European Journal of Biotechnology and Bioscience
European Journal of Biotechnology and Bioscience
Online ISSN: 2321-9122, Impact Factor: RJIF 5.44
www.biosciencejournals.com
Volume 4; Issue 6; June2016; Page No. 34-37
Isolation of catalase producing bacteria, production of catalase and its application to degrade hydrogen
peroxide from effuelent
1
MandeepKataria*, 2 Jyoti Saini, 3 Maninder Singh,4 Kuldeep Kumar
1
Department of biotechnology, CT institute of Pharmacy, Shahpur, Jalandhar, Punjab, India
2
Department of biotechnology, Govt. Mohindra College, Patiala, Punjab, India
3, 4
Department of biotechnology, M. M. Modi College, Patiala, Punjab, India
*Corresponding Author: [email protected]
Abstract
Catalase is very important enzyme in today scenario because of its applications in Milk, dye and other industry for removal of
H2O2. In the present study microbial production of catalase was carried out by taking isolate from paper mill effluent, soil and
industrial effluent from a drain. Best culture of microorganism is selected from out of three samples by comparing their enzyme
activity. Then enzyme was produced and extracted from cells. The crude enzyme and whole cells were compared for the removal
of hydrogen peroxide from synthetic effluent.
Keywords:Catalase, H2O2, synthetic effluent
1. Introduction
Catalase holds an important place in the enzymatic world
because of its use in various industries. Catalase has highest
turnover of all enzyme known to us 1 molecule of catalase can
convert 40000 molecule of H2O2 into water and molecular
oxygen. The main role of catalase in cells is to prevent
accumulation of H202 up to a toxic level formed as a biproduct of metabolic process occurs in a living system.
2 H2O2
Catalase
2 H2O + O2
Catalase is present in wide range of sources which includes
microorganisms, plants, and animals[1]. Catalase was first
noticed by Louis Jacques Thenard in 1818 and the name
‘Catalase’ was given by Oscar Loew in 1900. Catalase (EC
1.11.1.6) has tetramer of four identical subunits of 60kDa
exists as a dumb bell- shaped. Each monomer, at the catalytic
center, contains a heme prosthetic group, similar to heme
group found in hemoglobin. An iron atom is present in the
middle of each heme group.
Fig 1: Molecular (a) and Chemical structure (b) of Catalase enzyme[2]
Catalases are of 3 types Catalase contain heme group in their
structure so they are called as heme catalases. Catalase –
Peroxidase contain both peroxidase and catalase activity. MnCatalase contains Mn in place of Fe in its catalytic center so
they are called pseudo-catalase[3]. H2O2 is used in textile
industry for bleaching cloths in place of chlorine and it is less
toxic. Sodium bi-sulfate is used to remove traces of H2O2 but
know catalase is used to do this and it does not require
rinsing[4]. In the dyeing of cotton fabric can be improved when
the H2O2 bleached fabric is treated with catalase for the
removal H2O2 residues as compared to number of washing
steps to remove it and we can save lot of water [5]. In a De-ink
pulp mill H2O2 is used in pulper or post bleaching stage certain
amount of H2O2 residues remain in waste water after each
stage, catalase is used to remove H2O2 from water which is
very harmful to environment[6]. Catalase is used for treatment
milk to remove H2O2 before the cheese production[7]. Catalase
activity in milk as an Indicator of Thermization treatments
used in the production of Cheddar cheese. H2O2-catalase
treatment of milk before inoculation of starter culture contains
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European Journal of Biotechnology and Bioscience
aroma producing bacteria, increases the stabilization of diacetyl in cultured milk[8]. Because catalse has lots of
application the present study done to isolate the catalase
producing bacteria and production of catalase after that it is
applied on H2O2.
2.7 Enzyme Assay[9]
Enzyme assay was performed to check the activity of catalase
in the broth and was done by using Hydrogen peroxide
(100mM), Phosphatebuffer (pH 7.0), Potassium dichromate,
Distilled water by method explained in Table 1
2. Material and methods
2.1 Material
Hydrogen peroxide (30%), glacial acetic acid, phosphate
buffer, Sulphuric acid, Potassiumdichromate, Gram
stainingkit, urea, tryptone, beefextract, ferrous sulphate,
Ammoniumsulphate, Sodiumsulphate, Magnesiumsulphate,
Manganese sulphate, 1% (w/v), maltose, sucrose, lactose,
fructose and dextrose. all the chemicals used were of
analytical grade unless otherwise specified.
