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Abdollahzadeh Parsa M, Hosseinkhani S, jalili S, Rahmati F. Expression Optimization, Purification, and Fluorescence Spectroscopic Assessment of Creatinase Enzyme in the Presence of Gold Nanoparticles. JPMed 2022; 11 (1) : e18
URL: http://jpmed.ir/article-1-1069-en.html
1- Department of Biochemistry, Faculty of Biological Sciences, North-Tehran Branch, Islamic Azad University, Tehran, Iran
2- Department of Biochemistry, Faculty of Biological Sciences, Tarbiat Modares University, Tehran, Iran
3- Research Center for Life & Health Sciences & Biotechnology in the Police, Directorate of Health, Rescue & Treatment, Police Headquarter, Tehran, Iran
4- Department of Biochemistry, Faculty of Biological Sciences, North-Tehran Branch, Islamic Azad University, Tehran, Iran. , noora_1232003@yahoo.com
English Extended Abstract:   (1426 Views)
... [1]. Nowadays, the use of laboratory methods and kits based on enzymatic reactions to monitor the health of individuals, especially in the field of military forces, has become widespread. The use of the creatinase enzyme is one of the enzymatic methods for the clinical assay of creatine and creatinine in body fluids [2]. The use of recombinant proteins and enzymes in scientific research has increased. Molecular cloning of DNA is one way to produce recombinant proteins into bacterial chromosomal DNA. Today, this technology is used to produce recombinant proteins in clinical and industrial research [3]. ... [4]. One of these enzymes is creatinase (creatine aminohydrolase) [5]. Due to the role of creatine in increasing the synthesis of adenosine triphosphate (ATP) and its equilibrium reaction with creatinine, this enzyme is an important indicator for measuring the health of the kidneys as well as muscles. Therefore, measuring the level of creatine and creatinine in blood serum and urine can indicate the function of the kidneys and muscles [6]. Pseudomonas putida is one of the bacteria that produce this enzyme. Genetic engineering is used for the mass production of this enzyme [7]. ... [8-12]. Due to the instability of the enzyme produced, it is necessary to use a method to stabilize the enzyme. Various methods are used to stabilize enzymes, including the addition of nanoparticles [13]. ... [14-16]. In 2012, Yadav et al. investigated the effect of iron oxide and chitosan nanoparticles on keratinase stability [17]. Also in 2017, Afshari et al. examined the activity and expression of the keratinase enzyme in a liquid LB growth medium at different expression temperatures [18].
Aim (s)
This study aimed to optimize the induction temperature for mass production of more active keratinase enzyme and also to investigate the effect of gold nanoparticle stability on the structure of keratinase enzyme for use in serum and urinary creatinine and creatinine kits for faster and more accurate diagnosis of kidney and muscle diseases.
Research Society, Place, and Time
This research was conducted in the spring and summer of 2021 in the Electrochemical Institute at the University of Tehran, Iran.
Used Devices & Materials
The materials used were growth medium (Merck), IPTG (Fermentas), kanamycin (Duchefa), SDS-PAGE (Merk), BSA (Merck), Ni-NTA Sepharose (QIAGEN), and buffer (Merck). To use the enzyme creatinase to measure creatine and creatinine in body fluids enzymatically, it is necessary to express the enzymes highly and purify them. For this purpose, E. coli strain BL21 (expression vector pET28a containing Pseudomonas putida creatinase gene has been transformed) was first cultured in a liquid TB medium. After induction of the gene of the enzyme for its high expression (Over Expression), the bacterial cell wall was destroyed by a sonicator (20s sonike and 40s rest up to 15 pulses). To purify the enzyme, the affinity chromatography method (using a nickel-Sepharz column) was used. The expressed recombinant enzymes have a histidine sequence (His6-tag) at the amine end (N-Terminal). This histidine sequence has a strong affinity for nickel and interacts strongly with it. Finally, to separate the proteins attached to the column,( Elution Buffer) containing 300 mM NaCl, Imidazole 250 mM, Tris / HCl 50 mM with pH=8 was used. Then, SDS-PAGE electrophoresis was used to confirm the presence and purity of the samples, and finally, the Bradford method was used to measure the protein concentration. The concentration of enzyme samples was determined by drawing a standard curve using