Isolation of L-Asparaginase from Cannabis Sativa and Development of Biosensor for Detection of Asparagine in Leukemic Serum Samples
Pathak Teena1, Kumar Raman1, Kaur Jagjit1 and Kumar Kuldeep2*
1Department of Biotechnology, M.M. University, Mullana, Haryana, India
2Department of Biotechnology, Multani Mal Modi College, Patiala, Pujnab, India
*Corresponding Author E-mail: kuldeepbio@gmail.com
ABSTRACT:
Cannabis sativa Linnaeus (Cannabaceae) is a widely used plant of therapeutic and industrial importance. It produces two pigments: cannabinoids and tetrahydrocannabinol (THC), THC is responsible for its psychoactive properties which make its use important in medical field. The main objective of present work was to construct a biosensor using L-asparaginase extracted from C. sativa, the biosensor will be used for the measurement of asparagine levels in leukemic serum samples. Different immobilizing techniques such as Whatman filter paper, gelatin, agarose, agar, calcium alginate and hydrosol gel on nylon membrane were applied to develop a biosensor. Phenol red indicator was co immobilized with the enzyme and the change in color was noted which gave the indication of asparagine level. Hydrosol gel technique was the most suitable one with the fastest response time of 6-10 seconds, the detection range was 10-1 M to 10-10 M. The response time for the developed biosensor was lesser as compared to the earlier works. The biosensor fabricated from C. sativa is an efficient one as it gives fast response and a higher detection range.
KEYWORDS: L-Asparaginase, Immobilization, Phenol red indicator, Biosensor, leukemia.
INTRODUCTION:
L-asparaginase was the first enzyme to be studied in humans for its antitumor potential1. It is a tetrameric protein and is widely used in the treatment of Acute Lymphoblastic Leukaemia (ALL)2,3. It helps in the hydrolysis of non-essential amino acid, L-asparagine to aspartic acid and ammonia4. Since, L-asparaginase breaks down L-asparagine in the blood, the malignant cells become deprived of its supply and hence die as they cannot accomplish their protein synthesis 5,6. Asparaginase activity was first detected by Lang7 in beef tissues and then reported by Kidd8 in Guinea pig serum.
L-asparaginase is found in a number of microorganisms such as E. coli (Enterobacteriaceae)9, Erwinia cartovora (Enterobacteriaceae)10,
Thermus thermophiles (Thermaceae)11 and Candida utilis (Saccharomycetaceae)12. It has also been found in various plant species such as Glycine max L. (Fabaceae)13, Capsicum annum L. (Solanaceae)14, Pisum sativum L. (Fabaceae)15, Lupin arboreus Sims (Fabaceae) and L. angustiplius L. (Fabaceae)16. From Withania somnifera L. Dunal (Solanaceae) L-asparaginase has been reported to have high specific activity17. This seems to be the first report where C. sativa has been used for isolation of L-asparaginase. C. sativa is used mainly for medicines and as a psychoactive agent. It produces cannabinoids and tetrahydrocannabinol (THC). THC contributes to the psychoactive properties of Cannabis18.
Earlier also number of biosensors have been developed for the detection of asparagine levels in leukemic serum samples. A biosensor was developed from a thermo stable recombinant Archaeoglobus fulgidus (Archaeoglobaceae) for the detection of L-asparaginase19. Ammonia was detected with the help of a potentiometric biosensor with an online gas analyser20. L-asparaginase based biosensor was constructed from garlic tissue cells in which hydrolyzed ammonia was detected by the ammonium gas electrode21. Garlic (Allium sativum L.) (Amaryllidaceae) has also been used in the fabrication of biosensor immobilized on chitosan matrix, used for monitoring the levels of hydrogen peroxidase22. For the detection of oxalate in urine samples, an amperometric biosensor was developed from the tissue extracts of spinach (Spinacia aleracea L.) (Amaranthaceae)23. Oxalate oxidase was detected by an amperometric biosensor fabricated by immobilized sorghum leaf extracts on carboxylate multi-walled carbon nano tubes and conducting polymers24. Biogenic amines in wine and beer samples were detected via electrochemical biosensor developed with the help of diamine oxidase isolated from Lathyrus sativus L. (Fabaceae)25. A biosensor was developed by polyphenol oxidase isolated from banana tissues for detection of acetaminophen26. Oxalate oxidase isolated from barley roots was immobilized on the hybrid formed from gold nanoparticles and porous CaCO3 microspheres to develop an amperometric biosensor27. A fluorescent biosensor was made for the detection of target DNA fragment of the transgene cauliflower mosaic virus 35S (CaMV 35S)28. A plant biosensor was developed by immobilizing L-asparaginase isolated from Withania somnifera for the detection of asparagine levels in leukemic serum samples29. Using L-asparaginase extracted from green chillies (Capsicum annum L.) Kumar and Walia30 developed a potentiometeric biosensor by immobilizing the biocomponent to Ion Sensing Electrode (ISE). The developed biosensor was used to detect asparagine levels in the blood of normal and leukemic patients. Using the L-asparaginase from Citrus lemon asparagine biosensor was fabricated by Kumar et al., 201331. In these biosensors the enzyme was extracted from different plants and then immobilized using matrixes such as agar, agarose, gelatin, polyacrylamide, calcium alginate, etc. The enzyme (L-asparaginase) extracted from Withania somnifera (L) Dunal was immobilized into TEOS hydrosol gel-chitosan matrix and was used to monitor the levels of L-asparagine in various fruit juice samples32.
