Purification and characterization of Brevibacterium sp. amylase
PARTIAL PURIFICATION AND CHARACTERIZATION OF
Brevibacterium sp. AMYLASE
WIDYO TRI PUTRANTO
DEPARTEMENT OF BIOLOGY
FACULTY OF MATHEMATICS AND NATURAL SCIENCES
BOGOR AGRICULTURAL UNIVERSITY
BOGOR
2012
ABSTRACT
WIDYO TRI PUTRANTO. Purification and characterization of Brevibacterium sp. amylase.
Under supervision of ANJA MERYANDINI and YOPI.
Amylase is an enzyme that used widely in various industrial field. This enzyme
specifically catalyze the hydrolysis of -1,4-glycosidic bond on starch. To fulfill industrial need, the
search for a new promising source of amylase is important. One of amylase source that has not
been well explored yet is marine microbe. The purpose of this research was to purify and
characterize the amylase from Brevibacterium sp. isolated from Jakarta Bay. The Brevibacterium
sp. was cultured in Artificial Sea Water (ASW) medium. Measurement of specific amylase
activity was performed by modified Miller method. The highest crude enzyme activity was
produced 96 hours after the cultivation which is reaching 1,2 U/mL. After precipitation using
ammonium sulphate, the crude enzyme solution were purified by gel filtration chromatography.
SDS-polyacrylamide analysis result showed that the molecular weight of the enzymes were 79
kDa, 55,7 kDa, and 33,6 kDa. The partial purified enzyme has optimum activity on temperature
50oC and pH 6,4.
Key words: amylase, purification, characterization, Brevibacterium.
ABSTRAK
WIDYO TRI PUTRANTO. Purifikasi dan karakterisasi enzim amilase dari Brevibacterium sp.
Dibimbing oleh ANJA MERYANDINI dan YOPI.
Amilase adalah enzim yang digunakan di berbagai bidang industri. Enzim ini
mengkatalisis hidrolisis dari ikatan 1,4-glikosidik pada pati. Untuk memenuhi kebutuhan industri,
pencarian terhadap sumber amilase baru yang menjanjikan sangat penting. Salah satu sumber
amilase yang belum banyak dieksplorasi adalah mikroba laut. Penelitian ini bertujuan
mempurifikasi dan mengkarakterisasi enzim amilase dari Brevibacterium sp., yang diisolasi dari
perairan Teluk Jakarta. Isolat Brevibacterium sp. dikulturkan di media Artificial Sea Water
(ASW). Pengukuran aktivitas amilase dilakukan menggunakan metode Miller yang dimodifikasi.
Aktivitas amilase tertinggi pada enzim ekstrak kasar diproduksi pada jam ke-96 setelah
pengkulturan, yaitu mencapai 1,2 U/mL. Setelah presipitasi dengan menggunakan ammonium
sulfat, larutan enzim ekstrak kasar dimurnikan dengan kromatografi gel filtrasi. Hasil analisis
SDS-PAGE menunjukkan berat molekul protein adalah 79 kDa, 55,7 kDa, dan 33,6 kDa.
Berdasarkan karakterisasi, enzim bekerja optimum pada suhu 50oC dan pH 6,4.
Kata kunci: amilase, purifikasi, karakterisasi, Brevibacterium.
PARTIAL PURIFICATION AND CHARACTERIZATION OF
Brevibacterium sp. AMYLASE
WIDYO TRI PUTRANTO
An Undergraduate Thesis
Intended to Pursue Bachelor Degree of Science
In Faculty of Mathematics and Natural Sciences
Bogor Agricultural University
DEPARTEMENT OF BIOLOGY
FACULTY OF MATHEMATICS AND NATURAL SCIENCES
BOGOR AGRICULTURAL UNIVERSITY
BOGOR
2012
Title
Name
NIM
: Partial Purification and Characterization of Brevibacterium sp.
Amylase
: Widyo Tri Putranto
: G34080116
Approved by,
Supervisor I
(Prof. Dr. Anja Meryandini, M.Si)
NIP. 196203271987032001
Supervisor II
(Dr. Yopi)
NIP. 196912201989011001
Endorsed by :
Chief of Department
Department of Biology
Faculty of Mathematics and Natural Sciences
Bogor Agricultural University
(Dr. Ir. Ence Darmo Jaya Supena, M.Si)
NIP. 19641002 198903 1 002
Graduation date:
PREFACE
Praise to Almighty God for His blessing so this thesis can be done. The title of this thesis is
“Partial Purification and Characterization of Brevibacterium sp. Amylase. This research was taken
place at Biotechnology Research and Development Center, Indonesian Institute of Science,
Cibinong. This research was conducted from February until June 2012.
This thesis would not have been possible without the support of several thoughtful and
generous individuals. My acknowledgements to Prof. Dr. Anja Meryandini as my first supervisor
and Dr. Yopi as my second supervisor for the guidance along the process of this thesis. I also wish
to acknowledge Dr. Achmad Farajallah as examiner and representative of Department of Biology,
FMIPA, IPB for the advices and discussion for the completion of this thesis. I would like to thank
my beloved family, especially my parents for their supports and prayers. I would like to say thank
you to Mba Nanik, Mba Ade, Mas Ashif, Mba Lia, Mas Alex and Mas Dicky for every advices
they gave to me. I would like to say thank you as well to my comrades, Dewi, Viska, Yana,
Rohana, Yufi, Riza, Lady and Titi for their cooperation on laboratory, and also for all Biology 45
friends for their supports, prayers and motivations.
This research was supported and funded by bio catalyst and fermentation laboratory,
Bioproses division, Research Center Biotechnology Indonesian Institue of Science. I hope this
thesis will be usefull for all readers.
