Author/Authors :
Afshari, Elnaz Department of Microbiology - Islamic Azad University of Pharmaceutical Sciences Branch, Tehran, Iran , Amini-bayat, Zahra Department of Biotechnology - Iranian Research Organization for Science and Technology (IROST), Tehran, Iran , Hosseinkhani, Saman Department of Biochemistry - Faculty of Biological Sciences - Tarbiat Modares University, Tehran, Iran , Bakhtiari, Nahid Department of Biotechnology - Iranian Research Organization for Science and Technology (IROST), Tehran, Iran
Abstract :
Background: Pseudomonas putida (P. putida) ATCC12633 can produce creatinase. It
is a microbial enzyme which degrades creatinine in bacteria and provides source of
carbon and nitrogen. Also, this enzyme is used in the enzymatic measurement of
creatinine concentration for diagnosis of renal and muscles functions and diseases. Our
purpose was recombinant production of creatinase for using in clinical measurement
of serum or urine creatinine.
Methods: A 1209bp of open reading frame of creatinase was amplified by PCR from
P. putida ATCC12633 genome and cloned into pET28a expression vector which was
digested using NheI and XhoI restriction enzymes. Cloning was confirmed by colony
PCR, double digestion analysis and sequencing. Recombinant pET28a vector was
transformed to Escherichia coli (E. coli) BL21 (DE3). Creatinase expression was induced
in E.coli BL21 (DE3) using IPTG and confirmed by SDS-PAGE and western blotting.
Purification of creatinase was performed using Ni-NTA column. The specific activity of
this enzyme was also investigated.
Results: The creatinase gene cloning was confirmed by DNA sequencing. Successful
expression of creatinase was performed in E. coli (57.4% of total protein). SDS-PAGE
and western blot analysis showed a 45 kDa creatinase protein. Purification of creatinase
was done with high purity. The specific activity of recombinant enzyme is 26.54
unit/mg that is much higher than other creatinase used in the commercial kits (9
unit/mg).
Conclusion: The P. putida ATCC12633 recombinant creatinase was expressed efficiently
in E. coli BL21 and 57% of total protein was the recombinant creatinase. Also,
expressed creatinase has high solubility and also the enzyme has good activity compared
to enzymes used in commercial kits, so a new source of creatinase was produced
for creatinine assay kit in this study.