Data for high-throughput screening of enzyme mutants by comparison of their activity ratios to an enzyme tag

Data in this article are associated with the research article “High-throughput screening of enzyme mutants by comparison of their activity ratios to an enzyme tag” (Li et al., 2019) [1]. Data are provided on the development of a system for high-throughput (HTP) screening of mutants through the comparison of the activity ratios of an applicable enzyme and its mutants to a suitable tag enzyme in cell lysates of their fused forms, with Escherichia coli alkaline phosphatase (ECAP) as the tag fused to the N-terminus of Pseudomonas Aeruginosa arylsulfatase (PAAS) and its mutants via a flexible linker. Data were made publicly available for further analyses.


Data description
The data in this article provides information on how to develop an experimental system for HTP screening of mutants through the comparison of the activity ratios of an applicable enzyme and its mutants to a suitable enzyme tag in cell lysates of their fused forms (Figs. 1e4 and Tables 1e7). Data Specifications Table   Subject Chemistry, Biology Specific subject area Biomolecule engineering Type of data Figure, Table  How data were acquired The adsorbance change for enzyme activity assay was recorded with Biotek ELX 800 at room temperature. Data format Raw and Analyzed Parameters for data collection The mixture of cell lysate of 20 mL and substrate of 180 mL containing both or one of final 2.0 mM 4NPS and final 0.20 mM 4NNPP in a well was agitated for 2.0 min at room temperature, to record the absorbance in 30 min after the total lagging time of 4.0 min. Description of data collection Fusion expression of ECAP with PAAS and its mutants in DE3; Protein bands after SDS-PAGE staining with Coomassie brilliant blue or antibodies following standard protocol; SDESA or separate single assay of ECAP and PAAS/mutant to derive their activity ratios. Data  Value of the Data The dataset in this report will facilitate understanding the validation and application of a new high-throughput screening strategy to mutants of an enzyme in a library [1]. To recognize positive mutants in a library, this new screening strategy fuses enzyme/mutants with a tag enzyme to compare activity ratios of the enzyme/mutants to the tag enzyme in cell lysates of their fused forms, when such activity ratios have physical significance and are proportional to specific activities of the non-fused counterpart enzyme/mutants. Re-analyses of these data will benefit researchers to develop a practical system of the new high-throughput screening strategy for directed evolution of an applicable enzyme. The data in this report utilizes Escherichia coli alkaline phosphatase (ECAP) as the tag enzyme for fusion with Pseudomonas Aeruginosa arylsulfatase (PAAS) via a flexible linker. Through the analyses of the data of the activities of ECAP in lysates of both Escherichia coli BL21 (DE3) transformed with a blank plasmid and host cells transformed with the fused mutants of PAAS, a rational threshold of ECAP activities in cell lysates can be developed for physical significance of the activity ratios of their fused forms at a preset confidence limit. Meanwhile, with a focused library of PAAS mutants through saturated mutagenesis at M72, the data enable researchers to understand the proportionality between the activity ratios of PAAS/ mutants to ECAP in cell lysates of their fused forms and specific activities of the non-fused counterpart PAAS/mutants. The data in this report will provide insights on the application of the new screening strategy to the elucidation of sequence-activity relationship of an applicable enzyme.

Experimental design
The comparison of the activity ratios of an applicable enzyme and its mutants to a suitable enzyme tag in cell lysates of their fused forms for HTP screening of mutants requires both the negligible or  consistent impacts of the enzyme tag on the activities of enzyme/mutants and the proportionality of the activities of the enzyme tag in cell lysates of the fused enzyme/mutants to protein quantities of the active forms of the fused enzyme/mutants in both their native and partially-fragmented fused states. With ECAP as the tag fused to the N-terminus of PAAS and its mutants via a flexible linker, the proposed strategy was tested.

Materials and methods
For each fused enzyme/mutant, an individual clone was transferred into 0.50 mL LB medium in 48well microplate for amplification in 12 h at 37 C and 180 rpm till optical density of 0.4e0.6 at 600 nm. Afterwards, each enzyme/mutant was induced for expression with 1.0 mM IPTG for 21 h at 15 C. The lysates of fused mutants were prepared through alkaline lysis, unless otherwise stated. In detail, cell suspension of 20 mL from a well was transferred to a new well for mixing with 180 mL of the alkaline lysis buffer (1.0 M Tris-HCl at pH 9.0, plus 1.0 mM PMSF and 2.5 mM 4-aminobenzamidine) in 96-well microplates; the resulting mixture was agitated rapidly on Qilinbeier QB-9001 agitator for 4 h at room temperature to yield a cell lysate.
The substrate solution containing 2.0 mM 4NPS and/or 0.20 mM 4NNPP was utilized to monitor the absorbance change at 405 and/or 450 nm (The substrate solution containing both substrates was utilized for spectrophotometric-dual-enzyme-simultaneous-assay (SDESA) of ECAP and PAAS/mutant [2,3]). For HTP assay of enzyme activity(ies), cell lysate of 20 mL was mixed with a substrate solution of 180 mL in 96-well microplates. The mixtures in wells were agitated for 2.0 min at room tempearture; after a total lagging time of 4.0 min, the absorbance of each well was recorded in 30 min at room tempearture to estimate initial rate for enzyme activity by linear regression with the preset absorptivity of 12 (mmol) À1 $L À1 $cm À1 for 4-nitrophenol and 19 (mmol) À1 $L À1 $cm À1 for 4-nitro-1-naphthol considering the light path. The change of absorbance of no less than 0.003 in 10 min was taken as the detection limit of enzyme activity, after the correction of the overlapped absorbance of chromogenic products during SDESA.   a Activity ratio was the activity of PAAS/mutant to that of ECAP in their fused form.

Table 2
Activity ratios of three fused forms via different linkers.  The activity ratio of PAAS/mutant to ECAP was the percentage of PAAS/mutant activity on 4NPS to ECAP activity on 4NNPP. Receiver-operating-characteristic (ROC) analysis yielded the area-under-thecurve (AUC) for the recognition of the one of higher activity in a pair.