Bogor, September 2012
Widyo Tri Putranto
vi
CURRICULUM VITAE
Writer was born in Bogor at 8 August 1990 as the third child of three from the parents FX
Mugihardjo and Triasih Rudiatun.
On 2008, writer graduated from SMA Regina Pacis Bogor. On the same year, writer
successfully continued his education on Bogor Agricultural University through Seleksi Nasional
Mahasiswa Perguruan Tinggi Negeri (SNMPTN). Writer chose Biology major, Faculty of
Mathematics and Natural Sciences.
On 2010, writer join field study on Pangandaran Beach, Ciamis, West Java, and wrote a
report with title ‘Agarose-producing Bacteria Associated with Algae’. Writer was also conducted
practical field on Indonesian Institute of Science from July to August 2011, wrote about
Production of Amylase from Marine Bacteria.
TABLE OF CONTENTS
Page
LIST OF TABLES ............................................................................................... viii
LIST OF FIGURES ............................................................................................. viii
LIST OF APPENDIX .......................................................................................... viii
INTRODUCTION .................................................................................................. 1
Background ......................................................................................................... 1
Objective ............................................................................................................. 1
Place and Time .................................................................................................... 1
MATERIALS AND METHODS ............................................................................ 1
Preparation of Brevibacterium sp. culture .......................................................... 1
Production of enzyme. ....................................................................................... 2
Bioassay of Amylase ........................................................................................... 2
Ammonium Sulphate (NH4)2SO4 Precipitation .................................................. 2
Gel Filtration Chromatography ........................................................................... 2
Characterization of purified enzyme ................................................................... 2
Molecular Weight Determination ....................................................................... 2
RESULTS ............................................................................................................... 2
DISCUSSION ......................................................................................................... 4
CONCLUSION ....................................................................................................... 6
BIBLIOGRAPHY ................................................................................................... 6
APPENDIX ............................................................................................................. 8
LIST OF TABLES
Page
1 Purification of Brevibacterium sp. amylase ................................................................................... 4
LIST OF FIGURES
Page
1 Curve of amylase activity from Brevibacterium sp. on ASW medium with pH 8 at room
temperature ..................................................................................................................................... 2
2 Protein precipitation using various concentration of ammonium sulphate ..................................... 3
3 Amylase activity and protein profile of fraction from gel filtration chromatography .................... 3
4 Temperature effects on Brevibacterium sp. amylase activity using 0,5% substrate at pH 6,6. ...... 3
5 pH effects on Brevibacterium sp. amylase activity using 0,5% substrate at room temperature ... 3
6 SDS PAGE profile ......................................................................................................................... 4
7 Protein marker linear curve ............................................................................................................ 4
LIST OF APPENDIX
Page
1 Composition of bacteria growth medium ....................................................................................... 9
2 Composition of DNS solution ........................................................................................................ 9
3 Amylase activity on ASW medium with pH 8 on room temperature ............................................. 9
4 Precipitation using ammonium sulphate ........................................................................................ 9
5 Temperature effects on amylase activity ........................................................................................ 9
6 pH effects on amylase activity .................................................................................................... 10
7 Protein marker standard data ........................................................................................................ 10
8 Sediment of protein after precipitation with 50% concentration of ammonium sulphate ............ 10
1
INTRODUCTION
Background
Enzymes are catalysts that help to convert
other molecules called substrates into
products. An enzyme able to catalyze
chemical reaction at least until 1 million times
faster than reaction without catalizer (Yuwono
2005). Enzymes are giant molecules with
molecular weight varies from 5 kDa to 5000
kDa, with typical values in the range 20 kDa–
100 kDa (Bugg 2004). Enzymes work by
lowering the activation energy of a chemical
reaction, and thus dramatically accelerating
the rate of the reaction. Most enzyme catalyze
just one kind of reaction or a group of
reactions with high similarity. The specificity
was the consequence of the uniqueness of
each enzyme’s active site. Active site is part
of an enzyme where the reaction takes place.
The enzyme commission (EC) numbers
divided enzymes into six main groups
according to the type of reaction of catalysed:
oxidoreductases, transferases, hydrolases,
lyases, isomerases and ligases. Among the
groups, hydrolases are presently the most
commonly encountered class of enzymes
within the field of enzyme technology. One of
the hydrolases groups used widely in various
industries is amylase.
Amylase is an enzyme that specifically
catalyze the hydrolysis of glucosidic linkages
in polysaccharide. It catalizes the hydrolysis
of -1,4-glycosidic bond on starch (Liu et al.
2011). The amylases (α-amylases, β-amylases,
glucoamylases) are one of the most important
families of enzymes in the field of
biotechnology (Rodriguez et al. 2006). The αamylase belongs to a family of endo-amylases
that catalyses the initial hydrolysis of starch
into shorter oligosaccharides (van der Maarel
et al. 2002). This hydrolysis results in the
production of maltose and oligosaccharides.
Amylase also helps the carbohydrates in a
food break down more quickly and easily.
This makes amylase as popular enzyme for
food industry. Amylase is a highly demanded
industrial enzyme in various fields such as
pharmaceuticals, textiles, detergents, etc
(Sivaramakrishnan et al. 2006). The most
common industry using amylase is starch
industry. This kind of industry using amylase
for hidrolizing starch to produce fructose and
glucose syrup (Nielsen & Borchert 2000).
In human, alpha amylase is synthesized in
the acinar cells of the saliva glands and stored
in secretory granules inside the cells. Amylase
not only produced by human, but also by
animals, plants, and microorganisms (Kandra
2003). Although there are many sources of
amylase, producing amylase from bacteria
provides more advantages. Producing amylase
from bacteria requires less time and space for
production, easiness to modify, consistency of
the enzyme, and cheaper.
Marine bacteria is one of amylase source
that still has not been well explored yet. In
other words, there might be abundant of
promising amylase sources from marine.
