L-Asparaginase gives a great benefit in the cancer treatment, especially in acute\ud
lymphoblastic leukemia. L-Asparaginase is also proven to reduce the acrylamide\ud
content in the foods. The objective of this study was to perform immobilization and\ud
characterization L-Asparaginase produced from Bacillus licheniformis Strain HSA3-\ud
1a. The results showed that the free form L-Asparaginase from B.\ud
licheniformis HSA3-1a has optimum activity at pH 8 and 50oC, with a specific activity\ud
of 616.26 IU/mg protein and stabilized at the optimum pH and temperature for 60\ud
minutes. The immobilized L-Asparaginase with activated glutaraldehyde-carbon\ud
carrier has optimum activity at pH 7 and 60??C with a specific activity of 499.27 IU/mg\ud
protein and stabilized at the optimum pH and temperature for 60 minutes. The\ud
immobilized L-Asparaginase can retain its activity by 84.79% after 2 times repeated\ud
use

Ahmad, Ahyar

Hasanuddin University Repository




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Int J Pharm Bio Sci 2013 Oct; 4(4): (B) 274 - 280 
 
 
This article can be downloaded from www.ijpbs.net 
B - 274 
 
Research Article                                                                                                                         Biotechnology 
 
 
 
 
 
International Journal of Pharma and Bio Sciences 
ISSN 
0975-6299 
 
 
PURIFICATION AND IMMOBILIZATION OF  L-ASPARAGINASE  ENZYME 
FROM  THE THERMOPHILIC BACTERIA Bacillus licheniformis STRAIN HSA3-1a 
 
AHYAR AHMAD1*,  ABDUL MUIS PATTA2 AND  HASNAH NATSIR1 
 
                                            
1
Department of Chemistry, Hasanuddin University, Makassar, Indonesia. 
 2Vocational High Schooll of chemical Analyst, Ministry of Industry of Indonesia 
 
ABSTRACT 
 
L-Asparaginase gives a great benefit in the cancer treatment, especially in acute 
lymphoblastic leukemia. L-Asparaginase is also proven to reduce the acrylamide 
content in the foods.  The objective of this study was to perform immobilization and 
characterization L-Asparaginase produced from Bacillus licheniformis Strain HSA3-
1a. The results showed that the free form L-Asparaginase from B. 
licheniformis HSA3-1a has optimum activity at pH 8 and 50oC, with a specific activity 
of 616.26 IU/mg protein and stabilized at the optimum pH and temperature for 60 
minutes. The immobilized L-Asparaginase with activated glutaraldehyde-carbon 
carrier has optimum activity at pH 7 and 60°C with a specific activity of 499.27 IU/mg 
protein and stabilized at the optimum pH and temperature for 60 minutes. The 
immobilized L-Asparaginase can retain its activity by 84.79% after 2 times repeated 
use. 
 
KEYWORDS:  L-Asparaginase, Bacillus licheniformis Strain HSA3-1a, immobilization, and 
specific activity 
  
 
  
 
 
 
               
             *Corresponding author 
 
 
 
 
 
 
 
 
 
                                          AHYAR AHMAD  
Department of Chemistry, Hasanuddin University, Makassar, Indonesia. 
Int J Pharm Bio Sci 2013 Oct; 4(4): (B) 274 - 280 
 
 
This article can be downloaded from www.ijpbs.net 
B - 275 
 
INTRODUCTION 
 
L-Asparaginase has a great benefit in the 
cancer treatment, especially in acute 
lymphoblastic leukemia, given that the L-
Asparaginase can break down L-Asparagine, 
one of the nutritional components of cancer 
cells, which is expected to inhibit the growth of 
these cells 1.  L-Asparaginase is also proven to 
reduce the acrylamide content in the foods. L-
Asparaginase may prevent the formation of 
acrylamide by converting the amino acid of L-
Asparagine as its precursor which is naturally 
present in the food into another form of amino 
acid, namely aspartic acid that is also 
commonly found in the food 2. L-Asparaginase 
can be found in many animal tissues, bacteria, 
plants, and in the serum of mice, but is not 
found in humans. L-Asparaginase is produced 
in large quantities by some 
microorganisms; E. coli, Erwiniacartova, 
Enterobacteraerogenes, Corynebacterium 
glutamicum, Candida utilities, Bacillus sp., 
Pisumsativum, and Streptomyces sp. and 
these enzymes that exhibit antitumor activities 
1,3,4. Production of enzymes from thermophilic 
bacteria is increasingly becoming popular to be 
explored further to produce enzymes that have 
stability at high temperatures. Thermophile 
bacteria is often found in the extreme 
environment of hot springs at 60-80°C 
temperatures, such as Thermus, Bacillus, 
Clostridium, Thermoanaerabacter, and 
Desulfomaculum 5, 6, 7. The bacteria Bacillus sp 
used in the production of the enzyme L-
Asparaginase has been published by E-
Moharam (2010). One of the 
thermophile Bacillus bacteria isolated from the 
hot springs in South Sulawesi, Indonesia  
was B. licheniformis8. The use of thermostable 
enzymes can save some amount of money 
because these enzymes have longer shelf lives 
and higher activity at high temperatures. Some 
advantages of the use of immobilized enzymes 
compared with the free enzyme are increase in 
enzyme stability, less amount of enzyme used, 
facilitation of separation and re-use of the 
enzyme and in some cases it can increase the 
enzyme activity.  Seven media supporting the 
immobilization of L-Asparaginase from Bacillus 
sp have been investigated 1. This supporting 
media is previously activated by glutaraldehyde 
which may react differently to the terminal 
amino residues in the enzyme protein. The 
immobilization of L-Asparaginase can be 
through a covalent cross linking between 
enzymes with different carriers such as 
activated carbon, chitin, carboxymethyl 
cellulose, silica gel, tricalcium phosphate, and 
chitosan. As reported the highest activity of 
immobilized L-Asparaginase is active carbon 
as its carrier1. 
 
