Improving The Stability Of Creatinase Based On Chemical And Molecular Modification

As one kind of hydrolases,creatinase(EC 3.5.3.3,CRE)catalyzes the hydrolysis of creatine into urea and creatine,therefore plays important role in the metabolism of creatinine.CRE can be used as a diagnostic enzyme to detect creatinine levels in human serum and urine.The enzyme from pseudomonas putida was stable only under tempareture lower than 40?,which was not conducive to the industrial production of the CRE.In this study,the original cre sequence was taken from P.putida.The research works mainly involved the heterologo us expression of CRE in E.coli,the surface chemical modification of CRE,the analysis on the structure of CRE by bioinformatic software,and the construction of intersubunit disulfide bonds to improve the stability of the enzyme.The main results were listed as follows:(1)The optimization of cre gene sequence,heterologous expression and enzymat ic properties tests were implemented.By comparation of codon abundancy between P.putida and E.coli,significant differences was found and which directed the codon optimization without changing the amino acid sequence of CRE.The optimized cre gene in E.coli was expressed in soluble form,and the mass spectrometry showed that newly expressed CRE was in the form of homodimer,which was consistent with that in the original strain.The enzymatic properties of the purified CRE were analyzed.(2)The stability of the enzyme was improved by using poly-lysine to modify the surface of bi-subunit CRE.Based on the analysis of amino acid residues on CRE surface,poly-lys ine with appropriate length was selected as the modifying agent.After optimizing the modifica t io n conditions,CRE stability was improved and its Tm was raised by 2.07?.The suitable pH range meanwhile was broadened from pH 4.0 to pH 10.0.More than 50% of modified enzyme remained enzyme activity,while the enzyme activity in control group was almost lost.(3)Another method for improve enzyme stablity was designed by constructing disulfide bonds in the interface of two subunits of CRE by site-directed mutagenesis.Based on the structure of CRE,Disulfide by Design software was used to predict the possible formation of intersubunit disulfide bonds sites.Two disulfide bonds were created in four patterns after site-directed mutagenesis,one of the mutant named H125C-L130 C was obtained with improved stability.The formation of the disulfide bonds between the subunits of the mutant was confirmed by SDS-PAGE under non-reductive electropheresis running.The results showed that the three-dimensional structure of H125C-L130 C was not changed significantly by the software simulation and the analysis of Circular Dichroism.Tm value of H125C-L130 C was raised by 3.8? compared with the control.It confirms that the stability of CRE can be improved by construcion of artificial intersubunit disulfide bonds.