Use of bromelain isolated from pineapple (Ananas comosus) shoots in experimental design for practical Biochemistry teaching

HNUE JOURNAL OF SCIENCE DOI: 10.18173/2354-1075.2017-0129 Educational Sci., 2017, Vol. 62, Iss. 6, pp. 60-66 This paper is available online at USE OF BROMELAIN ISOLATED FROM PINEAPPLE (Ananas comosus) SHOOTS IN EXPERIMENTAL DESIGN FOR PRACTICAL BIOCHEMISTRY TEACHING Dao Văn Tan, Tran Trung Duc, Nguyen Sao Mai Faculty of Biology, Hanoi National University of Education Abstract. In the trend that students are required more and more practical skills in studying Biochemistry, a practical science, the improvement of experiments is essentially needed. There is a gap of experiments on protein separation based on electrophoresis or poor option for the content of enzyme kinetics in the practical Biochemistry for students in Vietnamese universities of education. In this paper, we report potential application of bromelain isolated from pineapple shoots to design an experiment for teaching Practical Biochemistry. Bromelain from 60% ethanolic fraction of pineapple shoots was used to design experiments related to enzyme and protein such as determining the isoelectric point of the protein, determining the activity of protease, electrophoresis, and enzyme kinetics. The appropriate amount of bromelain as well as other materials such as gelatin as the substrate and ratio of enzyme and substrate for each experiment has been determined. The experiments could be conducted in less than 3 hours, a period time suitable for practical lessons. These applications can be used to enrich contents of Practical Biochemistry and train students for taking national exams and International Biology Olympiad. Keywords: Bromelain, experimental practice, enzyme kinetics, electrophoresis, isoelectric point, Ananas comosus. 1. Introduction It is not easy for learners to train experimental practice skills by themselves while practical skills are more and more required nowadays [4]. Our understanding of molecular nature of life processes originates in the laboratory where data on biomolecules and their activities are collected. It is imperative that students gain appreciation for the link between laboratory activities and the growth and maturing of scientific knowledge base on biochemistry. Thus students in the discipline must be provided opportunities to learn and practice the experimental methods accompanying biochemistry [1]. Walddrop G.L. (2009) had a review of 20 Biochemistry textbooks used in undergraduate courses and revealed “Most of the understanding derived from kinetic studies comes from the patterns rather than the actual number” [12]. According to the Vietnamese textbooks used to teach experimental practices on biochemistry in Vietnamese universities [5, 10, 11], there is a lack of contents relating to electrophoresis and enzyme kinetics as well as poor options for the isoelectric point of proteins. Moreover, recently, experimental skills in analysis of enzyme and electrophoresis have been tested in Biological Olympiad exams. Received date: 10/4/2017. Published date: 20/5/2017. Contact: Dao Văn Tan, e-mail: daotanvn@yahoo.com 60 Use of bromelain isolated from pineapple (Ananas Comosus) shoots... Pineapple has been used as a medicinal plant in several native cultures and bromelain from pineapple has been identified chemically since 1876. In 1957, it was found in high concentrations in pineapple stems [9]. Bromelain isolated from pineapple has been used in various areas such as tenderizing meat, hydrolyzing cow’s liver, coagulating milk, cleaning silk. . . [14]. There are some advantages of using bromelain from pineapple: non-toxic compound with therapeutic values in modulating (1); high activity (2); inexpensive source (3), low molecular weight (4); and easy isolation (5). There have been some studies on the isolation of bromelain from pineapple recently. Bromelain can be isolated and purified with 40-80% ammonium sulfate precipitation [3]. However, using ammonium sulfate may cause the interaction between the enzyme and the salt. Based on column chromatography, bromelain was purified by H. B. Costa, et al. (2014) [6]. Chromatography technique requires expensive equipment and time. Previous experiments have tested bromelain activity using bovine serine albumin (BSA) and casein as the substrate. However BSA is quite expensive and casein is very difficult to dissolve. In a recent document, proteins from pineapple have been used to design an experiment on protein purification based on column chromatography [4]. Although there was a time limitation for this experiment type in teaching, we assume that there is a high potential of bromelain use in Biochemistry experiment teaching. Through reviewing Vietnamese practical textbooks, we found that there was no use of bromelain from pineapple in experiments. We also found that there were not experiments on protein purification using electrophoresis or poor experiments on protease detection, enzyme kinetics and the isoelectric point of protein. Therefore, our purpose is to find a simple and cheap method in order to isolate bromelain from cheap materials and thereafter to design simple experiments related to electrophoresis, enzyme kinetics as well as protein for Practical Biochemistry teaching. 