MATERIALS AND METHODS 
 
Purification of Bacteria B. licheniformis 
Purification is performed by growing the 
bacteria B. licheniformis stock in the collection 
of Biochemistry and Biotechnology Laboratory 
Department of Chemistry, Hasanuddin 
University Makassar Indonesia, into the growth 
medium plate.  The colonies that grow are then 
isolated, purified, and fertilized in a production 
medium culture of L-Asparaginase 8, 9, 10, 11. 
 
Production of L-Asparaginase Enzyme 
The production of  L-Asparaginase is carried 
out by fertilizing the isolates into 45 mL 
inoculum in 100 mL Erlenmeyer flask. The 
Erlenmeyer is incubated at 50°C in shaker 
incubator for 24 hours with 200 
rpm. Afterwards, 45 mL of inoculum is mixed 
with 500 mL of sterile production medium and 
then incubated for 48 hours at 50oC. The 
composition of the production medium are; 
0.05% yeast extract, 0.01% peptone, 0.1% 
NaCl, KH2PO4 0.01%, 0.01% MgSO4.7H2O, 2.0 
% L-Asparagine, 0.01% CaCl2 and 0.7% 
ammonium sulphate 8, 9, 10, 11. 
 
Isolation and Purification of L-Asparaginase 
Enzyme 
The medium liquid of cell culture results is 
separated from the cell mass by filtration. The 
filtrate containing ammonium sulphate enzyme 
is then added while stirring by the degree of 
saturation of ammonium sulphate 20-40%. 
After overnight settling, the precipitate formed 
is separated by centrifugation at 10,000 rpm for 
30 min with temperature of 4°C. The precipitate 
obtained is dissolved in 10 mL of each buffer 
0.1 M Tris-HCl pH 8. The enzyme solution of 
Int J Pharm Bio Sci 2013 Oct; 4(4): (B) 274 - 280 
 
 
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B - 276 
 
ammonium sulphate fractionation results is put 
in a cellophane bag and immersed into a 
vessel containing enough distilled water. The 
dialysis vessel is placed over a magnetic stirrer 
fitted with a low speed. Dialysis performed for 
48 hours by replacing the solution several 
times with 0.01 M Tris-HCl buffer solution of pH 
8 to show no more ammonia in buffer solution 
of dialysis results. The experiment is carried 
out with the addition of Nessler reagent that will 
react to form brown color if there is ammonia in 
the buffer solution. 
 
Determination of Protein Concentration 
The determination of protein concentration is 
carried out by using a modified method by 
Lowry; and using Bovine Serum Albumin (BSA) 
as standard solution. 
 
Assay of L-Asparaginase Enzyme Activity 
L-Asparaginase activity is determined by the 
colorimetric method12 at 50°C using UV-Visible 
Spectrophotometer (Shimadzu, UV-2600) by 
measuring the amount of ammonia produced 
from L-Asparaginase catalysis using Nessler 
reagent. The reaction mixture consisting of 0.1 
mL enzyme, 0.2 mL Tris-HCl buffer 0.05 M pH 
8.6 and 1.7 mL of 0.01 M L-asparagine is 
incubated at optimum temperature for 10 
minutes. The reaction is stopped by addition of 
0.5 mL of 1.5 M solution of trichloroacetic acid 
(TCA) after the centrifugation at 10,000 rpm for 
5 minutes, and 0.5 mL of the supernatant is 
diluted with 8.5 mL aquadest and then added 
with 1 mL Nessler reagent. The ammonia 
released by the hydrolysis of  L-Asparaginase 
enzyme reacts to the Nessler reagent. 
Afterwards, its level is determined using the 
standard ammonium chloride. One unit of L-
Asparaginase (IU) is defined as the amount of 
the  L-Asparaginase enzyme that catalyzes the 
release of one µmol of ammonia per minute at 
the test conditions. 
 