BSA protein and Bradford assay [19]. To determine the optimum temperature for enzyme expression, E. coli containing pET28a vector in which the keratinase gene was cloned and was cultured overnight in TB medium, and then expression induction was performed at 18, 22, 28, 32, and 37°C. After purifying and measuring the concentration of proteins, the results were compared and the optimal induction temperature was determined. The colorimetric method (Sigma-Aldrich) was used to evaluate the enzyme activity. For this purpose, the keratinase enzyme expressed at different temperatures was incubated with creatine solution in PBS as a substrate at 37°C for 5 minutes, and then concentrated dimethyl amino benzaldehyde and HCL were added and after 20 minutes incubated at room temperature, its absorption at 435 nanometers was read. With the help of adsorption, the activity of expressed enzymes at different temperatures was calculated and compared. Fluorescence spectroscopy was used to evaluate the structure of the protein. Since keratinase contains the amino acid tryptophan and this aromatic amino acid has the highest absorption, the excitation wavelength of 295 nm was used in this test to stimulate only the amino acid tryptophan and the resulting fluorescence spectrum was related to the tryptophan position in creatinase enzyme. Concentrations of 0.01, 0.02, 0.04, 0.06, 0.08, 0.1 and 0.2 nM gold nanoparticles were used to investigate the effect of gold nanoparticles on the structure of keratinase enzyme. After 5-minute incubating each concentration of gold nanoparticles with keratinase enzyme, the fluorescence spectrum of each was plotted at the excitation wavelength of 295 nm, and finally, the fluorescence spectra obtained in the presence and absence of gold nanoparticles were compared.
Finding by Text
After culturing the bacteria and inducing the expression of keratinase gene for its over-expression at 18, 22, 28, 32, and 37°C, purification of enzymes by inclination chromatography (using nickel Sepharz column) and confirmation of the presence and purity of proteins were determined using SDS-PAGE electrophoresis technique and the concentration of proteins was determined by Bradford method. By comparing the concentrations of purified proteins, the optimal induction temperature was determined. The results of measuring the concentration of proteins showed that the highest protein content was at the induction temperature of 28°C lasted for 16 hours (Table 1). Then, the structural study of the keratinase enzyme was performed by fluorescence spectroscopy in the presence and absence of gold nanoparticles and the effect of gold nanoparticles on the structure of the keratinase enzyme was determined. The activity of expressed enzymes was calculated at induction temperatures of 18, 22, 28, 32, and 37 under equal conditions. The results showed that the induction enzyme was more active at 28°C for 16 hours (Table 2). The emitted fluorescence spectrum of keratinase enzyme resulting from excitation at 295 nm was performed by Perkin Elmer Ls 45 spectroscopy fluorescence device with quartz cuvette with a diameter of 1 cm, the resulting fluorescence replication diagram is shown in Figure 1. The graph obtained from the effect of gold nanoparticles on the structure of the keratinase enzyme showed that with increasing the concentration of gold nanoparticles, the fluorescence intensity decreased. Figure 2 shows the fluorescence spectrum of the keratinase enzyme with different concentrations of gold nanoparticles. To compare the fluorescence spectroscopy of keratinase enzyme in the presence and absence of gold nanoparticles, a diagram of the emission spectrum in the presence and absence of gold nanoparticles was drawn. This diagram showed that the fluorescence intensity decreased in the presence of gold nanoparticles, which decreased further with the increasing concentration of gold nanoparticles (Figure 3).
Main Comparison to the Similar Studies