Many industrial applications need enzymes
that stable at extreme temperature, pH, and
salt concentration. In this case, marine
microbial enzymes may offer advantages. The
optimum activity of marine bacterial enzymes
usually occurs at high salinity, making these
enzymes utilisable in many harsh industrial
processes. In addition, most marine bacterial
enzymes are considerably thermotolerant,
remaining stable at room temperature over
long periods (Mohapatra e al. 2003).
Sea is a large saline water environment,
for consequence, in order to survive, marine
bacterias
must
have
salt
tolerance
characteristic. The example of bacteria with
that kind of characteristic is Brevibacterium
genus, gram positive bacterias from
Actinomycetales family. Brevibacteria are
short shaped, nonbranched, asporogenous,
obligately aerobic, rods which may exhibit a
marked rod-coccus cycle when cells become
older (Gruner et al. 1993). Brevibacterium is
known as soil bacteria which causing odor on
human feet. One of this genus member,
Brevibacterium linens , is known as the most
important surface bacteria in the cheesemaking process due its role in the colouring
surface and its typical flavouring activity
(Motta & Brandelli 2002).
Objective
The objective of this thesis was to purify
and
characterize
amylase
from
Brevibacterium sp., isolated from Jakarta Bay.
Place and Time
This research conducted from February
2012 until July 2012 in Research Center for
Biotechnology,
Bioproses
division,
Indonesian Institute of Science, Cibinong.
MATERIALS AND METHODS
Preparation of Brevibacterium sp.
culture. Brevibacterium sp. colony in petri
disk was inoculated to ASW solid medium.
2
Bioassay of Amylase. First, 250 µL of
substrate solution was added to all tube.
Substrate solution was made by mixing starch
with 0,2 M of buffer phosphate pH 6,6. The
substrate solution concentration was 0,5%.
Then 250 µL of enzyme was added into the
tubes. The addition of enzyme from one tube
to another was given some interval. This
interval was meant to synchronize the reaction
time to be exactly 30 minutes for each tube.
After the addition of enzyme, all tubes were
set aside for 30 minutes to react. The 3,5Dinitrosalicylic acid (DNS) solution then
added with the same interval and order. All of
the tube then heated in a boiling water for 20
minutes. All of tubes were then measured on
540 nm wavelength. The result in form of
absorban then converted to U/mL. Unit
protein defined as amount of enzyme that
catalyzes the transformation of 1 micromole
of substrate per minute.
Ammonium
Sulphate
(NH4)2SO4
Precipitation. The crude
enzyme was
precipitated using ammonium sulphate. This
step is to find out the best ammonium sulphate
concentration to precipitate the enzyme.
Different concentrations of ammonium
sulphate tested to enzyme. The analyzed
concentrations were 30%-70% with 10%
interval. Ammonium sulphate poured slowly
into crude enzyme. The solution stirred for 1
hour then centrifugated on 11000 rpm for 15
minutes to obtain its deposit. Finally the
protein deposit was diluted with 0,2 M of
buffer phosphate pH 6,6.
Gel Filtration Chromatography. The
result of ammonium sulphate precipitation
then applied to gel filtration chromatography.
First, the gel was washed with 0,2 M of
buffer phosphate pH 6,6 before applying the
enzyme. 1 ml of enzyme was applied and the
fractions of 1 ml were collected. The purpose
of this process was to gain the protein
gradually based on their size. Enzyme in each
fraction
then
measured
using
spectrophotometer on 280 nm wavelength.
The amylase activity was also measured using
modified Miller method on 540 nm of
wavelength.
Characterization of purified enzyme.
The characterization of purified enzyme
includes optimum pH and temperature. The
temperatures analyzed were 30oC - 80 oC with
10 oC interval. The pH optimization was also
performed with a modification.
The
modification was a change on the buffer that
used to dissolve the substrate and to dilute the
enzyme. The pHs analyzed were pH 6 until 7
with 0,2 interval.
Molecular Weight Determination. The
molecular
weight
determination
was
performed using sodium dodecyl sulfate
polyacrylamide gel electrophoresis (SDS
PAGE) method with 12,5% of polyacrilamide
gel. SDS-PAGE on this research was stained
with silver staining method.
RESULTS
Crude enzyme with the highest activity
was produced by Brevibacterium sp. 96 hours
after cultivation (Figure 1).
Enzyme activity (U/mL)
Production of enzyme. The production
of enzyme was using preculture method in
ASW liquid medium. Preculture medium
volume is 50 mL and the culture medium is
450 mL. Brevibacterium sp. cultured in solid
ASW medium was inoculated to the
preculture medium. Both the preculture and
culture medium were incubated in shaker
incubator with speed 150 rpm and temperature
26oC. The preculture medium was poured into
the culture medium after three days, and then
incubated again with the same condition for 8
days. The crude enzyme from the media was
separated every 24 hours from cell by cold
centrifuge with speed of 11000 rpm for 15
minutes. The amylase activity from each day
isolated crude enzyme was measured using
modified Miller method. The production then
performed once again to harvest the enzyme
with the highest activity.
Figure 1
Hour
Curve of amylase activity from
Brevibacterium sp. on ASW
3
medium with pH 8 at room
temperature.
Enzyme activity (U/mL)
Enzymeactivity
activity(U/mL)
(U/mL)
Enzyme
Enzyme activity (U/mL)
The enzyme activity in a variety of
ammonium sulphate saturation can be seen in
Figure 2. It showed amylase that posses the
highest specific activity was in fraction of
50%. The value of enzyme recovery
concentrated by ammonium sulphate 50%
reached to 41%, the highest among the others.
The temperature and pH effects on
Brevibacterium sp. graphs (Figure 4 & 5)
showed similar pattern. Amylase Activity
keep increasing until reached its peak and then
get lowering. Brevibacterium amylase showed
its activity on temperature 30oC - 80 oC and
high amylase activity only appear on
temperature 50oC. Analysis of pH optimum
showed Brevibacterium sp. has pH optimum
on pH 6,4.