2. Content 2.1. Materials and methods 2.1.1. Materials Pineapple shoots; hydrogen peroxide, gelatin, trichloroacetic acid, ethanol, citric acid and disodium hydrogen phosphate were purchased from China and tyrosine from Sigma. 2.1.2. Methods Bromelain extraction Thirty grams of pineapple shoots were sterilized with 0.1% H2O2 then homogenized in citric acid-sodium phosphate buffer pH = 6.0 for 5 minutes. The sample is then filtered through a clean and sterilized cloth to remove the residue. The filtrate is then centrifuged at a speed of 3000 rpm for 20 minutes at 4◦C. Bromelainisolation and purification Bromelain from the extractwere isolated and purified using 40% cold ethanol then centrifuged at speed of 4000 rpm for 15 min at 4 ◦C to collect the supernanant. The supernanant was then fractionated by adding ethanol up to final concentration of 60% and then centrifuged at speed of 4000 rpm for 15 min at 4 ◦C to collect the precipitate. Protein ditermination Protein contents were determined by Bradford method (1976) [2] using BSA as the calibration standard. The optical density of samples was measured with Biotek spectrophotometer 61 Dao Văn Tan, Tran Trung Duc, Nguyen Sao Mai at 595 nm. Enzyme assay The substrate used to determine bromelain activity is 5% gelatin. The enzyme was incubated with the substrate at 30◦C for 15 or 30 min and inactivated by precipitation with 10% TCA (trichloroacetic acid) solution. The samples were then centrifuged at speed of 4000 rpm for 15 minutes at 4◦C. The optical density of supernatants then was measured at 275 nm. The enzyme activity is defined as the amount of tyrosine (µg) released per mg protein per minute. Principles of experimental designing for Practical Biochemistry teaching For designing experiments, we followed the protocol of R. Boyer and A.Wilfson (2009) [1]. According to these authors, the best way to begin the design of a lab lesson plan is to answer five following questions: (1) How can lab session be constructed so as to give students practice in designing experiments, interpreting data, and understanding the limits of the experimental approach? (2) What technical skills and procedures should be practiced and mastered by students? (3) What instrumentation should undergraduate students become familiar with? (4) What teaching styles work best to most effectively train students in the lab? (5) What is the importance of the “other lab skills” such as communication, team work, ethics and responsibility? And how are these best taught. Data analysis: Data were analyzed using Microsoft Excel and SPSS software. 2.2. Results and discussion 2.2.1. Relationship between enzyme amount and enzyme activity Fig.1.Relationship between enzyme amount and enzyme activity, Gelatin at 5% were use as the substrate, Reaction times were 15 minutes (A) and 30 minutes (B). Understanding enzyme activity is very important in designing experiments related to enzyme. The activity of isolated bromelain was investigated using different enzyme amounts for 15 min and 30 min (Fig 1.). In these experiments, different enzyme amounts were used for the same substrate volume (3 mL of 5% gelatin) at 30◦C. It was found that there was strong relationship between the amount and enzyme activity. When using 2 or 4 µg of enzyme, bromelain activities were quite high in both reaction periods (15 and 30 minutes). The activity for each of these enzyme amounts for 15 min reactions were quite similar when compared with the 30 min reactions. Enzyme activities were observed to suddenly drop when using the amount of enzyme greater than 4 µg for both reaction periods. These results indicated that using 2- 4 µg of enzyme to hydrolyze 3 mL of 5% gelatin was suitable for enzyme assay. Because of time limitation in lessons, a short-time experiment was recommended. 62 Use of bromelain isolated from pineapple (Ananas Comosus) shoots... 