MATERIALS AND METHODS:
The chemicals and reagents used in the study were of analytical grade and purchased from HiMedia Laboratories Pvt. Ltd., India.
Preparation of crude extract:
The leaves from the plants were collected and then washed with distilled water. The leaves were homogenized with 0.15 M KCl buffer and centrifuged at 8000 rpm for 20 min at 4°C. The supernatant thus obtained was taken as the crude extract33. To check the presence of L-asparaginase in the crude extract, it was coimmobilized with phenol red indicator. Following the interaction between the indicator and the substrate solution of L-asparagine, the extract changed from partly orange colored solution to bright purple colored solution. It was due to the deamination reaction in which L-asparaginase break down L-asparagine to aspartic acid and ammonia.
Immobilization methods:
Immobilization on whatman filter paper:
Whatman filter papers (0.2 µm, Millipore) were cut into small pieces of approximately 1x1 cm. Then 10 µl of phenol red and 20 µl enzyme (0.5 U) was put on these pieces. 10-10 to 10-1 M concentrations of asparagine were prepared and 2-3 pieces were put in each of the concentration. The time taken for the color change was recorded.
Immobilization in gelatin:
10 % aqueous solution of gelatin was prepared by dissolving 1.0 g gelatin in distilled water. The solution was heated mildly so that the gelatin dissolves properly. A hardening solution was prepared by mixing 20 % Formaldehyde, 50 % Ethanol and 30 % Water. After this 20 µl of enzyme (0.5 U), 2 ml of hardening solution and 10 µl of phenol red indicator were added to the gelatin solution. It was placed at -20°C for 4 h to facilitate its solidification. The gel was warmed to room temperature and cut into small pieces of 1 cm per side34. The pieces were put into varying concentrations of L-asparagine from 10-10 to 10-1 M. The time taken for change in color was recorded.
Immobilization in agarose:
1.5 % agarose solution was prepared in 25 mM Tris-acetate buffer of pH 7.2 containing 2 mM CaCl2 by heating for 10 min. Then 20 µl of enzyme (0.5 U) (per 10 ml of above solution) and 10 µl of phenol red indicator were added to the above solution. This was followed by pouring the solution into the petri plates and allowed to solidify. The gel was cut into small pieces of 1.0 x 1.0 cm35. The pieces were put into varying concentrations of L-asparagine from 10-10 to 10-1 M. The reaction time was noted.
Immobilization in agar:
4% agar solution in distilled water was prepared by boiling and then cooling it to 45-50°C. 20 µl of enzyme (0.5 U) and 10 µl of phenol red indicator were added. The solution was then poured into the petri plates for solidification. The solidified gel was cut into small square pieces of approximately 1.0x 1.0 cm size36. Different concentrations of L-asparagine from 10-10 to 10-1 M were prepared. The cut pieces of gel were put in the varying concentrations of L-asparagine and the reaction time was noted.
Immobilization in calcium alginate:
Chilled CaCl2 solution (0.075 M) was prepared. The slurry was prepared by mixing 3 % sodium alginate with 20 µl of enzyme (0.5 U) and 10 µl of phenol red indicator. The prepared CaCl2 solution was placed on the magnetic stirrer and the slurry was added drop-wise. The orange colored calcium alginate beads were formed with the help of 2.5 ml syringe without needle37. The beads were hardened by placing them at room temperature for nearly half an hour. Beads were put in each test tube containing different concentration of L-asparagine i.e. from 10-10 to 10-1 M. The color of the beads changed from partly orange to bright purple. Time was noted for color change in every test tube.