Immobilization of L-Asparaginase Enzyme 
Immobilization enzyme was carried out using 
the medium supporting active carbon. 200 mg 
of the medium supporting active carbon is 
mixed with 2 mL of Tris-HCl buffer 0.01 mol of 
pH 8 which contains 2.5% glutaraldehyde at a 
room temperature of 4°C for 2 hours. A carrier 
that formed is then filtered and washed with 
distilled water to remove unbound 
glutaraldehyde. The carrier that formed is 
incubated with 5 mL of 0.05 M Tris-HCl buffer 
pH 8  containing 1 mL enzyme. After 4 hours of 
shaking in temperature of 4°C, unbound 
enzyme is released by washing with 0.05 M 
Tris-HCl buffer pH 8. No activity of the proteins 
is found in rinse buffer. 
 
Characterization of L-Asparaginase Enzyme 
Enzyme characterization is performed on the 
L-Asparaginase in free and immobilized forms.  
a. Effect of pH on activity of L-
Asparaginase 
L-Asparaginase activity is evaluated in 
several pH condition. Free and 
immobilized enzymes are each incubated 
in 0.05 M buffer of pH 6-10. In each 
condition, the amount of ammonia that is 
released is measured. Buffer that is used 
is potassium phosphate (pH 6.0 to 
7.0), Tris-HCl (pH 8.0 to 9.0) and Glycine-
NaOH (pH 10). 
b. Effect of temperature on activity of L-
Asparaginase 
The L-Asparaginase activities are 
evaluated with various temperatures at 
optimum pH. Free and immobilized 
enzymes are respectively incubated at 
temperatures between 30-70°C with 
intervals of 10°C. 
c. Determination of the pH and 
Temperature Stability on enzyme 
activity 
The stability of pH on enzyme activity is 
determined by pre-incubation of enzyme 
and buffer at optimum pH. Pre-incubation 
is performed for 2 hours and activity of L-
Asparaginase is tested at 30 minutes 
intervals at the optimum 
temperature. Determination of tempera-
ture stability is performed by pre-incubation 
of enzyme and buffer of optimum pH at 
optimum temperature for 2 hours and its 
activity tested at 30 minutes intervals 1,8. 
 
d. Determination of Stability of 
Immobilized L-Asparaginase Enzyme 
Operation 
The operational stability of the immobilized 
L-Asparaginase enzyme activity is 
performed with a test of the activity of L-
Int J Pharm Bio Sci 2013 Oct; 4(4): (B) 274 - 280 
 
 
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B - 277 
 
Asparaginase to enzyme solution 
repeatedly up to 5 repetitions at optimum 
pH and temperature. 
 
 
 
 
 
RESULTS AND DISCUSSION 
 
Characterization of L-Asparaginase Enzyme 
Some characteristics of the L-Asparaginase 
enzyme in free and immobilized form studied in 
this research include: the influence of pH, 
temperature, pH stability, temperature stability, 
and operational stability of immobilized L-
Asparaginase enzymes. 
 
 
 
Figure 1 
Determination the Optimum pH of free and Immobilized L-Asparaginase 
 
a. Effect of pH on L-Asparaginase Activity 
Figure 1 shows the optimum pH of free L-
Asparaginase is pH 8; while that of the 
immobilized L-Asparaginase enzyme is pH 
7. This difference is caused by the 
molecules capacity of enzyme protein and 
its matrix carrier, i.e. active carbon that 
leads to a decrease in optimum 
pH. Specific activity of the free  L-
Asparaginase enzyme increases in line 
with the increase of pH 7 up to pH 
8. Specific activity of free L-Asparaginase 
at pH 6 is 345.8 IU/mg protein, 506.64 
IU/mg protein at pH 7 and optimum at pH 8 
with a specific activity of 543.19 IU/mg 
protein which decreases at pH 9 and 10. 
Mean while, the specific activity of the 
immobilized L-Asparaginase enzyme 
increases up to pH 7. Specific activity of 
immobilized L-Asparaginase at pH 6 is 
255.19 IU/mg protein and optimum at pH 7 
with a specific activity of 436.46 IU/mg 
protein, the activity decreases at pH 8, 9, 
and 10. 
 
b. Effect of Temperature on L-
Asparaginase activity 
Figure 2 shows the activity of L-
Asparaginase to the temperature changes. 
L-Asparaginase activity is directly 
proportional to the increase in temperature 
from 30°C up to optimum temperature of 
50oC and then decreased at a temperature 
of 60oC and 70oC. Specific activity of free 
L-Asparaginase is 616.26 IU/mg protein. 
 
