3.4 Identification of the effect of each mutation on MPH
The SPAprE and MPH coding region of mutants MT1 and MT2 was sequenced. The mutation sites were shown in Figure 4a. Mutant MT1 contained three mutations, with two (T47C and G81T) located in SPAprE coding region and one (G938A) located in the MPH coding region. Among these three mutations, two led to amino acid change (Leu16Ser and Arg313His), while G81T (Ala27Ala) is a synonymous mutation. All three mutations of mutant MT2 located in MPH coding region (T806A, C821T and C892T), resulting in the amino acid substitutions of Ile269Ser, Ala274Val and Pro298Ser.
To determine the contribution of each mutation to the enhanced extracellular MPH activity of mutants MT1 and MT2, each mutation was individually introduced into strain BPC1, generating mutants MT-16, MT-27, MT-269, MT-274, MT-298 and MT-313. All six mutations did not affect the growth of their hosts compared with strain BPC1 (data not shown). The extracellular MPH activity of mutants MT-16, MT-27, MT-269, MT-298 and MT-313 were 1.73, 1.40, 1.75, 1.48 and 2.03 fold that of strain BPC1, while mutant MT-274 showed negative effect on the extracellular MPH activity (Figure 4b). The extracellular MPH of mutants MT-16, MT-27, MT-269, MT-298 and MT-313 were purified through Ni-NTA affinity chromatography. The MPH protein yield of MT-269, MT-298 and MT-313 were close to that of strain BPC1, while the MPH protein yield of MT-16 and MT-27 were 1.70 and 1.46 fold that of strain BPC1 (Figure 4d). Moreover, the specific activity of variants T806A, C892T and G938A were 1.53, 1.43 and 1.52 fold of wild-type MPH, while the variants T47C and G81T were comparable with that of wild-type MPH (Figure 4c). Taken together, mutations T47C and G81T in the SPAprE coding region enhanced extracellular MPH activity through increasing MPH protein yield, while the contribution of mutations T806A, C892T and G938A was enhancing the catalytic activity of MPH against chlorpyrifos. Additionally, to further enhance the extracellular MPH activity, two new mutants were created through recombining the mutations. Mutant MT-C2 harbors mutations T47C and T806A, and mutant MT-C3 contains mutations of T47C, T806A and G938A. The extracellular MPH activities of mutants MT-C2 and MT-C3 were 2.78 and 3.32 fold that of strain BPC1 (Figure 4e and 4f), respectively, indicating that the contribution of these mutations could be accumulated when combined together.