Construction And Screening Of Pseudomonas Aeruginosa Arylsulfatase Mutant Library

Pseudomonas aeruginosa arylsulfatase(PAAS)is a biopharmaceutical tool enzyme and potential labeling enzyme for immunoassay.However,the wild type of PAAS possesses poor catalytic activity and Enzyme engineering to improve catalytic activity of PAAS is required.High-throughput(HTP)screening of mutant libraries has been established through the fusion expression of two enzymes for determining the activity ratios of mutants to the tag enzyme by SDESA for comparison.The fused form of ECAP-PAAS was thus constructed for the screening of mutant libraries of PAAS by the determination of activity ratios of PAAS/mutants to ECAP as the indices.The lysates of fused M72 mutants were prepared with both alkaline lysis and ultrasonication treatment separately,to determine the activity ratios.There was no statistically significant difference between results by the two lysis methods,and thus the alkaline lysis was utilized for HTP preparation of lysate.The threshold for HTP screening was determined by measuring the inherent ECAP expression of ECAP lysates and the fusion expression of ECAP-PAAS was recognized when ECAP activity ? 0.0053 kU/L.The fusion expression of ECAP-PAAS in combination with 48-well plate culture,alkaline lysis,and 96-well microplate assay of activity ratios was applicable for HTP screening of PAAS mutants.To explore the reliability of the activity ratios of the fusion mutants for HTP screening of libraries and elucidation of sequence-activity relationship,the apparent specific activities of non-fused M72 mutants were determined by ITA for comparison with those determined as activity ratios for the fused mutants.Fortunately,their excellent correlation supported a high consistency of results between two methods.HTP screening based on the fusion expression of two enzymes for determining their activity ratios was thus promising for HTP screening of mutant libraries of PAAS and analysis of sequence-activity relationship.Iterative saturation mutation(ISM)is an effective strategy for directed evolution of enzymes.The formyl group(DDZ)of C51 is a putative catalytic residue of PAAS.Residues R55/M72/G138/D317/K375 were thus chosen for ISM since they located in close proximity to the active site of PAAS.Site-directed saturation mutagenesis libraries were constructed using the random primers(NNN)of a selected site,an equal-mole mixture of precise primers and a single purified precise primer each time for PCR amplification to reveal the suitable approach to libraries with site-directed saturation mutagenesis of M72 as the model.Results showed that the use of random primers for PCR resulted in just 10 among 20 expected mutants after the screening of over 600 monoclones,besides obvious codon bias;the use of an equal-mole mixture of precise primers yielded all of the 19 expected mutants after the screening of no more than 190 monoclones;site-directed saturation mutagenesis of M72 with the purified precise primers for PCR one-by-one gave all of the 19 expected mutants after the screening of just 2 monoclones for every expected mutant.The use of the precise primers for PCR to generate the complete library required fewer number of monoclones screened,less overall time and cost.Consequently,during site-directed saturation mutagenesis,the use of equal-mole mixture of precise primers for PCR was preferable for HTP screening of positive mutants based on the fusion expression for determining the activity ratios;while the use of precise primers for PCR one-by-one was more practical when the burden for screening is too heavy or the sequence-activity relationship is needed to be elucidated.There were no mutants with higher activity than wild type of PAAS after screening of the site-directed saturation mutagenesis libraries of R55/M72/G138/D317/K375 constructed with the purified precise primers.It's possible that the sites above are not the suitable candidate sites for ISM,or the synergic mutations can improve the activity of PAAS.In conclusion,for the construction and HTP screening of site-directed saturation mutagenesis library,(a)if positive mutants of high activity are desired only,the equal-mole mixture of precise primers combined with HTP screening based on the fusion expression of two enzymes is favorable;(b)if the sequence-activity relationship besides positive mutants of higher activity of PAAS is needed,the purified precise primers for PCR one-by-one in combination with HTP screening based on the fusion expression of two enzymes is preferable.