Extraction and Immobilization of Proteolytic Enzymes from Local Yemeni Bean Seeds (Dolichos Lablab L.)
Pdf : Views Download
Citation: Extraction and Immobilization of Proteolytic Enzymes from Local Yemeni Bean Seeds (Dolichos Lablab L.). American Research Journal of Biosciences; vol 5, no 1; pp: 1-6.
Copyright This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Abstract:
The aim of this study was to prepare immobilized protease with high hydrolytic activity for biotechnological applications. Yemeni Bean Seeds (Dolichos Lablab L.) were used for extraction of acid protease using tris-buffer, pH 4.5 as enzyme solvent. Free acidic protease was immobilized on entrapment in calcium alginate gel (in situ activated) by covalent binding method. Their activity and immobilization efficiency for hemoglobin hydrolysis was investigated. Temperature and pH maxima of the immobilized protease showed no changes before and after immobilization.The immobilized protease exhibited good thermal stability and re-usability.
Keywords: Acid protease, Yemeni Bean Seeds, Immobilization, Optimization.
Description:
INTRODUCTION
Enzymes are very efficient catalysts which serve to accelerate the biochemical reactions of living cells and catalyze a variety of chemical reactions. They speed up biochemical reactions by lowering the energy of activation without themselves appearing in the reaction products[1]. Enzymes may be immobilized by a variety of methods, as shown in figure 1, which may be broadly classified as Physical methods; where weak interactions between enzyme and support material exist and to Chemical methods; where covalent bonds are formed between the enzyme and the supporting material [2].
Immobilization on solid carriers is perhaps the most used strategy to improve the operational stability of biocatalysts [5,6]. Seeds of (Dolichos lablab L.)is a species of bean in the family Fabaceae. It is native to Africa and it is cultivated throughout the tropics for food. It is the only species in the monotypic genus Lablab[7]. Yemeni bean (Dolichos lablab) both in tender green and mature dry stages is consumed after cooking in Yemen, India and parts of South America.
The efficiency of the free enzyme and the immobilized protease will be determined by estimating some physicochemical properties such as reuse.
MATERIALS AND METHODS
Plant Material and
Chemicals
The Dolichos lablab L. seeds were obtain from local market in republic of Yemen. All the chemicals were of analytical or electrophoresis grade. All chemicals were purchase from Sigma Aldrich, Himedia (Indian company). Deionized water was use throughout the experiments.
Enzyme Assay
Proteolytic activity was assayed by following the modified
method of [10] for soluble enzyme using casein (0.65%) as substrate, 10%
trichloroacetic aid (TCA) for stop reaction, after separate supernatant by
centrifuge mix with 500 mM Na2CO3 and double times diluted
folinciocatored reagent (FCR). After vigorous mixing, the color was allow
developing for 30 min and was monitored using spectrophotometer at 732 nm.
One unit (U) protease was define as the amount of enzyme
that hydrolyzed casein to releases 1µmol of tyrosine per minutes at 37o C. The
specific activity was express in the units of enzyme activity per milligram of
protein (u/mg). The enzyme activity computation in (u/ml) were made using the
following equation.
Electrophoresis
Purity of enzyme preparation was assessed through with
sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) system
(Adjustable vertical gel system) using slab gel (0.5 mm thick, 10%
polyacrylamide) by following the method [11]. The protein in the gel were
stained with Coomassie Brilliant blue R-250.
Determination of
Molecular Weight by SDS-PAGE
Sodium dodecyl sulphate-Polyacrylamide gel electrophoresis
of purified protease was carried out according method of [12.].
Immobilization of
Protease
Calcium alginate [13] Sodium alginate suspension (3%) was
prepared by suspending 0.9 g sodium alginate in 30 ml boiling water and
autoclaved at 121˚C for 15 min. The suspension was cooled to room temperature
and 47 µl cell suspensions (equivalent to 0.03 g dry cell weight) was added and
mixed for 10 min by stirring with a glass rod. This was taken in a sterile
syringe and added drop wise into chilled 0.2 M CaCl solution from 5 cm height
with constant stirring. The beads obtained were kept for curing at 4˚C for 1 h
in refrigerator. The cured beads so formed were washed with sterile distilled
water and preserved in 0.9% NaCl solution at 4°C. All the operations were
carried out aseptically under a laminar flow hood.
Characteristics of
Immobilized Enzyme
Effect of pH to
Immobilized Enzyme Activity
Immobilized enzyme reacts to Casein in different pH values
within 10 minutes, at 450 C. After reaction, we determine the immobilized
enzyme activity in these pH values (3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5,
7.0)
Effect of Temperature
on Immobilized Protease Activity
Immobilized enzyme reacts to Case in different temperature
values within 10 minutes, at determined pH. After reaction, we determine
immobilized enzyme activity in these temperature values (30, 40, 50, 55, 60,
70, 80, 90°C).
RESULTS AND
DISCUSSION
In the present study, we have purified and characterized
protolytic enzyme from seeds of Dolichos lablab L.by ammonium sulfate
precipitation, DEAE-cellulose and sephadex G-70 column.
Our data showed that the 0-80% ammonium sulfate saturation fraction correlated with highest protolytic enzyme and specific activities compared with the crude protolytic enzyme and other fractions.(16) The purification procedures of the protolytic enzyme secreted by tested seeds are summarized in (table 1). The results showed that the protolytic enzyme was purified 1.074 folds with a specific activity of 40.82u.mg-1 enzyme after ammonium sulfate fractionation. The protolytic enzyme was then purified using DEAE-cellulose column and resulted in 1.422 folds purification with a specific activity of 215.4u.mg-1 . The final purification step presented 7 fold enzyme purification with a specific activity of 266.34u.mg-1 . The yield of the enzyme after purification was found to be 54.06%.
