Improving the acidic stability of a methyl parathion hydrolase by changing basic residues to acidic residues
Lu Huanget al
Supplementary materials(table and figures)
Contents
Supplementary Table 1 Primers used in this study
SupplementaryFigure 1 SDS-PAGE analysis of the purified WT and mutant enzymes with or without His-tag.
SupplementaryFigure 2 Effects of pH on the enzyme activity of the WT（Ochr-MPH）and mutants.
SupplementaryFigure 3The stability of the WT and mutant enzymes (K208D, K277E)under various pH values ranging from 4.0–9.0.
SupplementaryFigure 4The half-lives of the WT and its mutants (K208D, K277E) at pH 5.0 (A) and 6.0 (B).
SupplementaryTable 1 Primers used in this study
Mutant / Oligonucleotide sequenceK79E_F a / AACCAACCAGCTCCTGAAACTCAGTCTGCTTTG
K79E_R b / TTCAGGAGCTGGTTGGTTCAATCT
K208E_F a / CTTAACCCATACGTTGAAGCCGGTAAGTTCAAG
K208E_R b / TTCAACGTATGGGTTAAGGGAAGC
K277D_F a / TTGGACTCTGACTCCGATTCTGCTGCCGTTGAG
K277D_R b / ATCGGAGTCAGAGTCCAATTGGTT
K208D_F a / CTTAACCCATACGTTGATGCCGGTAAGTTCAAG
K208D_R b / ATCAACGTATGGGTTAAGGGAAGC
K277E_F a / TTGGACTCTGACTCCGAATCTGCTGCCGTTGAG
K277E_R b / TTCGGAGTCAGAGTCCAATTGGTT
The mutation sites were indicated by the underlined, bold sequences.a Forward primer.
b Reverse primer.
SupplementaryFigure 1 SDS-PAGE analysis of the purified WT and mutant enzymes with or without His-tag. Lane 1, Standard protein molecular weight;Lane 2, WT with His-tag; Lane 3, His-tag-free WT; Lane 4, K208E with His-tag; Lane; 5,His-tag-free K208E; Lane 6, K277D with His-tag; Lane 7, His-tag-free K277D; Lane 8, K208E/ K277D with His-tag; Lane 9, His-tag-free K208E/ K277D. The concentration of the proteinswas approximately 200 µg/mL for Lane 2, 3, 6,7,8,9 and 120 µg/ml for Lane 4,5. 10 µL sampleswereloaded for each protein.
SupplementaryFigure 2Effects of pH on the enzyme activity of the WT（Ochr-MPH）and mutants. The displayed activities were normalized relative to the activities measured at optimal pH, which were expressed as 100%. (A) The pH profile of Ochr-MPH, K208E, K277D and K208E/K277D. (B) The pH profile of Ochr-MPH, K208D, K277E.The enzyme activity was determined at pH 5.0 to 8.0 using McIlvaine buffer, pH9.0 using Tris-HCl buffer, and pH10.0 to 10.6 using Glycine-NaOH buffer.Means and standard deviations of triplicate experiments were shown.
SupplementaryFigure 3The stability of the WT and mutant enzymes (K208D, K277E)under various pH values ranging from 4.0-9.0.Activity was measured after the proteins were incubated for 1h under pH 4.0 to 8.0 using McIlvaine buffer and pH 9.0 using Tris-HCl buffer. A non-treated enzyme served as thecontrol (100%). Data points correspond to the mean values of three independent experiments.
SupplementaryFigure 4The half-lives of the WT and its mutants (K208D, K277E)at pH 5.0 (A) and 6.0 (B). Plot of Ln percent residual activity versus time (min) from which the rate constants (k = -slope) and half-lives could be estimated (t1/2 = 0.693/k). Activity was measured after the proteins were incubated for 10-80 min under pH5.0 (A) and 6.0 (B). A non-treated enzyme served as thecontrol (100%). Means and standard deviations of triplicate experiments were shown.
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