Table 1: Enzyme Assay procedure
2.2 Sample
Sample was collected from two places, industrial effluent was
taken from drain near Rajpura road, Patiala and soil sample
from Modi College garden.
2.3 Isolation
Sample collected from different places were serially diluted up
to 103 times.Loopful of mixture was streaked nutrient agar
medium and labeled as drain (DS) and soil sample (SS).
Inoculated plates were incubated at 37ºC for 24 hours. After
incubation small colonies of microorganism was observed on
petriplates. Then these colonies were transferred to nutrient
broth to obtain a cultural broth and again incubated at 37ºC for
24 hours.
2.4 Screening
Screening of catalase positive microorganism is done by
bubble test. Culture from the different samples was
centrifuged at 12000 rpm for 10 minutes. Supernatant and
pellet from centrifuged culture was taken into different test
tubes. Few drops of supernatant was put on clean slide and
then add few drops of H2O2over it,if formation of bubbles
takes place then it shows the presence of catalase in it. If it
doesn’t show any bubble formation then our sample is catalase
negative. The catalase positive colonies were compared for
catalase activity.
2.5 Growth characteristics
Culture was characterized morphologically using Gram’s
Stain. Prepared a thin smear of the culture on a clean glass
slide and heat-fixed the culture using Gram’s iodine and
safarnin dye. Air dried the slide and examined under
microscope.
Solution
Hydrogenperoxide(100mM)
Phosphate buffer (pH 7.0)
Enzyme
Potassium dichromate
Distilled Water
Test Sample
5 ml
4.9 ml
0.1 ml
4 ml
Blank Sample
4.9 ml
0.1 ml
4 ml
5 ml
2.8 Extraction of catalase
Extraction of extracellular enzyme 100 ml medium was
pelleted by centrifugation at 12000 rpm for 10 min having
temperature 4ºC. The supernatant was used as the crude
enzyme. Extraction of intracellular enzyme from pellet of
cells: - Cell pellet was disrupted by sonicating it for 10 min. at
pulse on and off 15 sec and 10 sec respectively having 50%
amplitude Again centrifugation of sample was done to collect
the supernatant containing desired intracellular.
2.9 Production of Catalase
The selected colony was grown on LB medium for 24 hrs at
37C. The enzyme activity was checked for both extra cellular
and intracellular enzyme as above said.
2.10 Application of Catalase(Amorim, 2002)[5]
Crude enzyme as well as cells was immobilized in 3 % sodium
alginate slurry separately and beads were prepared. These
beads were treated with synthetic effluent which contains 30%
H2O2. After regular time interval the remaining H2O2 was
calculated
3. Results and Discussion
3.1 Isolation of catalase producing bacteria
Spread plating of serially diluted effluent was done on
petriplates containing nutrient agar, petriplates were incubated
at 37°C for 24 hours in a BOD incubator. After 24 hours the
petriplates were examined to check the growth of bacteria.
Figure below shows the colonies appeared after spreading on
nutrient agar. The colonies obtained from drain sample are
denoted by DC and those from soil sample are denoted by DS.
2.6 Stanadard curve for H2O2
H2O2 concentrations of 100mM were taken in test tubes and
4.0 ml of dichromate acetic acid solution was added to each.
Addition of reagent to H2O2 instantaneously produced an
unstable blue precipitate of perchromic acid. On subsequent
heating for 10 min in boiling water bath, the colour of the
solution changed to stable green due to the formation of
chromic acetate. After cooling the absorbance was taken at
570 nm[9].
Fig 2: Petriplate shows the colonies of microorganism after
spreading.
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European Journal of Biotechnology and Bioscience
3.2 Bubble test
Then different colonies were transfer to nutrient broth after the
incubation of 24 hr thee bubble test was performedby put
some drops of H2O2 over it, the colonies produced bubble is
catalase positive and colonies that doesn’t produce bubbles are
catalase positive. There were 5 catlase positive (DC1, DC3,
DC4, SC1, SC2) and 3 catalase negative colonies (DC2, DC5,
and SC3) (Table 2)
3.4 Characterization of bacterial isolate
The microorganisms were now heat fixed on a glass slide to
know its characteristics. The isolated microorganisms were
blue colored gram positive, rod shaped that occur either as a
single rod or in a chain when observed under microscope after
gram staining (Fig 4) is supposed to be bacillus species
Table 2: Shows catalase positive and catalase negative colonies and
enzyme activity
Name of
colony
DC1
DC2
DC3
DC4
DC5
SC1
SC2
SC3
Catalase
positive/negative
+
+
+
+
+
-
Enzyme activity
(units/ml/min.)