SDS-PAGE analysis of the proteins at each step of purification shows that revealed a monomer band with a molecular weight of 50 KDa (figure 2).
IMMOBILIZATION OF
ACID PROTEASE
Purified protease enzyme after immobilization show in figure 3.
CHARACTERIZATION OF
IMMOBILIZED ENZYME
Effect of pH to
Immobilized Enzyme Activity
In this experiment, immobilized enzyme with Casein solution in pH range (4-7), within 10 minutes, 45o C. We also perform the control test using free enzyme. Results of immobilized enzyme activity on Casein at different pH values are shown in figure 4.
Effect of Temperature on Immobilized Enzyme Activity
In this experiment, immobilized enzyme with Casein in the temperature rang (10 to 90o C), in 10 minutes, at pH 4.5. We also conduct the control tests with free enzyme. Results of immobilized enzyme activity on Casein at different temperature values are shown in figure 5.
CONCLUSION
The immobilized enzymes on calcium alginate gel are prepared
for purpose of repeated use and the possibilities of continuous reaction
system. One of the most important properties is the stability of proteins when
they are used in some medical and industrial applications. The immobilization
of the enzymes improves this property as well as many other properties. In this
study, protease was purified and immobilized on calcium alginate gel. protease
was used in this study for its biological and industrial applications It is
used in paper textile, pharmaceutical applications, food, and detergent
industries. The reuse efficiency of the free and immobilized enzymes showed the
immobilized enzymes showed same in the relative activity.
References
1. Emel, E., Sibel, S. and Ural, A., (2006): Polyacrylamide–gelatine carrier system used for invertase immobilization. Food Chemistry, 97: 591-597.
2. Arıca, Y. and Bayramoglu, G., (2004): Reversible immobilization of tyrosinase onto polyethyleneiminegrafted and Cu (II) chelated poly (HEMAco-GMA) reactive membranes, Journal of Molecular Catalysis B: Enzymatic, 27: 255-265.
3. Saleem, M., Rashid, M., Jabbar, A., Perveen, R., Khalid, A. and Rajoka, M., (2005): Kinetic and thermodynamic properties of an immobilized endoglucanase from Arachniotus citrinus. Process Biochemistry, 40: 849- 855.
4. Abdel-Fattah, A., Osman, M. and Abdel-Naby, M., (1997):
Production and immobilization of cellobiase from Aspergillus niger A20,
Chemical. Engineering., 68:189-96.
5. Garcia-Galan C, Berenguer-Murcia A, Fernandez-Lafuente R,
Rodrigues RC (2011) Potential of different enzyme immobilization strategies to
improve enzyme performance. Adv Synth Catal 353: 2885-2904.
6. Aissaoui N, Landoulsi J, Bergaoui L, Boujday S, Lambert
JF (2013) Catalytic activity and thermostability of enzymes immobilized on
silanized surface: influence of the crosslinking agent. Enzyme Microb Technol
52: 336-343.
7. Hedstrom, L. (2002):Serine protease mechanism and
specificity. Chem Rev 102(12): 4501-24.
8. Akhtaruzzaman, M. N.H.M,Rubel, R. Jamal, A. Rahman and
Rahman, T.(2012): Isolation and characterization protease enzyme from
leguminous seeds. Agricultural Science 2(8): 434-440.
9. Lowry, O.H., Rosebrough, N. J., Farr, A. F. andRandall,
R. J. (1951): Protein measurement with Folin Phenol Reagent. Biological
chemistry 193: 265-275.
10. Anson, M.L. (1938): The Estimation of pepsin, Tripsin,
Papain and cathepsinwith Hemoglobin. General Physiology 22: 79-89.
11. Laemmli, UK (1970): Cleavage of structural proteins
during the assembly of the head of bacteriophage T4. Nature 227(5259): 680-685.
12. Smith, B.J. (1984): SDS Polyacrylamide gel
electrophoresis of proteins. Method in molecular Biology 41-56.
13. Johnsen A and Flink JM. Influence of alginate properties
and gel reinforcement on fermentation characteristics of immobilized yeast
cells. Enz. Microb. Technol. 1986, 8:737-748.
14. Mateo, C.; Palomo, J.M.; Fernandez-Lorente, G.; Guisan,
J.M.; Fernandez-Lafuente, R. Improvement of enzyme activity, stability and
selectivity via immobilization techniques. Enzyme Microbiology Technology. 40,
2007, 1451–1463.
15. Kumar, A.G.; Swarnalatha, S.; Kamatchia, P.; Sekaran, G.
Immobilization of high catalytic acid protease on functionalized mesoporous
activated carbon particles. Biochemistry Eng. J. 43, 2009, 185–190.
16. Geethanjali, S. and Anitha, S. Optimization and
immobilization of purified Labeo rohita visceral protease by entrapment method.
Enzyme research, 2013, 874050.
17. Abida A., Shah A., Aliya R., Samina I. and Abid A..
Calcium Alginate: A Support Material for Immobilization of Proteases from Newly
Isolated Strain of Bacillus subtilis KIBGE-HAS. World Applied Sciences Journal
7 (10), 2009, 1281-1286.
18. Rafat, R., Przemystaw, T., Matgorzata, D. and Klaudia,
K. The evidence in sprouted seeds and their application for animal protein
digestion. Chemical papers, 72, 2018, 1213-1221.