2.2.2. Use of bromelain in designing experiment on the isoelectric point of protein Protein separation can be based on the isoelectric point. Understanding of the isoelectric point is of great help in protein purification by ion exchange chromatography. Therefore, isoelectric point is a very important term in Biochemistry. Theoretically, the isoelectric point can be calculated based on peptide sequence [7]. However, practically, protein can be modified after synthesis. Previous research attempted to design protein fractionation experiment based on isoelectric point. This experiment took less than an hour to complete [13]. In practical lessons (protein properties) for Vietnamese students, a simpler experiment on isoelectric point of protein was designed using albumin [5]. This protein has an acidic isoelectric point. In order to bring the overview of isoelectric point term, there is essential need for an experiment on alkaline isoelectric point. Bromelain is such a candidate. Therefore, the experiment was designed as the following: Fig.2. Turbidity of bromelain in different pH Step 1. Prepare solutions: citric acid-disodium hydrogen phosphate buffers pH= 6.0, 7.0, 7.5 and 8.0, 450 µg/mL bromelain solution and 96% ethanol. Step 2. Add 1 mL of 450 µg/mL bromelain solution to the glass tubes containing 2 mL of buffer with different pH (6.0, 7.0, 7.5 and 8.0). Then mix well. Step 3. Add 1 mL of 96% ethanol in each tube. Then mix well. Step 4. Observe and compare the turbidity of the solution in each tube. A result of the above experiment was shown in Fig.2. There are some merits of this experiment in teaching biochemistry: the signal of experiment is quite clear; it can be very simply carried out; it does not require expensive equipment and chemicals; and it takes very short time to complete. 2.2.3. Use of bromelain in designing experiment on protease detection In practical lessons, experiments on enzyme including enzyme prediction experiments such as urease, amylase, invertase prediction were designed for students [5]. Although proteases are very important enzymes in the cell, there is a lack of simple experiments on protease prediction in the textbook. Therefore, such a simple experiments should be designed. To design an experiment on protease detection, we carried out as followings: prepare 5 test tubes labeled from 1 to 6; control tubes labeled from 1’ to 6’. Three milliliters of 1% gelatin were added into each tube. For the test tubes 1, 2, 3, 4 and 5, 100 µl of 0.02 mg/mL, 0.04 mg/mL, 0.08 mg/mL, 0.16 mg/ml and 0.32 mg/mL bromelain solutions, respectively, were added and incubated at 30 ◦C for 15 min. For the control tubes 1’, 2’, 3’, 4’, 5’ and 6’ after incubation at 30 ◦C for 15 min, 100 µL of 0.02 mg/mL, 0.04 mg/mL, 0.08 mg/mL, 0.16 mg/mL and 0.32 mg/mL bromelain solutions and water were added, respectively. Each tube then was added with 3 ml of 10% TCA and mixed well. The results (Fig. 3) showed that the turbidity of the solution in control tubes 3’, 4 ’, 5’ could be distinctly distinguishable from that in test tubes 3; 4; 5. No difference in turbidity of the solution in control tubes. We centrifuged the test tubes after the reaction and found that there were no precipitates in test tubes 3, 4, 5 and there were white precipitates in the remaining tubes. 63 Dao Văn Tan, Tran Trung Duc, Nguyen Sao Mai Fig.3. Turbidity of gelatin solutions hydrolyzed with bromelain at different concentrations. Tested tubes 1, 2, 3, 4 and 5 were added with 2, 4, 8, 16 and 32 µg of bromelain, respectively and control tubes 1’, 2’, 3’, 4’ and 5’ were also added with with 2, 4, 8, 16 and 32 µg of bromelain, respectively. Tube 6’ was 100 µL of distilled water. These results indicated that aliquots of 8 to 32 µg of bromelain could be used to hydrolyze 3 mL of 1% gelatin for 15 minutes at 30◦C to design protease detection experiments. Based on these experimental results we designed protease detection experiments as follows: Material and equipment Tubes; a marker pen; 0.08 -0.32 mg/mL bromelain; 1% gelatin; 10% TCA. Procedure Preparing the substrate: Take 2 glass tubes and label them as T and C. Add 3 mL of 1% gelatin in each tube. Reaction: Add 100 µL (or 2 drops) of distilled water and 0.08 -0.32 mg/mL bromelain solution to tube C and tube T, respectively, mix well then and incubate them for 15 minutes at 30◦C. Stopping reaction: Add 3 mL of 10% TCA to each tube. Mix well. Results and observation: Observe the turbidity of the solution in both tubes. Analyzing the results: Based on the turbidity of the solution in two tubes, draw conclusions. Similar to the experiment on the isoelectric point of protein, the advantages of this experiment are: clear experimental signal; simple perform; not require expensive equipment and chemicals; taking place in a short time. 2.2.4. Use of bromelain in designing experiment on enzyme kinetics We designed the enzyme kinetic experiment using bromelain as follows: Use 4 µg of bromelain for hydrolyzing 1 mL of gelatin solution at concentrations of 5, 10, 15, 20 and 50 mg/mL in buffer pH = 6.0 for 15 min. One milliliter of 20% TCA solutions then was added to stop the reaction and precipitate the proteins. After centrifugation to remove the protein, the optical density of the supernatants was measured to determine the enzyme activity. Based on the substrate concentration and enzyme activity, a Lineweaver-Burk plot was built (Fig.4). 