Biochemical Characterization of CTX-M-15 ESβL purified from clinical strain of Klebsiella pneumoniae SJ16
Layla T. Yassen1, Saad L. Hamed2, Ban O. Abdulsattar3, Asmaa A. Hussein4
1,2,3Mustansiriyah University, College of Science, Department of Biology, Baghdad, Iraq.
4Al-Nahrain University, College of Science, Department of Biotechnology, Baghdad, Iraq.
*Corresponding Author E-mail: laylataha.23@yahoo.com
ABSTRACT:
The aim of this study was purification and characterization of CTX-M-15 as a medically important enzyme from locally Klebsiella pneumoniae isolate, CTX-M-15 enzyme was subjected to two purification steps including: precipitation with 80% ammonium sulfate saturation and gel filtration chromatography by using Sepharose -6B column. Specific activity of purified enzyme has been increment up to 21.9 IU/mg with 7.3 purification folds and 69% enzyme recapture. Characterization study of purified enzyme demonstrated that the M.wt. of CTX-M-15 produced by K. pneumoniae was almost 32.2 kDa. The maximal enzyme activity at (pH 7.0), and enzyme settled at pH 6-7. The enzyme also revealed a full activity at a range of temperature between 30-37oC. Enzyme activity has inhibited powerfully in the existence of EDTA and calcium chloride, when added separately at a constant concentration. Moreover, copper chloride, and ferric chloride also caused a strong inhibition to the enzyme activity while cloxacillin showed a minor effect on enzyme activity.
KEYWORDS: CTX-M-15 ESβL; clinical strain; Klebsiella pneumoniae; SJ16.
INTRODUCTION:
Klebsiella pneumoniae is one of the most significant multi-drug resistant microorganism causing nosocomial infections that may lead to high morbidity and mortality 1,2. K. pneumoniae strains have several mechanisms of antibiotic resistance, the most frequent of which is beta-lactamase production3. ESβLs are enzymes belong to class A beta-lactamases that hydrolyze and cause resistance to various types of beta-lactam antibiotics4,5. The bla CTX-M-15 is a prevailing gene giving ESβL-production in Enterobacteriaceae causing community-acquired infections6,7. Over the last two decades, many studies revealed that the CTX-M enzymes have almost displaced TEM and SHV ESβLs variants in Enterobacteriaceae8. Within the CTX-M enzymes, the CTX-M-15 is the most significant ones, which attacking all human and animal compartments in addition to the environment all parts of the world9.
MATERIAL AND METHODS:
Bacterial isolate:
Previously, we have transformed E.coli BL21-pLysS (Invitrogene) with cloned CTX-M-15 from K. pneumoniae clinical isolate SJ16 which isolated from blood. The bla CTX-M-15 from this strain has been identified and submitted its DNA sequence in GeneBank under accession number MN20076710.
Enzyme Extraction:
The enzyme was prepared by destruction of the cell pellet using lysozyme (Sigma/USA) and ultra-sonication (Soniprep 150/UK) for 5 min (30 sec on: 30 sec off) on ice, then 100µl of Triton 100x (Fisher/USA) was added, then centrifuged at 4500rpm for 20 min by cooling centrifuge(Eppendorf/Germany). Soluble fraction was filtered through 0.45µm syringe filter unit and the supernatant has used as a crude enzyme.
Precipitation of enzyme by ammonium sulfate:
Ammonium sulfate has been added to cell free supernatant with 80% proportion of saturation. The combination has been mixed softly on magnetic stirrer at 4oC for 20 minutes. After that centrifuge at 6000rpm for 20 min at 4oC. The deposited protein was melted with appropriate volume of potassium phosphate buffer (pH 7.0).
Detection of enzymatic activity:
Beta- lactamase (CTX-M-15) was detected by measuring an enzyme activity according to a micro-iodometric assay11,12. The bla CTX-M-15 activity was assayed using reaction mixture containing penicillin G (25µl), starch solution (200µl), crude enzyme solution (1000µl), and (1000µl) of starch-iodine. The mixture was measured at 620 nm and rapid decolorization indicted β-lactamase (CTX-M-15) production.