The results of this study showed better expression and higher activity of the keratinase enzyme at an induction temperature of 28 ° C for 16 hours in a liquid TB medium. Compared to previous studies on the expression of keratinase enzyme in liquid LB medium and similar temperature conditions [18], the enzyme expressed in this study showed a higher concentration and higher activity, which indicates a more favorable expression of keratinase rather than previous methods. ... [19-25]. In the past, studies have been performed to stabilize the enzyme keratinase such as evolvement of single, pair, and triple random mutations on the enzyme keratinase in which the triple mutations created caused relative stability of the enzyme but had an adverse effect on enzyme activity [26]. In another study, the effect of silver ions on the keratinase enzyme was investigated, which indicates inhibition of keratinase activity [27]. Another study investigated the effect of iron oxide and chitosan nanoparticles on the keratinase enzyme, which caused relative stability in the structure and activity of the enzyme [28]. The results of this study showed a decrease in the intensity of fluorescence in the presence of gold nanoparticles, and with increasing the concentration of gold nanoparticles, a greater decrease in fluorescence intensity was seen. This may indicate a change in the fluorescent amino acid microenvironment in the structure of the enzyme keratinase. As a result, the use of gold nanoparticles to stabilize the enzyme keratinase does not seem to be a good option.
There was a time limit to evaluate the activity and structure of this enzyme due to the instability of the keratinase enzyme. Therefore, in this study, the activity and structure of the enzyme were investigated immediately after its purification of the enzyme.
Considering the results of the effect of gold nanoparticles on the keratinase enzyme, other methods for stabilization of keratinase enzyme are proposed, including study on the effect of inhibitors and activators on the keratinase enzyme, targeted mutations in enzyme sequence and evaluation of enzyme stability and activity, consideration of enzyme stability versus various other factors as well as examining the crystallographic structure of the enzyme keratinase for a better understanding of the enzyme and selecting a better option for enzyme stabilization for commercial use.
The results of this study showed that in the culture of E. coli BL21 containing pET28a amplification vector in liquid TB medium and its induction at 28 ° C for 16 hours, the keratinase enzyme was more active with higher expression for use in creatine and creatinine assessment kits. Further study of the effect of gold nanoparticles on the structure of the keratinase enzyme has shown that due to the possibility of changes in the fluorescent amino acid microenvironment in the structure of the keratinase enzyme in the presence of gold nanoparticles, these compounds are not a good option for keratinase enzyme stability. Therefore, the use of other methods and compounds to stabilize the creatinase enzyme is recommended for use in commercial creatine and creatinine assay kits.
Clinical & Practical Tips in Police Medicine
The use of accurate and rapid creatine and creatinine measurement kits in military laboratories can monitor the health of military personnel as well as future employees in this field.
This article is taken from the Ph.D. thesis in Health Psychology, Islamic Azad University, Central Tehran Branch, code 10120709981008. We sincerely thank all the people who helped us in this research.
Conflict of Interest
The authors state that there is no conflict of interest in the present study.
Funding Sources
The present study had no financial support
Table 1) Protein concentrations at different induction temperatures (volume of each vial collected is 1 ml)
expression (ºC)
Protein concentration in vials collected from purification (mg/ml)
E1 E2 E3 E4 E5 E6 E7
18 1.37 1.16 0.93 0.74 0.48 0.26 0.18
22 1.53 1.28 1.05 0.85 0.54 0.38 0.25
28 2.13 1.98 1.78 1.53 0.82 0.76 0.64
32 1.92 1.73 1.48 1.11 0.68 0.49 0.32
37 2.04 1.85 1.68 1.25 0.73 0.57 0.38

Table 2) Creatinase activity at different induction temperatures
Temperature induced protein expression(ºC) 18 22 28 32 37
Absorption at 435 nm (dilution 1.2) 0.623 0.665 0.758 0.736 0.683
Creatinase Enzyme activity 11.64 U/ml 12.42 U/ml 14.17 U/ml 13.27 U/ml 12.76 U/ml

Figure 1) Fluorescence spectrum of Ceratinase enzyme at excitation wavelength (295 nm)

Figure 2) Fluorescence spectrum of Ceratinase enzyme at the excitation wavelength (295 nm)

Figure 3) Fluorescence spectrum of Ceratinase enzyme at the excitation wavelength (295 nm) in the presence and absence of AuNPs

Article number: e18
Full-Text [PDF 985 kb]   (776 Downloads)    
Article Type: Original Research | Subject: Police Medicine Related Technologies
Received: 2022/01/7 | Accepted: 2022/02/21 | Published: 2022/04/13

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