Int J Pharm Bio Sci 2013 Oct; 4(4): (B) 274 - 280 
 
 
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B - 278 
 
 
 
Figure 2 
Determination the Optimum Temperature of Free and Immobilized L-Asparaginase 
 
Specific activity of the immobilized L-
Asparaginase enzyme increases to a 
temperature of 60°C. Specific activity of 
immobilized L-Asparaginase at 50°C is 
464.36 IU/mg protein and the optimum 
temperature of 60°C with a specific activity 
of 520.14 IU/mg protein. 
 
c. Stability of L-Asparaginase on pH and 
Temperature 
L-Asparaginase in free form is stable in 
conditions of optimum pH (pH 8), it can be 
stable for 60 minutes with an 89.1% 
relative activity then decreases again to 
68.6% at pre-incubation of 90 minutes and 
44.8% remaining for its relative activity at 
120 min pre-incubation. This suggests that 
L-Asparaginase in free form can retain the 
active enzymes and are able to work well 
even after storage for 1 hour at optimum 
temperature as shown in Figure 3. The 
stability of the immobilized L-Asparaginase 
enzyme form to the optimum pH (pH 7) as 
shown in Figure 4 has a relative activity at 
30 min (85.35%), 60 minutes (78.02%), 90 
minutes (59.71%), and 120 min (45.05 %) 
of incubation. 
  
 
 
Figure 3 
Determination of Stability at Optimum pH of Free (pH 8) and  
Immobilized L-Asparaginase (pH 8) 
 
 
Int J Pharm Bio Sci 2013 Oct; 4(4): (B) 274 - 280 
 
 
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B - 279 
 
 
 
Figure 4 
Determination of Stability at Optimum Temperature of Free (50°C) and 
Immobilized L-Asparaginase (60°C) 
 
Figure 4 shows the stability of the free L-
Asparaginase to pre-incubation at optimum 
temperature (50°C). Pre-incubation up to 60 
minutes displays has 82.9% relative activity.  
Its relative activity declines at the 90 minutes 
and 120 minutes pre-incubation with relative 
activity of 59.9% and 45.32%, respectively. The 
stability of immobilized L-Asparaginase form to 
the pre-incubation at optimum temperature 
(60°C) shows the relative activity of 77.64% at 
the 60 minute pre-incubation, and a decline in 
relative activity at the 90 minutes and 120 
minutes pre-incubation with relative activity of 
61.66 and 55.27%, respectively. 
 
 
 
Figure 5 
Operational Stability (Repeated Use) Immobilized L-Asparaginase 
 
The operational stability of the immobilized L-Asparaginase enzyme activity is performed with a 
test of the activity of L-Asparaginase to enzyme solution repeatedly up to 5 repetitions at optimum 
pH and temperature. The immobilized L-Asparaginase enzyme can be used again as shown in 
Figure 5. The immobilized L-Asparaginase can be used twice with relative activity of 84.79%. 
 
CONCLUSION 
 
L-Asparaginase from B. licheniformis HSA3-1a in a free form has optimum activity at pH 8 and 
temperature of 50oC with a specific activity of 616.26 IU/mg protein and is stable at the optimum 
pH and temperature for 60 minutes. The immobilized L-Asparaginase with active glutaraldehyde-
carbon carrier has optimum activity at pH 7 at 60°C with a specific activity of 499.27 IU/mg protein 
and is stable at the optimum pH and temperature for 60 minutes. The Immobilized   L-
Asparaginase can retain its activity by 84.79% after 2 times repeated use. 
Int J Pharm Bio Sci 2013 Oct; 4(4): (B) 274 - 280 
 
 
This article can be downloaded from www.ijpbs.net 
B - 280 
 
ACKNOWLEDGEMENT  
 
The authors thank the Head of Biochemistry and Biotechnology Laboratory of Hasanuddin 
University, Indonesia for sample preparation and enzymatics assay. The authors also  thank to 
Madhyastha Radha for editorial reading of the manuscript. 
 
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PURIFICATION AND IMMOBILIZATION OF L-ASPARAGINASE ENZYME FROM THE THERMOPHILIC BACTERIA Bacillus licheniformis STRAIN HSA3-1a

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PURIFICATION AND IMMOBILIZATION OF L-ASPARAGINASE ENZYME FROM THE THERMOPHILIC BACTERIA Bacillus licheniformis STRAIN HSA3-1a

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