Ammonium sulphate concentration
Figure 2 Protein precipitation using various
concentration
of
ammonium
sulphate.
Figure
4
Temperature
effects
on
Brevibacterium sp. amylase
activity using 0,5% substrate
at pH 6,6.
Enzyme activity (U/mL)
U/mL
OD 280
The protein pattern (measured on 280 nm
of wavelength) and activity of amylase
obtained from gel filtration chromatography
can be seen on Figure 4. The result showed
the absorban on 280 nm and 540 nm of
wavelength shared a same pattern. The pattern
showed that protein and amylase activity were
at peak on fraction number 48-52. The
enzyme on fraction number 48-52 were
collected to be characterized.
Temperature (oC)
pH
Fraction number
Figure 5 pH effects on Brevibacterium sp.
amylase activity using 0,5%
substrate at room temperature.
Figure 3 Amylase activity and protein profile
of fraction from gel filtration
chromatography.
Purification process using ammonium
sulphate precipitation and gel filtration
chromatography method (Table 1) indicated
the decreasing of crude enzyme total activity.
4
Table 1 Purification of Brevibacterium sp. amylase
Total
Total Protein
Purification Step
Activity
(mg)
(Unit)
Crude enzyme
629.5
55
Ammonium sulphate
257.46
7.28
precipitation
Sephadex G-75
31.12
0.35
Purification
(fold)
Yield
(%)
1
100
35.37
3.1
40.9
88.91
7.8
4.9
was made with Rf as vertical axis and log
MW as horizontal axis.
Rf
Protein precipitation using ammonium
sulphate made specific enzyme activity
reduced by 59,1% and caused total protein
decreased sharply from 55 mg to 7,28 mg.
After all the precipitation and purification
process, the overall yield of 4,9% was
obtained with the improvement of specific
enzyme activity 7,8 times.
Specific
Activity
(U/mg)
11.4
(1)
(2)
(3)
260
260 kDa
kDa
140 kDa
238 kDa
100 kDa
79 kDa
Log MW
Figure 7 Protein marker linear curve.
70 kDa
40 kDa
55,7 kDa
35 kDa
33,6 kDa
25 kDa
Figure 6 SDS PAGE profile (1) marker, (2)
crude enzyme, (3) purified enzyme.
The SDS PAGE with silver staining
showed there were four bands of protein. To
determine the enzyme molecular weight, the
first step was to find the Rf (Retardation
factor) value of the protein marker. The
migration distance of substance and the
migration distance of solven front must be
measured in order to find the Rf value.
Rf =
migration distance of substance
migration distance of solven front
After obtaining Rf and log MW value of
protein marker, the standard curve was made
to find thelinier equation. The standard curve
According to the analysis of molecular
weight and retardation factor of marker, the
linear equation was: y = -1.049x + 2.251.
Molecular weight of the enzyme was
determined with this equation. X is Rf, and Y
is the logaritma value of the enzyme’s
molecular weight. From the calculation using
this equation, the molecular weight of the
purified proteins were 238 kDa, 79 kDa, 55,7
kDa and 33,6 kDa.
DISCUSSION
Brevibacterium sp. crude enzyme with the
highest activity was produced 96 hours after
incubation. Different incubation time can be
seen on a research conducted by Nouadri
(2010). In his research he found that
Penicillium camemberti PL21 produced crude
enzyme with the highest activity on 168 hours
after incubation. Each bacteria produced crude
enzyme with the highest activity on different
time, depends on many factors, such as the
adaptability of bacteria in their growth
medium.
Purification was very important to study a
particular enzyme because in analysis there
5
might be interference from other enzymes in
the extract that use the same substrate or
cofactor. Purified enzyme also very important
for industrial use, especially in pharmaceutical
and clinical sectors. The crude enzyme usually
purified using chromatography methods.
Among the other precipitation methods,
precipitation by ammonium sulphate and
aseton was the most common method
(Seftiono 2001). Ammonium sulphate used
oftenly because of its high solubility, low cost,
and the stability of protein in ammonium
sulphate solution can last for years. The salt in
the solution was then removed by dialysis.
Salt precipitation does not usually lead to a
highly purified protein, but can assist in
eliminating some unwanted proteins in a
mixture and concentrating the sample. That is
why this precipitation was followed by gel
filtration chromatography method.
The enzyme was purified using gel
filtration chromatography. Gel filtration
chromatography separates biomolecules based
on their molecular size differences (Stanton
2004). Gel-filtration chromatography is a
popular and versatile technique that permits
the effective separation of proteins and other
biological molecules in high yield. Gel on gel
filtration consisted series of three dimension
molecule that crosslinked each other in form
of beads (Harsono 2001). In the column, pores
that formed inside the gel selected the
molecules based on their size. Small
molecules diffuse freely into the pores and
their movement through the column is
retarded, whereas large molecules are unable
to enter the pores and are therefore eluted
earlier. This selection makes the larger
molecule will pass through the gel faster than
the small one. The separating media used in
this research was Sephadex G-75, which is
effectively separate proteins with 3.00080.000 Da size.
Purification process made total protein
decrease because each process eliminates
unwanted substances from enzyme solution
and this eliminated substances also could be in
form of protein. Specific activity is enzyme
activity value per milligram protein.
Purification factor is the comparation of
specific activity value of every purification
process with the crude enzyme specific
activity. The purification factor indicated the
purity of enzyme as the result of purification
process compared to crude enzyme.