Immobilization in hydrosol gel on nylon membrane:
A homogenous stock sol-gel solution was prepared by mixing 600 µl ethanol, 10 µl 5 mM NaOH, 50 µl tertra ethyle orthro silicate (TEOS) and 60 µl distilled water. This solution was kept overnight in the refrigerator at 4°C. On the nylon membrane of size 1x1 cm the stock sol-gel solution was applied followed by 20 µl of enzyme and 10 µl of phenol red indicator. The membrane was dried at room temperature for 30 min. Varying concentrations of L-asparagine were poured on the nylon membrane and the response time was noted38.
Testing of L-asparagine levels in normal and leukemic blood samples:
The immobilized enzyme with different techniques was tested by putting the paper discs, agar, agarose, gelatin pieces, calcium alginate beads, and nylon membrane pieces in the normal and leukemic blood samples. The response time for change in color for all the samples was noted. 10-10 to 10-1 M concentrations of L-asparagine were prepared and the response time of normal and leukemic blood samples was noted.
Storage stability:
The immobilized enzyme is checked for its stability after immobilizing by placing it in the refrigerator at 4°C and then monitoring its activity time to time.
RESULTS AND DISCUSSION:
Immobilization on whatman filter paper:
The response time obtained for different concentrations of L-asparagine was shown in graph 1. The maximum response time was 23 seconds for 10-1 M concentration. The response time range falls between 23-11 seconds When the paper discs were put in various concentrations of L-asparagine the color changed from pale yellow to light pink (Figure 1). The leukemic blood sample showed change in color in 21 seconds while normal blood sample gave color change in 16 seconds.
Figure1: A.- Comparison of color of Whatman Filter paper (before and after the reaction) ; B.-Leukemic Blood Sample
Immobilization in gelatin:
The response time for 10-1 M concentration for L-asparagine was 21 seconds while the response time range for other concentrations was 8-19 seconds (Graph1). Color of gelatin pieces changes from partly orange to bright purple as shown in figure 2. The response time is directly proportional to the change in the concentration levels. The leukemic blood sample gives the response time of 19 seconds (10-2 M concentration) in comparison to normal blood sample which gives the response time of 15 seconds (10-5 M).
Figure 2: A.- Comparison of color of gelatin blocks (before and after the reaction) ; B.-Leukemic Blood Sample
Immobilization in agarose:
Figure 3 shows the color of agarose pieces before and after the reaction with L-asparaginase. The detection range for 10-9-10-1 M was 10-16 seconds and for 10-10 was 9.17 seconds With the decrease in concentration of L-asparagine the time taken for color change also decreased. The time taken for change in color by leukemic and normal blood samples was compared. The response time for leukemic blood sample was 15.8 seconds and for normal blood sample was 12.9 seconds corresponding to 10-2 M and 10-5 M concentrations respectively.
Figure 3: A.- Comparison of color of agarose gel pieces (before and after the reaction) ; B.-Leukemic Blood Sample
Immobilization in agar:
Graph 1 show the response time of different concentrations of L-asparagine ranging from 10-10-10-1 M. The response time falls in the range of 7-15 seconds with a minimum of 7.3 seconds for 10-1M concentration. The color of the pieces changed after reaction with L-asparagine as seen in figure 4. When we compared the response time of leukemic blood sample with normal blood sample it was found that normal blood sample gives lower response time i.e. 10 seconds for 10-5 M concentration of asparagine than leukemic blood sample which gave the response time of 13.3 seconds for 10-2 M concentration.
Figure 4: A.- Comparison of color of agar blocks (before and after the reaction) ; B.-Leukemic Blood Sample
Immobilization in calcium alginate:
Detection limit of asparagine achieved was 10-1-10-1 M. Response time for 10-1 M concentration was 11 seconds whereas for the next concentrations the response time was between 10-7 seconds (Graph 1). Normal blood sample has the response time of 8.9 seconds and leukemic sample has response time of 11.1 seconds Figure 5 shows that the color of the beads changed from partly orange to bright purple after reaction with L-asparagine.
Figure 5: A.- Comparison of color of Calcium alginaqte beads (before and after the reaction) ; B.-Leukemic Blood Sample
Immobilization in hydrosol gel on nylon membrane:
Detection limit of asparagine achieved was 10-10_10-1 M. In the hydrosol gel approach the response time for 10-1 M asparagine concentration was 10 seconds (Graph 1). The response time was higher than the response time for other techniques due to the mass transfer restriction. The color change for the lower concentrations is very fast as the pH was not adjusted initially. The response time for normal blood sample was 7.25 seconds for 10-5 M concentration whereas for leukemic blood sample it was 9 seconds for 10-2 M concentration of asparagine (Figure 6).
