Additionally, degradation half-lives of polyester-polyurethane nanoparticles were reduced from 41.8 h to 6.2 h (6.7-fold) in a diluted polyester-polyurethane suspension (0.04% w/v).

KnowVolution Campaign of an Aryl Sulfotransferase Increases Activity toward Cellobiose.

Sulfated polysaccharides such as cellulose can mimic the functionalities of pathophysiologically important glycosaminoglycans. Enzymatic sulfation offers a green chemistry route to selective (mono)sulfation of oligosaccharides (e.g., cellobiose as a building block of cellulose) in aqueous solution, at ambient temperature, and high chemoselectivity. Here, we report the first KnowVolution campaign for the aryl sulfotransferase B (ASTB) from Desulfitobacterium hafniense to advance ASTB toward a synthetically attractive biocatalyst. The generated final recombination variant (ASTB-M5) carries two amino acid substitutions (Leu446Pro and Val579Lys) leading to an up to 7.6-fold increase in specific activity (6.15 U mg-1 ) that was obtained with one round of KnowVolution. Mass spectrometry analysis confirmed a monosulfated product of cellobiose and structure elucidation by NMR confirmed the sulfation at the positions C-3 or C-4 of GlcNAc-linker-tBoc as opposed to the preferred C-6 by chemical means. Computational analysis suggested an important role of Leu446Pro in substrate-binding and recognized Val579Lys as a distal substitution.

A robust protocol for directed aryl sulfotransferase evolution toward the carbohydrate building block GlcNAc.

Bacterial aryl sulfotransferases (AST) utilize p-nitrophenylsulfate (pNPS) 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 pNPS 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.