Yield value is total amount of enzyme
after purification compared to total amount of
enzyme before purification. Compared to
another bacterias amylase purified with same
method, yield value of Brevibacterium sp.
amylase is very low. Purification of
Penicillium camemberti (Nouadri et al. 2010)
and Pseudomonas sp. (Liu et al. 2011)
resulting enzyme with yield value 34,6% and
44%, while purified Brevibacterium sp.
amylase only 4,9%.
Characterization of enzyme conducted to
find out the enzyme’s characteristics. The test
of temperature effects on amylase activity
showed that amylase activity increasing until
the optimum temperature on 50 oC. Low
activity on lower temperature was caused by
the lack of activation energy available.
Amylase activity was then decreased
significantly on 60 oC, and keep decreasing on
higher temperature. Higher temperature
affected the conformation of substrate. It
makes the active site of substrate inhibited to
enter the enzyme’s active site which makes
the enzyme activity low. Beside that, the
higher temperature was also caused enzyme
denaturation. Denaturation makes the folding
structure of the enzyme opened on the surface
and change the active site and causing low
enzyme activity (Hames & Hooper 2000).
The effects of pH showed the same pattern
as temperature effects on amylase activity.
Amylase activity increasing until its optimum
pH on 6,4. An extreme pH change could make
the enzyme denaturated. pH change can also
disturb non kovalen interaction that keep the
stability of enzyme’s three dimension
structure (Hames & Hooper 2000). The
purified amylase has optimum activity on pH
6,4 and temperature 50oC.
Each bacteria has unique characteristic
based on their living environment. Therefore,
each of them produce enzyme with different
characteristic as well. In his research, Ashwini
et al. (2011) found that the amylase extracted
from Bacillus sp. marini has optimum activity
on 40 oC and pH 7. This result indicated a
similarity with the characterization result of
Brevibacterium sp. amylase. Both bacteria
have a similar type of living environment.
Both bacteria live on marine environment,
near the land.
In another research, amylase purified and
characterized from Pyrococcus furious has
optimum activity on 100 oC (Laderman et al.
1993). This various result were also caused by
the differences of the bacterias living
environment. Pyrococcus furious which is
living on high temperature environment,
secreted enzymes that able to work optimum
on high temperature.
6
Various result of amylase optimum pH can
be seen in many researches as well. The
differences of the amylase optimum pH of
each bacteria was caused by the differences of
its living environment. pH of living
environment known to affect the synthesis and
secretion of amylase just like its stability
(Sivaramakrishnan et al. 2006). The research
about amylase production from Aspergillus
sp. JGI 12 (Alva et al. 2007) showed that the
optimum pH of medium growth for the
bacteria was same to the optimum pH of its
crude enzyme. The optimum pH of medium
growth and crude enzyme were 5,8 ; 7,5 and
9. In another research,
Penicillium
camemberti has optimum pH on pH 5, which
is an acidic environment (Nouadri et al.
2010). This variety of amylase characteristic
is the reason why amylase has been widely
used in various industrial fields.
Molecular weight of the enzyme was
determined using SDS PAGE method. It is an
electrophoresis
technique
that
use
polyacrilamide as its separation material. The
SDS PAGE result showed that there were four
bands obtained. The appearance of four bands
occured possibly because the purified enzyme
was an isozyme. Isozyme was enzymes that
have different structure but catalyze the same
reaction. Another possibility is because
another proteins with different molecular
weight that entered the same fraction with the
enzyme fraction. Among the others, the
second band was the thickest. According the
analysis and calculation, this band has
molecular weight 79 kDa, while the other
three have 238 kDa, 55,7 kDa and 33,6 kDa of
molecular weight.
CONCLUSION
The purification and characterization
process of Brevibacterium sp. amylase were
performed
using
amonium
sulphate
precipitation
and
gel
filtration
chromatography method. The purification
process resulting an enzyme with optimum
activity on temperature 50oC and pH 6,4.
SUGGESTION
Further purification and characterization
need to be conducted to get more about
Brevibacterium sp. amylase character.
Zymogram is also necessary to know which
protein band that possess the catalytic feature.
BIBLIOGRAPHY
Alva
S et al. 2007. Production and
characterization of fungal amylase
enzyme isolated from Aspergillus sp.
JGI 12 in solid state culture. African
Journal of Biotechnology 6:576-581.
Ashwini K, Gaurav K, Karthik L, Bhaskara
RKV. 2011. Optimization, production
and partial purification of extracellular
α-amylase from Bacillus sp. Marini.
Archives of Applied Science Research
3:33-42.
Bugg TDH. 2004. Introduction to Enzyme and
Coenzyme
Chemistry.
Oxford:
Blackwell Publishing.
Gruner E, Pfyffer GE, Graevenitz A. 1993.
Characterization of Brevibacterium
spp. from clinical specimens. Journal
of Clinical Microbiology 31:14081412.
Hames BD, Hooper NM. 2000, Biochemistry:
The Instant Notes. Hongkong:SpringerVerlag.
Harsono Y. 2001. Pemurnian enzim α-amilase
dengan menggunakan filtrasi gel.
[skripsi]. Bogor: Fakultas Matematika
dan Ilmu Pengetahuan Alam, Institut
Pertanian Bogor.
Kandra L. 2003. α-Amylases of medical and
industrial importance. Journal of
Molecular Structure THEOCHEM 666667:487-498.
Laderman et al 1993. The purification and
characterization of an extremely
thermostable α-amylase from the
hyperthermophilic
Archaebacterium
Pyrococcus furiosus. The Journal of
Biological Chemistry
268:2439424401.
Liu
J et al. 2011. Isolation and
characterization of α-amylase from
marine Pseudomonas sp. K6-28-040.
African Journal of Biotechnology
10:2733-2740.
7
Miller GL. 1959. Use of dinitrosalicylic acid
reagent for determination of reducing
sugar. Analytical Chemistry 31(3):426–
428.
Mohapatra BR, Bapuji M, Sree A. 2003.
