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.
7 trang |
Chia sẻ: thanhle95 | Lượt xem: 138 | Lượt tải: 0
Bạn đang xem nội dung tài liệu Use of bromelain isolated from pineapple (Ananas comosus) shoots in experimental design for practical Biochemistry teaching, để tải tài liệu về máy bạn click vào nút DOWNLOAD ở trên
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