3.2 ×105
4.4 ×106
1.245 ×106
2.123 ×104
2.38 × 105
Fig 4: Gram positive, rod shaped cells isolated from water effluent
collected from sewage drain.
3.5 Extraction of enzyme
The activity of crude enzyme obtained after extraction was
checked by using enzyme assay and was found 4.6 ×10 6
units/ml/min. The activity of cells obtained after extraction
was checked by using enzyme assay and was found
148units/ml/min (Table 3).
Fig 3: Slide showing the formation of bubbles due to presence of
catalase
3.3 Identifications
After identifying catalase positive bacterial colonies, each
catalase positive colony was further streaked on freshly
prepared nutrient medium and then incubated at 37°C at 24
hours and then activity was checked and bacterial colony
having more activity was used for the production of catalase.
Activity in DC3 colony was more than the other colonies
therefore bacteria from DC3 colony was used for the
production of catalase.
(A)
Table 3: Comparing activities of extracellular (crude) and
intracellular enzyme (cells).
Source
Extracellular enzyme
Intracellular enzyme
Activity (units/ml/min.)
4.6 ×106
148
By comparing the enzymatic activity of extracellular and
intracellular enzyme, crude enzyme shows higher activity than
cells and therefore catalase produced by microorganism is
extracellular in nature.
3.6 Applications of immobilizes beads
Beads obtained by immobilization of Crude enzyme and cells
(Fig5).
(B)
Fig 5: Immobilized beads of cells (a) and crude enzyme (b)
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European Journal of Biotechnology and Bioscience
Table 4: Immobilized beads containing crude enzyme and cells and their comparison for removal of H2O2
Time(hours)
11/2
3
4½
Decreased concentration (%) of synthetic effluent
(H2O2) by crude enzyme
80 %
67 %
54 %
Decreased concentration (%) of synthetic
effluent (H2O2) by cells
97 %
90 %
86 %
From above (Table 4) we can say that our enzyme was
extracellular because the immobilized beads containing crude
enzyme degrade the synthetic effluent more rapidly and
decrease the concentration of H2O2 as the time passes than the
beads containing cells. The beads of crude enzyme degrade
the effluent up to 54 %.
4. Conclusion
Isolated bacteria may be Bacillus sp. because it appears rod
like and gram positive. The enzyme produced is extra cellular
and enzyme reduced the H2O2 more efficiently then cells.
5. References
1. Chance B, Sies H, Broveris A. Hydroperoxide
metabolism in mammalian organs. Physical. Rev. 1979;
59(3):527-625.
2. Maleki LA, Salehi B, Behfar R, Saeidmanesh H,
Ahmadian F, Sarebanhassanabadi M, et al. Designing a
hydrogen peroxide biosensor using catalase and modified
electrode with magnesium oxide nanoparticles. Int. J.
Electrochem. Sci. 2014; 9:257-271.
3. Allgood GS, Perry JJ. Characterization of a manganesecontaining catalase from the obligate thermophile
Thermoleophilumalbum. Journal of Bacteriology. 1986;
168(2):563-567.
4. Doshi R, Shelke V. Enzyme in textile industry-An
environment friendly approach. Indian Journal Of Fibre &
Textile Research. 2001; 26:202-205.
5. Amorim AM, Gasques MD, Andreaus J, Scharf M. The
application of catalase for the elimination of hydrogen
peroxide residues after bleaching of cotton fabrics. Anais
da Academia Brasileira de Ciências., 2002; 74(3):433436.
6. Bobby N, et al. Method for Controlling Catalase in a
Deink Pupl Mill. Tappi Journal, 1997; 80:198-199.
7. Hirvi Y, Griffiths MW. Milk catalase activity as an
indicator of thermization treatments used in the
manufacture of Cheddar cheese. J Dairy Sci. 1998;
81(2):338-345.
8. Pack MY, Vedamuthu ER, Sandine WE, Elinker PR.
Hydrogen Peroxide-Catalase Milk Treatment for
Enhancement and Stabilization of Diacetyl in Lactic
Starter Cultures.Journal of Dairy Science. 1968;
51(4):511-516.
9. Sinha AK. Colorimetric assay of catalase. Anal Biochem.
1972; 47:389-394.
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