Production of Industrial Enzymes
(Amylase, Carboxymethylcellulase and
Protease) by Bacteria Isolated from
Marine Sedentary Organisms. Acta
Biotechnologica 23:75-84.
Motta
AS,
Brandelli
A.
2002.
Characterization of an antibacterial
peptide produced by Brevibacterium
linens.
Journal
of
Applied
Microbiology 92:63-70.
Nielsen JE, Borchert TV. 2000. Protein
engineering
of
bacterial
alphaamylases. Biochimica et Biophysica
Acta 1543:253-274.
Nouadri T, Meraihi Z, Shahrazaed DD, Leila
B.
2010
Purification
and
characterization of the α-amylase
isolated from Penicillium camemberti
PL21. African Journal of Biochemistry
Research 4(6):155-162.
Rodriguez VB, Alameda EJ, Gallegos JFM,
Requena AR, Lopez AIG. 2006.
Enzymatic hydrolysis of soluble starch
witn an α-amylase from Bacillus
licheniformis. Biotechnology Progress
22:718-722.
Sivaramakrishnan S, Gangadharan D,
Nampoothiri KM, Soccol CR, Pandey
A. 2006. α -Amylases from microbial
sources – an overview on recent
developments. Food Technology and
Biotechnology 44 (2): 173–184.
Seftiono H. 2001. Pemurnian dan karakterisasi
mananase
dari
Streptacidiphilus
luteoalbus [skripsi]. Bogor: Fakultas
Matematika dan Ilmu Pengetahuan
Alam, Institut Pertanian Bogor.
Stanton P. 2004. HPLC of Peptides and
Proteins, Methods in Molecular
Biology. New York: Humana Press.
van der Maarel MJ, van der Veen B,
Uitdehaag JC, Leemhuis H, Dijkhuizen
L. 2002. Properties and applications of
starch-converting enzymes of the
alpha-amylase family. Journal of
Biotechnology 94:137-155.
Yuwono T. 2005. Biologi Molekular. Jakarta :
Erlangga.
8
APPENDIX
9
Appendix 1 Composition of bacteria growth medium :
1. Solid Artificial Sea Water (ASW)
- 3.8 g ASW
- 0.1 g yeast extract
- 0.5 g pepton
- 1.5 g agar
- 1.5 g starch
- 100 ml akuades
2. Liquid ASW
- 3.8 g ASW
- 0.1 g yeast extract
- 0.5 g pepton
- 1.5 g starch
- 100 ml akuades
Appendix 2 Composition of DNS solution :
- 1 g DNS
- 30 g Na K tartat
- 1.6 g NaOH
- 100 ml akuades
Appendix 3 Amylase activity on ASW medium with pH 8 on room temperature
Hour
Amylase
Activity (U/ml)
24
48
72
96
120
144
168
0,44
0,5
0,61
1,26
1,01
0,62
0,16
Appendix 4 Precipitation using Ammonium Sulphate
Ammonium suphate
concentration
Amylase
Activity (U/ml)
30%
0,2
40%
1,07
50%
3,36
60%
1,02
70%
0,63
Appendix 5 Temperature effects on amylase activity
Temperature
30 oC
Amylase Acivity
(U/ml)
0,77
10
40 oC
0,87
o
4,43
o
1,67
o
1,69
o
1,54
50 C
60 C
70 C
80 C
Appendix 6 pH effects on amylase activity
pH
Amylase Activity
(U/ml)
6
0,46
6,2
1,03
6,4
1,27
6,6
0,63
6,8
0,71
7
0,69
Appendix 7 Protein marker standard data
Marker
No.
1
2
3
4
5
6
7
8
9
10
Marker Tracking Distance
B
Rf
Log BM
BM
1,5
5,5
9
18,5
21
27
34
41
54
63
68
68
68
68
68
68
68
68
68
68
0,02
0,08
0,13
0,27
0,31
0,4
0,5
0,60
0,79
0,93
2,41
2,15
2
1,85
1,7
1,60
1,54
1,4
1,18
1
260
140
100
70
50
40
35
25
15
10
Appendix 8 Sediment of protein after precipitation with 50% concentration of ammonium sulphate
Brevibacterium sp. AMYLASE
WIDYO TRI PUTRANTO
DEPARTEMENT OF BIOLOGY
FACULTY OF MATHEMATICS AND NATURAL SCIENCES
BOGOR AGRICULTURAL UNIVERSITY
BOGOR
2012
ABSTRACT
WIDYO TRI PUTRANTO. Purification and characterization of Brevibacterium sp. amylase.
Under supervision of ANJA MERYANDINI and YOPI.
Amylase is an enzyme that used widely in various industrial field. This enzyme
specifically catalyze the hydrolysis of -1,4-glycosidic bond on starch. To fulfill industrial need, the
search for a new promising source of amylase is important. One of amylase source that has not
been well explored yet is marine microbe. The purpose of this research was to purify and
characterize the amylase from Brevibacterium sp. isolated from Jakarta Bay. The Brevibacterium
sp. was cultured in Artificial Sea Water (ASW) medium. Measurement of specific amylase
activity was performed by modified Miller method. The highest crude enzyme activity was
produced 96 hours after the cultivation which is reaching 1,2 U/mL. After precipitation using
ammonium sulphate, the crude enzyme solution were purified by gel filtration chromatography.
SDS-polyacrylamide analysis result showed that the molecular weight of the enzymes were 79
kDa, 55,7 kDa, and 33,6 kDa. The partial purified enzyme has optimum activity on temperature
50oC and pH 6,4.
Key words: amylase, purification, characterization, Brevibacterium.
ABSTRAK
WIDYO TRI PUTRANTO. Purifikasi dan karakterisasi enzim amilase dari Brevibacterium sp.
