Activity across temperature and pH of PET hydrolase candidates.

These data are a condensed version of data from our upcoming manuscript re. identification of novel PET hydroloases with activity across temperature and pH. PLACEHOLDER. It is used in our paper describing AIDE to showcase the utility of the software for rapidly developing supervised homolog predictors. Included: csv of sequences and activity across conditions HMM61, the null model for PETase activity predicted crystal structures from AF2 for each sequence Experimental for expression and activity measurement of PET hydrolases: Genes were codon optimized for E. coli expression, synthesized (Twist Biosciences), and cloned into pCDB179 vector (Addgene #91960) with N-terminal 10xHis-SUMO fusion. Proteins were expressed in E. coli C41(DE3) using autoinduction media in 24-deep well plates. Expression, lysis, and nickel affinity purification used an OT-2 robotic platform as previously described. SUMO tags were cleaved with His-tagged SUMO protease. Protein concentration was measured by BCA assay and normalized to 0.1 mg/mL. PET substrates (amorphous film or crystalline powder from Goodfellow) were loaded into 96 deep-well plates (3.5 mg/well). 450 µL buffer (50 mM NaCitrate for pH 4.5/5.5; 50 mM NaPhosphate for pH 6.5/7.5) and 50 µL enzyme solution (5 µg) were added per well. Plates were sealed and incubated at 40°C or 60°C for 48 h. Product release was quantified by measuring absorbance at 260/280 nm of reaction supernatant, with powder reactions filtered through MultiScreenHTS GV Filter Plates prior to analysis.