|Year : 2019 | Volume
| Issue : 4 | Page : 228-232
Screening and optimization of staphylokinase from Staphylococcus aureus isolated from nasal swab of healthy students in Himachal Pradesh University, India
Kumari Deepa1, Sameer Singh Faujdar1, Wamik Azmi2, Priya Mehrishi1, Seema Solanki1
1 Department of Microbiology, Maharishi Markandeshwar Medical College and Hospital, Solan, Himachal Pradesh, India
2 Department of Biotechnology, Himachal Pradesh University, Shimla, Himachal Pradesh, India
|Date of Submission||28-Aug-2019|
|Date of Acceptance||28-Oct-2019|
|Date of Web Publication||03-Dec-2019|
Dr. Sameer Singh Faujdar
Department of Microbiology, Maharishi Markandeshwar Medical College and Hospital, Kumarhatti, Solan - 173 229, Himachal Pradesh
Source of Support: None, Conflict of Interest: None
Background: One of virulence factors produced by Staphylococcus aureus is staphylokinase (SAK), which enhances their proteolytic activity leading to tissue damage and improving bacterial invasiveness. In the present study, we estimated the ability to produce SAK by S. aureus isolates from nasal carriers. We would like to verify relationship between SAK production and different S. aureus isolates. Methods: In this study, all nasal swab samples from healthy nasal carriers were collected and further processed in the Department of Biotechnology, Himachal Pradesh University. All S. aureus isolates were screened for SAK and optimization was done. Results: Out of all S. aureus isolates 20% isolates were positive for SAK production. Isolate SAK-24 shown increase in SAK production after optimization and response surface methodology (RSM). Conclusion: Production of SAK varies from strain to strains. SAK production can be increased by optimization and RSM.
Keywords: Optimization, response surface methodology, Staphylococcus aureus, staphylokinase
|How to cite this article:|
Deepa K, Faujdar SS, Azmi W, Mehrishi P, Solanki S. Screening and optimization of staphylokinase from Staphylococcus aureus isolated from nasal swab of healthy students in Himachal Pradesh University, India. Biomed Biotechnol Res J 2019;3:228-32
|How to cite this URL:|
Deepa K, Faujdar SS, Azmi W, Mehrishi P, Solanki S. Screening and optimization of staphylokinase from Staphylococcus aureus isolated from nasal swab of healthy students in Himachal Pradesh University, India. Biomed Biotechnol Res J [serial online] 2019 [cited 2019 Dec 9];3:228-32. Available from: http://www.bmbtrj.org/text.asp?2019/3/4/228/272188
| Introduction|| |
Enzymes are biocatalysts that are produced by the living cells to bring about specific biochemical reactions and metabolic processes of the cells. These are increasingly finding applications in industry, medicine, and environment management. Some of the widely used enzymes in therapeutics include protease, collagenase, staphylokinase (SAK), etc. Protease is a group of enzymes whose catalytic function is to hydrolyzes peptide bond of proteins and break it down into polypeptides or free amino acids. They are derived from microorganisms and animals. SAK is also a protease that plays a major contribution in the field of medicine; it is an extracellular protein that helps in dissolving fibrin meshwork to inactive proenzyme plasmin, thus acting as clot busters. However, microorganisms serve as a preferred source of these enzymes because of their rapid growth, limited space requirement for cultivation, and the ease with which they can be genetically manipulated to generate new enzymes with altered properties that are desirable for their extended application. Thrombolysis can lead to life-threatening diseases like vascular blockage, pulmonary embolism, deep vein thrombolysis and acute myocardial acute infraction which are commonly known as heart attack due to blood clot. As a therapeutic enzyme, SAK can be used in the treatment of thrombolic disorders where it dissolves a blood clot by the activation of plasminogen to plasmin which, in turn, degrades fibrin to soluble products and establish normal blood flow., Plasmin is the active fibrinolytic component of the circulatory system, solubilizing the fibrin network in blood clots through limited proteolysis., SAK (E. C 126.96.36.199) is a bacterial kinase or fibrinolytic protein produced by Staphylococcus aureus. It is one of the virulence factors which breaks down fibrin clots and facilitate the spread of infection.
| Methods|| |
Isolation and identification of Staphylococcus aureus
Totally 50 nasal swab samples from healthy students with their consent were collected in Himachal Pradesh University. Ethics Committee Approval number: HPU/BT/15/1766 and date of Approval: 15-01-2015. The swabs were intimated in sterile physiological saline (0.9% NaCl) for at least 15 min. Then, samples were cultured on nutrient agar and blood agar plates then incubated at 37°C for 24 h to obtain pure colonies of S. aureus. The identification of S. aureus was done by using the standard biochemical tests (gram stain, 3% catalase test, slide/tube coagulase, mannitol fermentation, DNase).