Dibimbing oleh ANJA MERYANDINI dan YOPI.
Amilase adalah enzim yang digunakan di berbagai bidang industri. Enzim ini
mengkatalisis hidrolisis dari ikatan 1,4-glikosidik pada pati. Untuk memenuhi kebutuhan industri,
pencarian terhadap sumber amilase baru yang menjanjikan sangat penting. Salah satu sumber
amilase yang belum banyak dieksplorasi adalah mikroba laut. Penelitian ini bertujuan
mempurifikasi dan mengkarakterisasi enzim amilase dari Brevibacterium sp., yang diisolasi dari
perairan Teluk Jakarta. Isolat Brevibacterium sp. dikulturkan di media Artificial Sea Water
(ASW). Pengukuran aktivitas amilase dilakukan menggunakan metode Miller yang dimodifikasi.
Aktivitas amilase tertinggi pada enzim ekstrak kasar diproduksi pada jam ke-96 setelah
pengkulturan, yaitu mencapai 1,2 U/mL. Setelah presipitasi dengan menggunakan ammonium
sulfat, larutan enzim ekstrak kasar dimurnikan dengan kromatografi gel filtrasi. Hasil analisis
SDS-PAGE menunjukkan berat molekul protein adalah 79 kDa, 55,7 kDa, dan 33,6 kDa.
Berdasarkan karakterisasi, enzim bekerja optimum pada suhu 50oC dan pH 6,4.
Kata kunci: amilase, purifikasi, karakterisasi, Brevibacterium.
PARTIAL PURIFICATION AND CHARACTERIZATION OF
Brevibacterium sp. AMYLASE
WIDYO TRI PUTRANTO
An Undergraduate Thesis
Intended to Pursue Bachelor Degree of Science
In Faculty of Mathematics and Natural Sciences
Bogor Agricultural University
DEPARTEMENT OF BIOLOGY
FACULTY OF MATHEMATICS AND NATURAL SCIENCES
BOGOR AGRICULTURAL UNIVERSITY
BOGOR
2012
Title
Name
NIM
: Partial Purification and Characterization of Brevibacterium sp.
Amylase
: Widyo Tri Putranto
: G34080116
Approved by,
Supervisor I
(Prof. Dr. Anja Meryandini, M.Si)
NIP. 196203271987032001
Supervisor II
(Dr. Yopi)
NIP. 196912201989011001
Endorsed by :
Chief of Department
Department of Biology
Faculty of Mathematics and Natural Sciences
Bogor Agricultural University
(Dr. Ir. Ence Darmo Jaya Supena, M.Si)
NIP. 19641002 198903 1 002
Graduation date:
PREFACE
Praise to Almighty God for His blessing so this thesis can be done. The title of this thesis is
“Partial Purification and Characterization of Brevibacterium sp. Amylase. This research was taken
place at Biotechnology Research and Development Center, Indonesian Institute of Science,
Cibinong. This research was conducted from February until June 2012.
This thesis would not have been possible without the support of several thoughtful and
generous individuals. My acknowledgements to Prof. Dr. Anja Meryandini as my first supervisor
and Dr. Yopi as my second supervisor for the guidance along the process of this thesis. I also wish
to acknowledge Dr. Achmad Farajallah as examiner and representative of Department of Biology,
FMIPA, IPB for the advices and discussion for the completion of this thesis. I would like to thank
my beloved family, especially my parents for their supports and prayers. I would like to say thank
you to Mba Nanik, Mba Ade, Mas Ashif, Mba Lia, Mas Alex and Mas Dicky for every advices
they gave to me. I would like to say thank you as well to my comrades, Dewi, Viska, Yana,
Rohana, Yufi, Riza, Lady and Titi for their cooperation on laboratory, and also for all Biology 45
friends for their supports, prayers and motivations.
This research was supported and funded by bio catalyst and fermentation laboratory,
Bioproses division, Research Center Biotechnology Indonesian Institue of Science. I hope this
thesis will be usefull for all readers.
Bogor, September 2012
Widyo Tri Putranto
vi
CURRICULUM VITAE
Writer was born in Bogor at 8 August 1990 as the third child of three from the parents FX
Mugihardjo and Triasih Rudiatun.
On 2008, writer graduated from SMA Regina Pacis Bogor. On the same year, writer
successfully continued his education on Bogor Agricultural University through Seleksi Nasional
Mahasiswa Perguruan Tinggi Negeri (SNMPTN). Writer chose Biology major, Faculty of
Mathematics and Natural Sciences.
On 2010, writer join field study on Pangandaran Beach, Ciamis, West Java, and wrote a
report with title ‘Agarose-producing Bacteria Associated with Algae’. Writer was also conducted
practical field on Indonesian Institute of Science from July to August 2011, wrote about
Production of Amylase from Marine Bacteria.
