β d glucose pentaacetate

Restriction endonucleases, HindIII, BamHI, PstI, Phusion DNA polymerase, aLICator™ LIC Cloning and Expression System Kit 3, PageRuler^TM Prestained Protein Ladder were purchased from Thermo Fisher Scientific, Vilnius, Lithuania, Pierce™ Coomassie Plus (Bradford, UK) Assay Reagent and HisPur™ Ni‐NTA spin column were purchased from Thermo Fisher Scientific, Rockford, IL. pET21a Vector was purchased from Novagen (Madison, USA). Nutrient medium were purchased Roth, Germany. pNP‐acyl esters, tributyrin, β‐d‐glucose pentaacetate, β‐d‐galactose pentaacetate, and uridine were purchased from Sigma‐Aldrich. ‘ZR Soil Microbe DNA MidiPrep™’ was purchased from Zymo Research, Freiburg, Germany. Nitrocefin was purchased from Oxoid, UK. 3′‐O‐benzoyl‐2′‐deoxyuridine were purchased from Carbosynth, UK. 3′‐O‐acetyl‐2′‐deoxyuridine, 3′‐O‐acetyl‐N⁴‐benzoyl‐2′‐deoxycytidine, 3′‐O‐levulinyl‐N⁴‐benzoyl‐2′‐deocytidine, and 5′‐O‐levulinyl‐N⁴‐benzoyl‐2′‐deocytidine were purchased from Jena Bioscience, Jena, Germany.

MZ0003 deacetylase activity was detected using the Acetate Colorimetric Assay Kit (Sigma-Aldrich, St Louis, MO, USA). The substrates used, purchased from Sigma-Aldrich, were: 4-methylumbelliferyl acetate and 7-aminocephalosporanic acid (dissolved in DMSO); α-naphtyl acetate, β-D-glucose pentaacetate and β-D-galactose pentaacetate (dissolved in methanol); 5-bromo-4-chloro-3-indoyl acetate (dissolved in ethanol); p-NP acetate and cellulose acetate (dissolved in acetonitrile). The reaction mixtures, each containing 10 μg of protein and substrate, were pre-incubated in 5 mM Na-phosphate buffer pH 7.3 for 30 min at 35°C. Thereafter the assay was developed according to the manufacturer’s instructions and the absorbance was read at 450 nm. Values for each substrate were taken as the mean of three independent measurements after subtraction of a blank sample, without MZ0003, to correct for background degradation. The amount of acetate liberated was calculated in nM μl^-1 from a standard curve according to the manufacturer’s instructions.

The slug gut metagenome DNA was provided by Dr Natalie Ferry, University of Salford, Manchester, United Kingdom. The substrates—4-nitrophenyl acetate, 4-nitrophenyl butyrate, 4-nitrophenyl octanoate, 4-nitrophenyl decanoate, and 4-nitrophenyl palmitate, β-D-glucose pentaacetate, pectin, chlorogenic acid, benzyl cinnamate, and 4-methylumbelliferyl acetate—were purchased from Sigma-Aldrich (Gillingham, United Kingdom). Xylan (birchwood, partially acetylated) was purchased from Megazyme (Wicklow, Ireland). Other chemicals were of molecular grade and purchased from Sigma-Aldrich (Gillingham, United Kingdom). Primers for PCR were synthesised and sequenced by Eurofins (Konstanz, Germany). BL21 and TOP10 competent cells were purchased from ThermoFisher Scientific (Leicester, United Kingdom).