Cloning, Expression And Characterization Of A Creatinase From Bacillus Sp. BSD-8 In Escherichia Coli

Creatinase(creatine amidinohydrolase,EC 3.5.3.3)catalyzes the hydrolysis of creatine to sarcosine and urea.Creatinase,together with creatinine amidohydrolase (EC 3.5.2.10;creatininase)and sarcosine oxidase(EC 1.5.3.1),has been used in clinics for the determination of creatinine in the samples of serum and urine,which is an important parameter to estimate kidney function.It has been found in some bacteria,but few characteristics of these enzymes have been published.The results of this research were reported as following.(1)The gene coding for creatinase(creatine amidinohydrolase,EC 3.5.3.3)was isolated from Bacillus sp.BSD-8.The creatinase gene(NCBI accession no. EU546226)has an open reading frame of 1,236 bp and codes for a polypeptide of 411 residues with a molecular mass of 46.96 kDa predicted by the gene sequence.The -10 and -35 regions of the putative promoter and the inverted repeats are indicated.(2)The creatinase gene was over-expressed in Escherichia coli BL21(DE3)and BL21(DE3)pLysS.The recombinant creatinase(rCRE)was over 20.0%of the total protein.(3)The rCRE was purified to homogeneity and was examined by SDS-PAGE. And a clear single band was observed.(4)The properties of the rCRE were studied.The rCRE was stable below 45℃at pH 7.5 and pH between 6.0 and 7.0 at 25℃.The optimal temperature was 37℃and the optimal pH was 7.5.The Km and Kcat of the enzyme were 20.49 mM and 12.2/s, respectively.None of the cations studied enhanced the activity and it was inhibited by Cu²⁺,Hg²⁺,Ag⁺,Co²⁺and Zn²⁺.The creatinase from Bacillus sp.BSD-8 was one of the most thermostable creatinases bublished.