Measurement of protein concentration:
Protein concentration has been measured by Bradford technique with bovine serum albumin as standard13.
Purification of protein by gel filtration chromatography:
The concentrated enzyme is applied to the Sepharose-6B column (2×35cm). It was equipped as prescribed by Bioworld company; it that was preequilibrated with (50mM) potassium phosphate buffer. The enzyme has been eluted with 50mM potassium phosphate buffer, and then elution has been accomplished at a flow rate 3ml/ fraction with the similar solution for equilibration. The absorbance for fraction has been calculated at 280nm, and enzyme activity has been measured in the peaks and vigorous fractions have been collected.
Determine the enzyme purity by SDS-PAGE:
Purity of (bla CTX-M-15) has been determined by electrophoresis on SDS polyacrylamide gel as per14 by 4% stacking and 12% resolving gel in the existence of standard protein (10-180kDa) (Invitrogen, USA). Distance of protein migrated from cathode to anode have been estimated after electrophoresis to calculate the protein molecular weight.
Determination of the optimum pH for CTX-M-15 activity and stability:
Purified enzyme has been added to 0.34µM penicillin G and 0.05M buffers of various pH (3-9). This can be achieved by using different buffers including: acetate buffer with pH (3, 3.5, 4, 4.5 and 5), potassium phosphate buffer pH (5.5, 6, 6.5, 7, 7.5), and Tris-HCl buffer for pH values (8, 8.5 and 9). The enzyme was incubated at various pH (3-9) for 30 min. at 37oC, then the tubes have been transferred and cooled in an ice bath, after that enzymatic activity has been assayed.
Determination of the optimum temperature for CTX-M-15 activity and thermal stability:
The bla CTX-M-15 activity has been determined after incubation of reaction solution at different temperatures (25, 30, 35, 37, 40, 45, 50 and 55)°C. While for stability, purified bla CTX-M-15 has been incubated in a water-bath at (25, 30, 35, 37, 40, 45, 50 and 55)°C for 30 min., and instantly transferred into an ice-bath. Enzymatic activity has been assayed for each treatment.
Effect of inhibitors and metal ions on CTX-M-15 activity:
The effect of various divalent ions and inhibitors on enzyme activity has been assayed with incubating the enzyme with diverse ions and inhibitors (CuCl2, FeCl2, CaCl2, Cloxacillin and EDTA) equipped at the concentrations of (5mM and 10mM) at 37°C for 30 min. The enzyme activity has been assayed for each treatment. The control was the enzyme solution without any of these compounds. The remaining activity was assayed for each treatment.
RESULTS:
Purification of CTX-M-15
A two-step purification method is made up ammonium sulfate precipitation, and gel filtration chromatography was used to obtain a highly purified CTX-M-15 from Klebsiella pneumoniae (SJ16), were summarized in Table (1). The enzyme has been deposition with (80%) ammonium sulfate saturation. It has been found that this ratio showed specific activity of 3.2 IU/mg proteins with 1.07fold of purification Table (1). During gel filtration chromatography on Sepharose - 6B, one peak contained most bla CTX-M-15 activity Figure (1). In this step, the enzyme revealed a specific activity of 21.9 U/mg protein and the fold of purification was 7.3 with a 69% yield.