Figure 6: A.- Comparison of color of Hydrosol gel (before and after the reaction) ; B.-Leukemic Blood Sample
Graph 1: Performance of biosensor with L-asparagine standards (10-1-10-10 M) using different matrix
Storage stability:
L-asparaginase was found to be active after the storage. In air tight containers the nylon membrane pieces can be stored for a period of more than 4 months after drying.
CONCLUSIONS:
Hydrosol gel technique was the best among the six techniques used above. The detection range was 10-10 M to 10-1 M, while in the earlier works it was 10-5 M 16. The plant based biosensor gave fast response time as compared to the E. coli biosensor18. It shows that the biosensor developed using plant sources are cost effective, sensitive, reliable, easy to handle and cheap. The response time is lower than the response time observed with biosensor developed using Withania somnifera29, chilli30 and Citrus lemon31.
ACKNOWLEDGEMENTS:
The authors wish to thank Modi Education Society and Dr. Khushvinder Kumar, Principal, M.M. Modi College, Patiala for encouragements.
REFERENCES:
1. Stecher L. et al. Stability of L-asparaginase: an enzyme used in leukaemia treatment. Pharmaceutica Acta Helvetiae. 74; 1999: 1-9.
2. Schemer G and Holcenberg JS. Enzyme as drugs. Wiley Interscience, New York. 1981: 455-473.
3. Kumar K. et al. L-Asparaginase: An Effective Agent in the Treatment of Acute lymphoblastic Leukemia. Leukemia and Lymphoma. DOI: 10.3109/10428194.2013.803224; 2013.
4. Hendriksen HV et al. Evaluating the potential for enzymatic acrylamide mitigation in the range of food products using an asparaginase from Aspergillus oryzae. Journal of Agricultural and Food Chemistry. 57 (10); 2009: 4168-4176.
5. Kotzia GA and Labrou NE. Cloning expression and characterization of Erwinia carotovora L-asparaginase. Journal of Biotechnology. 119; 2005: 309-323.
6. Ghosh S. et al. Polyaniline nanofibres as a novel immobilization matrix for the anti-leukaemia enzyme L-asparaginase. Journal of Molecular Catalysis B: Enzymatic. 74 (1-2); 2011: 132-137.
7. Lang S. Uberdesamidierungim Tierkorper. Beitr. Chem. Physiology and Pathology. 5; 1904: 321-345.
8. Kidd J. Regression of transplanted lymphomas induced in vivo by means of normal guinea pig serum. Journal of Biological Chemistry. 98; 1953: 565-582.
9. Derst C et al. States and functions of tyrosine residues in Escherichia coli asparaginase II. European Journal of Biochemistry. 224 (2); 1994: 533-540.
10. Prista AA and Kyriakidis DA. L-asparaginase of Thermus-thermophilus: purification, properties and identification of essential amino acids for catalytic activity. Molecular and Cellular Biochemistry. 216; 2001: 93-101.
11. Maladkar N. et al. Fermentative production and isolation of L-asparaginase from Erwinia carotovora. Hindustan Antibiotics Bulletin. 35 (1-2); 1993: 77-86.
12. Kil O. et al. Extraction of extracellular L-asparaginase from Candida utilis. Bioscience, Biotechnology and Biochemistry. 59 (4); 1995: 749-750.
13. Streeter G. Asparaginase and L-asparagine Transaminase in Soybean Leaves and root nodules. Plant Physiology. 60; 1977: 235-239.
14. Mozeena V and Sivaramakrisnan M. Preparation and properties of L-asparaginase from green chillies (Capsicum annum L.). Journal of Biosciences. 2; 1980: 291-297.
15. Siechiechowicz K and Ireland R. Isolation and properties of an Asparaginase from leaves of Pisum sativum. Phytochemistry. 28; 1989: 2275.
16. Borek D et al. Isolation and characterization of cDNA encoding L-asparaginase from Lupinus luteus. Plant Physiology. 119; 1999: 1568-1570.
17. Oza P. et al. Withania somnifera (ashwagandha): Novel source of L-asparaginase. Journal of Integrative Plant Biology. 51 (2); 2009: 201-206.