Screening of isolates for staphylokinase activity
The S. aureus isolates were screened for proteolytic activity by both qualitative and quantitative methods.,
Quantitative estimation of staphylokinase activity
The bacterial isolates obtained above were inoculated on skimmed milk agar (SMA) plates and the ability of bacterial isolates to hydrolyze casein was recorded by the zone of digestion or radical caseinolytic activity around the colonies after incubating the plates at 30°C for 24 h.
Qualitative estimation of staphylokinase activity
The seed culture was prepared by using medium-contained 1% nutrient broth 4.5% yeast extract, 1% NaCl, 1.5% beef extract, and 1 ml glycerol pH 6.5 and was incubated for at 30°C for 24 h at 150 rpm.
Production of staphylokinase
To 50 ml of production medium in Erlenmeyer flask, 1% (v/v) of seed culture was added. This was incubated at 30°C for 24 h in an incubator shaker (150 rpm) the fermentation broth was centrifuged at 10,000 rpm for 10 min, and supernatant was assayed for extracellular activity.
The SAK activity of the enzymes was assayed spectrophotometrically, according to Kunitz (1947). The reaction mixture contained 450 μl of 0.3% (w/v) casein in 0.1 M Tris-HCL and 50 μl of the enzyme and was incubated for 20 min at 37°C. The reaction was stopped by adding 750 μl of trichloroacetic acid (TCA) solution containing TCA 5% (w/v), sodium acetate 9% (w/v), acetic acid 9% (w/v), followed by 30 min incubation at room temperature. The content was centrifuged at 15,000 rpm for 15 min. The absorbance of the soluble peptide (supernatant) was measured at 280 nm.
For SAK assay, one unit of enzyme activity was defined as the amount of enzyme required to release 1 μg of tyrosine per min under assay condition.
For blood clot-dissolving assay, the minimum concentration of the enzyme which completely dissolve 1 ml of clotted blood is considered as 1 enzyme unit.
Protein was estimated using the Bradford method.
Optimization of physicochemical parameter for maximum production of staphylokinase
Satoh medium was used for optimization of physicochemical parameter for production of SAK by bacterial isolates.
Optimization of production medium pH
To find the effect of medium pH on extracellular production of protease, it was grown on different pH range from 4.0 to 8.0. One ml of preculture was added to 50 ml of production medium prepared at different pH.
Effect of incubating temperature
A volume of 50 ml of culture was inoculated with 1 ml of preculture (24 h old) and incubated at different temperature (20°C–40°C), and the protease activity in the culture supernatant was assayed.
Effect of agitation rate
To find out the effect rate on extracellular production of protease it was grown on different rpm 50–200. One ml of preculture was added to 50 ml of the medium at different rpm.
Response surface methodology optimization of growth and production parameters for staphylokinase by Staphylococcus aureus
Optimization of the medium components for maximum production of SAK by S. aureus was performed in two stages. First, all the components having a significant effect on the enzyme production were identified. Second, the optimization values of these components for the production of SAK were determined.
Initial screening of the most important components affecting the production of SAK was performed by Plackett–Burman design. A total of eight components were selected for this study, with each being represented at two levels, high (+1) and low (−1) as shown in [Table 1]. In this design, it is assumed that the main factors have no interactions and a first-order multiple regression model is appropriate.
The response function (Y) for response surface methodology (RSM) is calculated by following formula (Design-Expert version 09 software):
Y = β° + iixβ ε(i = 1., k)
Where Y is response function (SAK production) and βi is the regression coefficient.
Central composite design
Central composite design (CCD) was employed to optimize the growth and production parameter including peptone, yeast extract, beef extract. The CCD contained a total of 20 experimental runs. The experiments were conducted, and enzyme activity (U/ml) was taken as the response (Y). The statistical software “Design-Expert 9.0” (StatEase INc., Minneapolis, MN, USA) was used to analyze the experimental results.