Table 1: Purification steps for CTX-M-15 enzyme from K. pneumoniae SJ16.
|
Yield (%) |
Purification (folds) |
Total activity (U) |
Specific activity (U/mg) |
Protein concentration (mg/ml) |
Enzyme activity (U/ml) |
Volume (ml) |
Purification step |
|
100 |
1 |
120 |
3 |
0.80 |
2.4 |
50 |
Crude enzyme |
|
72.5 |
1.07 |
87 |
3.2 |
0.9 |
2.9 |
30 |
Ammonium sulfate precipitation 80% |
|
69 |
7.3 |
82.8 |
21.9 |
0.21 |
4.6 |
18 |
Gel filtration chromatography Sepharose-6B |
Figure 1: Gel filtration chromatography of bla CTX-M-15 using sepharose-6B column (2×35cm) equilibrated with 50 mM potassium phosphate buffer
Determination of enzyme purity and estimation of its molecular weight:
The homogeneity of the purified (bla CTX-M-15) was investigated with 4-12% SDS-PAGE. For protein profile (Figure 2) showed appropriate single band with estimated molecular weight 32.2 kDa, that indicates for purity of the final preparation upon staining with coomassie stain.
Figure 2: SDS-PAGE of bla CTX-M-15 Lane 1: represent protein marker (10-180kDa) and lane 2: represent eluted protein in 32.2kDa predicted weight.
CTX-M-15 characteristics:
Optimum pH for enzyme activity and stability:
Enzyme activity can be either enhanced or inhibited depending on the change in the pH. The bla CTX-M-15 revealed lesser activity at acidic pH (3-5), although the highest activity has been noted at pH 7 (Figure 3).
Figure (3): Optimum pH for bla CTX-M-15 activity purified from locally isolated K. pneumoniae SJ16 at different pH values
After incubation of bla CTX-M-15 for 30min. the enzyme revealed highest stability in a range between pH 5-7, since at these pH, the enzyme gave maximal (90-100%) remaining activity (Figure 4). The majority of enzymes perhaps undergo irreparable denaturation in elevated acidic or alkaline solution15, If the pH is not suitable, microbial metabolism would be disturbed and its growth would be paused16.
Figure (4): Stability of purified bla CTX-M-15 from locally isolate K.pneumoniae SJ16.
Temperature effects on CTX-M-15 activity and stability:
Temperature can be considered as critical variable that determine the velocity of reaction, however, for biological systems; the effects of this factor are complicated with contributions from protein stability and enzyme catalysis17. Maximum enzyme activity of bla CTX-M-15 has been recorded at 37°C with an noted reduce in activity at less or a elevated temperature of incubation. The outcome revealed a rise in enzyme activity until it reached 37°C after that it initiate to decrease (Figure 5).
Figure (5): Optimium temperature of bla CTX-M-15 activity purified from local isolate K.penumoniae SJ16.
The purified bla CTX-M-15 was shown good stability and retained 100% of its primary activity next incubation for 30 min in a temperature ranged from 30-37°C, then the activity has been declined with increasing the degree of temperature and lost more than 60% of its activity at 50°C (Figure 6).
Figure (6): Thermal stability of bla CTX-M-15 purified from local isolate K.pneumoniae SJ16.
Effect of inhibitors and metal ions on bla CTX-M-15 activity:
Table (2) summarized the effect of various substances on bla CTX-M-15 activity. Enzyme activity was inhibited by 5mM of each of FeCl2, CaCl2 and CuCl2 by 96.08%, 87.6%, 90.5% respectively. Also, the enzyme showed 70% resistance to Cloxacillin at 10mM. EDTA have a powerful inhibitory effect than other inhibitors with 99.14% loss of enzyme activity, Whereas cloxacillin had little inhibitory effect on the enzyme (45%).
Table (2): Effect of metal ions and inhibitors on K. pneumoniea bla CTX-M-15
|
Inhibition (%) |
Remaining activity (%) |
Concentration (mg/ml) |
Divalent ions |
|
96.08 |
3.92 |
5 |
CaCl2 |
|
98.86 |
1.14 |
10 |
|
|
87.6 |
12.4 |
5 |
CuCl2 |
|
88 |
12 |
10 |
|
|
90.5 |
9.5 |
5 |
FeCl2 |
|
89.52 |
10.48 |
10 |
|
|
Inhibition (%) |
Remaining activity (%) |
Concentration (mg/ml) |
Inhibitor |
|
99.14 |
0.86 |
5 |
EDTA |
|
99.64 |
0.36 |
10 |
|
|
45 |
55 |
5 |
Cloxacillin |
|
30 |
70 |
10 |
|
|
------ |
100 |
------- |
Control |
DISCUSSION:
Upon purification of crude intracellular enzyme, the first step indicated that there was a rise in the specific activity (3.2) compared with crude extract (3U/mg proteins), Salting out using ammonium sulfate is one of the classical methods in protein biochemistry. Study carried out by18 showed the last concentration of the protein has been calculated as 2.8mg/ml, while the concentrations of the proteins have been calculated at 280nm and was found to be 2.5mg/ml19.