64 Use of bromelain isolated from pineapple (Ananas Comosus) shoots... Fig. 4. Lineweaver-Burk plot of bromelain kinetic Fig. 5. SDS-polyacrylamide gel electrophoresis image. Lanes 1,2,3,4,5, and 6 wereof sample buffer, 1250 µg/mL BSA, 2000 µg/mL bromelain, 1000 µg/mL bromelain, 250 µg/mL bromelain and a mixture containing 250 µg/mL bromelain and BSA 1250 µg/mL incubated at 30◦C for 5 min, respectively. G.L Waldrop (2009) used thymidylate synthetase and its substrate, N5, N10 methylene-tetrahydrofolate to design enzyme kinetic experiment. However the author did not describe the details of his experiment. Chemicals used in his experiment were quite expensive. The designed experiment can be finished in an hour. Through the experiment, students can practice a number of skills such as micropipetting, centrifugation, and enzyme assay to succeed in conducting this experiment. 2.2.5. Use of bromelain in designing experiment on electrophoresis Electrophoresis is a basic technique for protein separation, determination of peptide weight and detection of protein expression. This technique is widely used in every biochemistry lab in the world. However, after reviewing Vietnamese textbooks used to teach experimental practices on biochemistry in Vietnamese universities [5, 10, 11] we found that there is a lack of contents relating to electrophoresis. Therefore in this study the potential of applying bromelain for electrophoresis experiment using sodium dodecyl sulfate polyacrylamide gels was checked. The gel contained 12.5% acrylamide. Electrophoresis was run using a voltage of 250V, a current of 25mA for 60 minutes. Gels were stained with CBB (Coomassie Brilliant Blue Staining). For each well, 10 µL of samples were loaded. Gels were stained with CBB solution for 30 min and then were distained for 60 min. Total time for the experiment was less than 3 hours. Each wells 1,2,3, 4, 5 and 6 were loaded with 10 µL of sample buffer; 1250 µg/mL BSA, 2000 µg/mL bromelain, 1000 µg/mL bromelain, 250 µg/mL bromelain and a mixture containing 250 µg/mL bromelain and BSA 1250 µg/ml incubated at 30◦C for 5 min, respectively. 65 Dao Văn Tan, Tran Trung Duc, Nguyen Sao Mai The electrophoresis image (Fig. 5.) showed that there were no bands in buffer lane (lane 1), indicating no protein contamination. A major band can be observed in BSA lane (lane 2). Four distinctly distinguishable bands can be observed in lane 3 and 4 which contained 2000 µg/ml and 1000 µg/ml bromelain, respectively, indicating peptides presence in bromelain solutions. However, using low bromelain concentration (250 µg/mL) no bands were seen, indicating the under threshold of detection. Three clear bands were observed in lane 6 (mixture of BSA and bromelain), indicating the partly hydrolyzed BSA. Signal from the gel image were very clear. 3. Conclusion Bromelain isolated from pineapples can be used to design experiments in teaching enzyme kinetics; electrophoresis, protease detection, determination of isoelectric point to enrich contents of practical biochemistry. The designed experiment includes in some advantages such as clear experimental signals; simple performs; using inexpensive equipment and materials. REFERENCES [1] R. Boyer and A.Wolfson, 2009. Commetary: Innovation in the Biochemistry/Molecular Biology lab. Molecular Biochemistry Education. Vol. 37, pp. 11-15. [2] M.M. Bradford, 1976. Rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem.,72, pp. 248–254. [3] I. R. A. P. Bresolin , I. T. L. Bresolin , E.Silveira, .E. B. Tambourgi and P. G.Mazzola, 2013. Isolation and Purification of Bromelain from Waste Peel of Pineapple for Therapeutic Application. Braz. Arch. Biol. Technol. Vol.56, n.6: pp. 971-979. [4] P.T.T. Chau, L.T.P. Hoa, T.V. Khanh, T.Q.Phong, N.H.M. Quyen, N.Q. Uyen and J. Wang, 2011. Document on Some Techniques of Molecular Biology and Biochemistry. Vietnam Association of Biochemistry, 73 pages (in Vietnamese). [5] P.T. T. Chau, N.T. Hien and P.G. Tuong (1998), Practical Biochemistry. Educational Publisher. 132 pages (in Vietnamese). [6] H. B. Costa, P. M. B. Fernandes, W. Romão, J. A. Ventura, 2014. A new procedure based on column chromatography to purify bromelain by ion exchange plus gel filtration chromatographies. Industrial Crops and Products 59, pp. 163–168 [7] G. Hegyi, J. Kardos, M. Kovács, A. Málnási-Csizmadia, L. Nyitray, G.Pál, L. Radnai, A. Reményi, I.Venekei , 2003. Introduction to Practical Biochemistry. Eotvos Loránd University 204 pages. [8] P. T. A. Hong (2003), Biochemistry techniques. Vietnam National University Press, Ho Chi Minh City, 217 pages (in Vietnamese). [9] G. S. Kelly, 1996. Bromelain: A Literature Review and Discussion of its Therapeutic Applications. Alternative Medicine Review, 1, pp. 243-257. [10] N. V.Mui (2001) Practical Biochemistry. Vietnam National University Press, Hanoi, 173 pages (in Vietnamese). [11] N.Q.Vinh, B. P. Thuan and P.T.Nghia (2004), Practical Biochemistry. Vietnam National University Press, Hanoi 130 pages (in Vietnamese). [12] G. L. Waldrop, 2009. A Quanltitive Approach to Enzyme Inhib