Validation of experimental model: to validate the model equation, experiments were conducted for SAK production by Staphylococcus spp. under optimum conditions predicted by the model.
Blood clot-dissolving assay
Sterile empty microcentrifuge tubes were taken, labeled suitably and their weights were determined (W1). Human blood was freshly collected, and 500 μl of blood was transferred into each microcentrifuge tube and inoculated at 37°C for 45 min. After clot formation, serum was completely removed without disturbing the clot. The weights of the microcentrifuge tubes with the clots were noted (W2). To determine the clot weights, W1 was subtracted from W2. A volume of 500 μl of the respective cell-free supernatant were added to the respective tubes. Presterilized distilled water was added to one of the tubes containing clot and served as control. All the tubes were then incubated at 37°C for 90 min and observed for clot lysis.
Following incubation, the fluid on each tube was removed, and tubes were again weighed (W3) to observe the difference in clot weight. Percentage of clot lysis was calculated using the following equation.
Percentage lysis = 100 – ([(W3 − W1)/(W2 − W1)] × 100)
W1 = Weight of empty micro centrifuge tube.
W2 = Weight of micro centrifuge tube with clot.
W3 = Weight of micro centrifuge tube with remaining clot after incubation.
The concentration of the enzyme was varied from 100 to 2000 μl to perform the blood clot dissolving assay.
| Results|| |
Screening and selection of staphylokinase producing Staphylococcus aureus
Total 50 isolates were obtained, out of these three samples, only 10 isolates hydrolyzed the casein. The proteolytic activity of the above listed isolated was assayed in the culture supernatant after 24 h incubation at 30°C in SMA medium (7.0 pH) containing (%w/v) skimmed milk 2.8, casein enzymatic hydrolysate 0.5, dextrose 0.1, agar 0.15, and yeast extract 0.25. The result of screening is summarized in [Table 2].
The maximum zone of radial caseinolysis was observed in SAK-24 isolate, followed by isolates SAK-12 and SAK-20. The radial caseinolysis zone was less in other isolates. Isolates SAK-24 was selected for further study.
Optimization of individual physicochemical parameter for the maximum production of staphylokinase
The effect of various physicochemical parameter (temperature, pH, agitation rate on growth and production) on SAK by S. aureus were observed in Erlenmeyer flasks (250 ml) each containing 50 ml sterilized production medium (Satoh medium) which were incubated in temperature-controlled orbital shaker for 24 h at 37°C and 150 rpm.
Optimization of production medium pH for maximum growth and staphylokinase production
Variation of pH has a large impact on the uptake of the nutrients by the cells; hence, this phenomena makes it mandatory to optimize the pH of the medium. For this, pH of the medium was varied from 4.0 to 8.0. The maximum SAK activity (0.81 IU/ml) was observed at pH 6.5. However, with further increase in pH of the production medium, a gradual decrease in the SAK production was observed. Hence, it is evident from the results that the SAK production by Staphylococcus spp. was greatly affected at highly acidic and alkaline pH. An overall decline in the final pH of the fermentation broth was observed in each case irrespective of the initial pH.
Optimization of incubation temperature for maximum growth and staphylokinase production
To study the effect of incubation temperature, production medium incubated at different temperature 20°C–40°C for 24 h at 150 rpm. Maximum SAK (0.85 IU/ml) activity was observed at 30°C. However, further increase in the incubation temperature, a decrease in the enzyme activity was observed.
Optimization of the agitation rate for maximum growth and production of staphylokinase
The effect of agitation rate was studied by incubating the cells of SAK sp in production medium under shaking condition at varying rpm 50–200 rpm. Maximum SAK activity (0.88 IU/ml) was observed at an agitation rate 100 rpm.
Response surface methodology optimization of growth and production parameter for Staphylokinase
Screening of the most important components affecting the production of SAK was performed by Plackett–Burman design as shown in [Table 3]. A total of eight components were selected for this study, with each begin represented at two levels, high (+1) and low (−1).
|Table 3: Screening of the medium components on the basis of Plackett-Burman design|
Click here to view
A set of 12 combinations was generated by the software, in which the SAK activity was determined individually. Out of all the runs, maximum SAK activity (1.63 IU/ml) was observed in the twelve run. A Pareto graph showing the positive effect of some constituents has also been prepared.
CCD was employed to optimize the growth parameter/components namely, peptone, yeast extract, beef extract of the production medium. The CCD contained a total of 20 experimental run.