The second step on column purification resulted in specific activity 21.9 U/mg, 7.3 purification fold and 69% yield Table (1).20 reported the specific activities of purified CTX-M-15 was 185 U/mg of protein with a 50 fold of purification.
The molecular weight of the (CTX-M-15) has been determined by comparison of the standard marker protein, the molecular weight of the bla CTX- M-15 with 6x histidine was approximately 32.2kDa Figure (4).20 reported the relative molecular mass of bla CTX- M-15 determined by SDS–PAGE analysis was ∼29 kDa while 18 reported the purity of CTX-M-15 protein noted by a sole band of 31 kDa. Another study showed Purified proteins of bla CTX-M-15 was obtained in 97% purity, as seen in an one band of 31 kDa19.
The pH be able to exert its effect on enzyme activity in diverse ways; on the ionization of groups in the substrate, enzyme’s activity site and by affecting the conformation of either the enzyme or the substrate21. Another observation concerning the pH stability revealed that S. aureus RU57 β-lactamase retains 50% and 20% of its activity in the pH 8-922.
The rise in temperature degrees leads to imparting extra kinetic energy to the interactive molecules, leads to more productive clashes per unit time. This ought to be within the proper arrangement of tertiary structure of an enzyme kept up primarily by a huge number of frail non covalent bonds, However, if the molecule absorbs excessive energy, the tertiary structure will be crashed and the enzyme will be denaturated, that is loss of catalytic activity23,24. A local study by22 showed the purified β-lactamase had a good stability and retained about 100% of its initial activity after incubation for 30 min from range 25-40°C. Another study by25 showed an optimal temperature of β-lactamase isolated from local isolate from asopharyngeal region of healthy individuals from Basra city was, at 25, 30, and 35°C.
The purified bla CTX-M-15 of K.pneumoniae revealed a sole substrate profile with hydrolyzing the majority of cephalosporines and pencillines including cefotaxime and cloxacillin, that were reported to be resistant to decomposition with beta-lactamases of G-ve bacteria26, 27. The activity of the bla CTX-M-15 from K.pneumoniae has been inhibited by EDTA (99.64%) and cloxacillin (45%) when pencillin G was used as substrates as shown in table (2), also enzyme activity was inhibited by CaCl2, FeCl2 and CuCl2 (98.86%, 89.52%, and 88%), respectively. Local study by28 in Kirkuk in Iraq revealed that the activity of β-lactamase was inhibited to 93.75% by EDTA and by cloxacillin to 62.5%, also CaCl2 (76%), FeCl2 (91%) and CuCl2 (86%).
CONCLUSION:
The CTX-M-15 purified from locally isolated K. pneumoniae (SJ16) was stable at a broad range of temperature (30-37oC) and pH (5-7), EDTA and Calcium chloride strongly inhibited the enzyme activity.
ACKNOWLEDGMENT:
The authors thanks and appreciation to Mustansiriyah University (http://uomustansiriyah.edu.iq/) for its support during the tenure of this work.
CONFLICT OF INTEREST:
The author has no disclosures to report.
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Received on 24.10.2019 Modified on 16.12.2019
Accepted on 12.02.2020 © RJPT All right reserved
Research J. Pharm. and Tech. 2021; 14(8):4080-4084.
DOI: 10.52711/0974-360X.2021.00706