18. Techen N et al. Genomic profiling of Cannabis sativa L. Planta Medica. 10.1055/s-2009-1216447; 2009.
19. Wang J and Bachas J. Biosensor for L-asparagines using a thermostable recombinant asparaginase from Archaeoglobus fulgidus. Analytical Chemistry. 74 (14); 2002: 3336-3341.
20. Fraticelli Y and Meyerhoff M. Online gas analyser for automated enzymatic analysis with potentiometric ammonia detection. Analytical Chemistry. 55 (2); 1983: 359–364.
21. Kim J. et al. Determination of L-asparagine using a garlic tissue electrode. Yakhak Hoeji. 39 (2); 1995: 113-117.
22. El Ichi S et al. A new peroxidase from garlic (Allium sativum) bulb: its use in H2O2 biosensing. Biotechnology and Applied Biochemistry. 51; 2008: 33-41.
23. Sezginturk K and Dinckaya E. A novel amperometric biosensor based on spinach (Spinacia oleracea) tissue homogenate for urinary oxalate determination. Talant. 59 (3); 2003: 545-551.
24. Yadav S. et al. An amperometric oxalate biosensor based on sorghum oxalate oxidase bound carboxylated multiwalled carbon nanotubes–polyaniline composite film. Journal of Biotechnology. 151; 2011: 212–217.
25. Di Fusco M et al. Characterization and application of a diamine oxidase from Lathyrus sativus as component of an electrochemical biosensor for the determination of biogenic amines in wine and beer. Analytical and Bioanalytical Chemistry. 401; 2011: 707-716.
26. Rounaghi G and Kakhki RM. Preparation and electrochemical application of a new biosensor based on plant tissue/polypyrrole for determination of acetaminophen. Bulletin of Material Science. 35; 2012: 811–816.
27. Chauhan N et al. Immobilization of barley oxalate oxidase onto gold-nanoparticle-porous CaCO3 microsphere hybrid for amperometric determination of oxalate in biological materials. Clinical Biochemistry. 45; 2012: 253-258.
28. Qiu B. et al. A novel fluorescent biosensor for detection of target DNA fragment from the transgene cauliflower mosaic virus 35S promoter. Biosensors and Bioelectronics. 10.1016/j.bios.2012.08.017; 2012.
29. Kumar K. et al. Plant asparaginase-based asparagine biosensor for leukemia. Artificial Cells Blood Substitutes and Biotechnology. 10.3109/10731199.2012.716062; 2012.
30. Kumar K and Walia S. L-asparaginase Extracted From Capsicum annum L and Development of Asparagine Biosensor for Leukemia. Sensors and Transducers Journal. 144 (9); 2012: 192-200.
31. Kumar, K., S. Punia, J. Kaur and T. Pathak. 2013. Development of plant asparagine biosensor for detection of leukemia. J. Pharma. Biomed. Sci. 35(35):1796-1801.
32. Verma, N., M. Kataria, K. Kumar and J. Saini. 2013. TEOS hydrosol gel-chitosan matrix based biosensor for monitoring asparagine in various fruit juices. Ann. Biol. Res. 4(1):265-70.
33. Bano M and Sivaramakrishnan VM. Preparation and properties of L-asparaginase from green chillies (Capsicum annum L). Journal of Biosciences. 2 (4); 1980: 291-297.
34. Alteriis ED,et al. Enzyme immobilization with in insolubilized gelatin. Journal of Chemical Technology and Biotechnology. 35; 1985: 60.
35. Prakash O. et al. Immobilization of watermelon (Citrus vulgaris) urease in agarose gel for urea estimation. Biotechnology and Bioprocess Engineering. 12; 2007: 131-135.
36. Mahajan R. et al. Comparison and suitability of gel matrix for entrapping higher content of enzyme for commercial applications. Indian Journal of Pharmaceutical Sciences. 72 (2); 2010: 223-228.
37. Johansen A and Flink JM. Influence of alginate properties and gel reinforcement on fermentation characteristics of immobilized yeast cells. Enzyme and Microbial Technology. 8; 1986: 737-748.
38. Wang Q. et al. Hydrogen peroxide biosensor based on direct electrochemistry of hemoglobin immobilized on carbon paste electrode by a silica sol-gel film. Sensors and Actuators B. 99; 2004: 50-57.
Received on 16.06.2014 Modified on 22.06.2014
Accepted on 24.06.2014 © RJPT All right reserved
Research J. Pharm. and Tech. 7(8): August 2014 Page 850-855