The optimal concentration for the three components obtained from the maximum points of the model was calculated to be as peptone 2.1 (w/v %), yeast extract 2.65 (w/v %), beef extract 2.05(w/v %). The maximum production of SAK obtained experimentally by RSM was (5.28 IU/ml). On the basis of data obtained, graphs presenting predicted versus actual values, as well as curve of media component optimization, were also prepared. The effect of different variables on SAK production has been demonstrated through response surface plots.
Blood clot-dissolving assay
The blood clot was incubated with SAK-24 and observed the solubilization of blood clot for different time periods. After 40 min of incubation, the insoluble form of the clot was converted into soluble form. The ability of SAK to digest the blood clot is the major application of this enzyme as it plays major role in therapeutics. The concentration of protein was varied from 0.43 to 8.72 mg/ml. Maximum percentage lysis was observed in 4.36 mg/ml concentration of the protein used. With further increase in enzyme amount no increase in the percentage clot lysis was observed.
| Discussion|| |
The accumulation of fibrin in blood vessels leads to cardiovascular diseases. Microorganisms are important resources of thrombolytic agents. SAK is a bacterial protein which is produced by many strains of Staphylococcus species and S. aureus is one of the main species. They convert inactivated plasminogen into active plasmin form which is well known to have profibrinolytic effect. Hence, screening was done by casein hydrolysis assay because isolates utilized casein as a substrate which is considered as the most common substrate for the induction and assay of protease. In radical caseinolytic assay, the highest zone measuring the diameter of 3 cm was reported by Pulicherla et al. and similar, 2.9 cm diameter zone was reported by Shagufta et al. In the present study, the bacterial isolates SAK-24 utilized casein as a substrate showing zone of diameter of 2.5 cm. The S. aureus (SAK-24) under the present study showed maximum SAK activity at pH 6.5, at 30°C for 24 h and optimization under CCD of 20 experiments increased the yield to five-fold. The yeast extract acts as a major nitrogen source for the production of SAK and shows maximum activity at concentration 2.65% (w/v). The production of SAK by SAK-24 has reported to be maximum at pH 6.5, at 37°C for 24 h. Optimizations using RSM (CCD) and one factor at a time of 30 experiments increase the yield of 1.98-fold, Dunn and BeduAddo.,
A number of SAK producing bacterial isolates have been reported from the different habitat (soil, water, sewage, skin, throat, and intestine etc.). SAK can be used as good clot buster than other commercially available chemical such as heparin, ethylenediaminetetraacetic acid. Isolation of native Staphylococcus spp. from the clinical sample has ethical issue because it is highly pathogenic, but few strains have been isolated from environment safe to use. SAK is useful for cost-effective thrombolytic therapeutic purpose in clinical areas.
Doss HM et al. reported that 5.23 mg/ml of streptokinase dissolve 1 ml of clotted blood in 18 h, similarly in the present study, diluted 4.36 mg/ml SAK protein dissolve 0.5 ml of clotted blood in 60 min. The clot lysis capability of SAK was checkedin vitro clot lysis assay method or modified Holmstrom method reported by Prasad et al. The highest 72% clot lysis in 90 min was observed show the highest clearance zone of 3.1 cm diameter in the caseinolytic assay. Similarly, in the present study, the bacterial isolates Staphylococcus spp. showed a maximum of 85% of clot lysis in 60 min in 0.5 ml of blood at 4.36 mg/ml SAK protein. Future prospective of the current study includes strain improvement of the S. aureus sequencing and production of recombinant SAK gene.
| Conclusion|| |
The present study was carried out to explore the potential of S. aureus. Strain SAK-24 showed maximum SAK activity. To enhance the growth and production of SAK various individual physicochemical parameters were optimized. A 2.5-fold increase in production of SAK was observed after optimization. The RSM was performed to further production of SAK by using various medium components. A five-fold increase in SAK production was observed by SAK-24 after RSM. Our data confirm phenotypic dissimilarity in SAK production of S. aureus strains isolated from various types of infections. It is compatible with the biological role of SAK and with hypothetical model of SA- mediated bacterial invasion of host tissues. Thus, the estimation of SAK production by S. aureus isolates may be regarded as the parameter describing potential invasiveness of staphylococci and can be useful as a medical recommendation for the eradication of staphylococci carrier state.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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[Table 1], [Table 2], [Table 3]