Characterization Of Pseudomonas Aeruginosa Arylsulfatase By Site-directed Mutagenesis

Background: Pseudomonas Aeruginosa Arylsulfatase as a candidate label enzyme for SDESA-ELISA, which structure has been reported, is difficult to meet the needs of the application,because the specific activity and thermal stability at 37℃ of it is very low.Thus molecular engineering is needed, site-directed mutagenesis are more applied in practice, because of its high efficiency.Purpose:By site-directed mutagenesis assistanting the analysis of structure-function relationship, to obtain the desirable Pseudomonas Aeruginosa arylsulfatase mutants; having high specific activity and good thermal stability,which could meet the prerequisites of SDESA-ELISA.Method The recombinant expression and purification of arylsulfatase; around the active site,point at catalytic area; after confirmed by the sequencing, combined with PET-24 a containing 6-His label, construct the site–directed mutagenesis,after transformed,cultivated,induced,cracked and purified,then characterization of the mutants.Further analysis on the special phenomenon,based on it,design new site-directed mutagenesis and characterization again.Then characterization of the mutants such as SDS-PAGE and PAGE,specific activity, kinetic parameters,optimal pH, the effect of metal ion and so on;The exploration of inactivation in reaction : compared the kinetics reaction curve of PAAS mutants; explored inhibition of products and the stability in reaction period. Point at M72 K,using magnetic beads to adsorpt, separate, release and react; analysis the mechanism of M72 K by MALDI-TOF and LTQ Orbitrap to decide whether there are some modification in reaction.The thermal stability of PAAS mutants: investigate its half-life at 37 ℃ and 4 ℃; analysis the mechanism of special phenomena by comparing with the wild type at the same conditions,scanning absorbance and fluorescence emission spectrum,doing synchronous fluorescence and MALDI-TOF; Using different types of buffer storage,compared the thermal stability of PAAS at 37 ℃.Result Compared with the wild type,there were significant differences in kinetic parameters, specific activity,optimal pH,the effect of metal ion;especially R55K(almost inactivation), K375(change for the specificity of substrate), and M72(kinetic parameters down about two orders of magnitude).the inactivation phenomemon appeared obviously when mutants M72 X and its natural substrate PNS in reaction; Results show that may be sulphation modification effect happened in the process of reaction by MALDI-TOF;Besides when M72 K storage at 37℃ in pH7.4 different kinds of buffer,the thermal activation existed obviously, that is to say,when M72 K in pH7.4 buffer at 37℃,the specific activity for PNS increased more than two orders of magnitude within a certain period of time; Compared with the wild type,both absorbance and fluorescence emission spectrum of M72 K have significant differences. While, in the aspect of thermal stability of PAAS, there have differences between different kinds of storage buffer. when in 10 mM pH7.4 sodium borate at 37℃, the half-life time of PAAS extended.Conclusion: There are a great change of the mutants in properties, and special phenomenon appeared. This provides a certain basis in preliminary explore structure-activity relationship.