TABLE OF CONTENTS
Page
LIST OF TABLES ............................................................................................... viii
LIST OF FIGURES ............................................................................................. viii
LIST OF APPENDIX .......................................................................................... viii
INTRODUCTION .................................................................................................. 1
Background ......................................................................................................... 1
Objective ............................................................................................................. 1
Place and Time .................................................................................................... 1
MATERIALS AND METHODS ............................................................................ 1
Preparation of Brevibacterium sp. culture .......................................................... 1
Production of enzyme. ....................................................................................... 2
Bioassay of Amylase ........................................................................................... 2
Ammonium Sulphate (NH4)2SO4 Precipitation .................................................. 2
Gel Filtration Chromatography ........................................................................... 2
Characterization of purified enzyme ................................................................... 2
Molecular Weight Determination ....................................................................... 2
RESULTS ............................................................................................................... 2
DISCUSSION ......................................................................................................... 4
CONCLUSION ....................................................................................................... 6
BIBLIOGRAPHY ................................................................................................... 6
APPENDIX ............................................................................................................. 8
LIST OF TABLES
Page
1 Purification of Brevibacterium sp. amylase ................................................................................... 4
LIST OF FIGURES
Page
1 Curve of amylase activity from Brevibacterium sp. on ASW medium with pH 8 at room
temperature ..................................................................................................................................... 2
2 Protein precipitation using various concentration of ammonium sulphate ..................................... 3
3 Amylase activity and protein profile of fraction from gel filtration chromatography .................... 3
4 Temperature effects on Brevibacterium sp. amylase activity using 0,5% substrate at pH 6,6. ...... 3
5 pH effects on Brevibacterium sp. amylase activity using 0,5% substrate at room temperature ... 3
6 SDS PAGE profile ......................................................................................................................... 4
7 Protein marker linear curve ............................................................................................................ 4
LIST OF APPENDIX
Page
1 Composition of bacteria growth medium ....................................................................................... 9
2 Composition of DNS solution ........................................................................................................ 9
3 Amylase activity on ASW medium with pH 8 on room temperature ............................................. 9
4 Precipitation using ammonium sulphate ........................................................................................ 9
5 Temperature effects on amylase activity ........................................................................................ 9
6 pH effects on amylase activity .................................................................................................... 10
7 Protein marker standard data ........................................................................................................ 10
8 Sediment of protein after precipitation with 50% concentration of ammonium sulphate ............ 10
1
INTRODUCTION
Background
Enzymes are catalysts that help to convert
other molecules called substrates into
products. An enzyme able to catalyze
chemical reaction at least until 1 million times
faster than reaction without catalizer (Yuwono
2005). Enzymes are giant molecules with
molecular weight varies from 5 kDa to 5000
kDa, with typical values in the range 20 kDa–
100 kDa (Bugg 2004). Enzymes work by
lowering the activation energy of a chemical
reaction, and thus dramatically accelerating
the rate of the reaction. Most enzyme catalyze
just one kind of reaction or a group of
reactions with high similarity. The specificity
was the consequence of the uniqueness of
each enzyme’s active site. Active site is part
of an enzyme where the reaction takes place.
The enzyme commission (EC) numbers
divided enzymes into six main groups
according to the type of reaction of catalysed:
oxidoreductases, transferases, hydrolases,
lyases, isomerases and ligases. Among the
groups, hydrolases are presently the most
commonly encountered class of enzymes
within the field of enzyme technology. One of
the hydrolases groups used widely in various
industries is amylase.
Amylase is an enzyme that specifically
catalyze the hydrolysis of glucosidic linkages
in polysaccharide. It catalizes the hydrolysis
of -1,4-glycosidic bond on starch (Liu et al.
2011). The amylases (α-amylases, β-amylases,
glucoamylases) are one of the most important
families of enzymes in the field of
biotechnology (Rodriguez et al. 2006). The αamylase belongs to a family of endo-amylases
that catalyses the initial hydrolysis of starch
into shorter oligosaccharides (van der Maarel
et al. 2002). This hydrolysis results in the
production of maltose and oligosaccharides.
Amylase also helps the carbohydrates in a
food break down more quickly and easily.
This makes amylase as popular enzyme for
food industry. Amylase is a highly demanded
industrial enzyme in various fields such as
pharmaceuticals, textiles, detergents, etc
(Sivaramakrishnan et al. 2006). The most
common industry using amylase is starch
industry. This kind of industry using amylase
for hidrolizing starch to produce fructose and
glucose syrup (Nielsen & Borchert 2000).
In human, alpha amylase is synthesized in
the acinar cells of the saliva glands and stored
in secretory granules inside the cells. Amylase
not only produced by human, but also by
animals, plants, and microorganisms (Kandra
2003). Although there are many sources of
amylase, producing amylase from bacteria
provides more advantages. Producing amylase
from bacteria requires less time and space for
production, easiness to modify, consistency of
the enzyme, and cheaper.
Marine bacteria is one of amylase source
that still has not been well explored yet. In
other words, there might be abundant of
promising amylase sources from marine.
Many industrial applications need enzymes
that stable at extreme temperature, pH, and
salt concentration. In this case, marine
microbial enzymes may offer advantages. The
optimum activity of marine bacterial enzymes
usually occurs at high salinity, making these
enzymes utilisable in many harsh industrial
processes. In addition, most marine bacterial
enzymes are considerably thermotolerant,
remaining stable at room temperature over
long periods (Mohapatra e al. 2003).
Sea is a large saline water environment,
for consequence, in order to survive, marine
bacterias
must
have
salt
tolerance
characteristic. The example of bacteria with
that kind of characteristic is Brevibacterium
genus, gram positive bacterias from
Actinomycetales family. Brevibacteria are
short shaped, nonbranched, asporogenous,
obligately aerobic, rods which may exhibit a
marked rod-coccus cycle when cells become
older (Gruner et al. 1993). Brevibacterium is
known as soil bacteria which causing odor on
human feet. One of this genus member,
Brevibacterium linens , is known as the most
important surface bacteria in the cheesemaking process due its role in the colouring
surface and its typical flavouring activity
(Motta & Brandelli 2002).
Objective
The objective of this thesis was to purify
and
characterize
amylase
from
Brevibacterium sp., isolated from Jakarta Bay.
Place and Time
This research conducted from February
2012 until July 2012 in Research Center for
Biotechnology,
Bioproses
division,
Indonesian Institute of Science, Cibinong.
MATERIALS AND METHODS
Preparation of Brevibacterium sp.
culture. Brevibacterium sp. colony in petri
disk was inoculated to ASW solid medium.
2
Bioassay of Amylase. First, 250 µL of
substrate solution was added to all tube.
Substrate solution was made by mixing starch
with 0,2 M of buffer phosphate pH 6,6. The
substrate solution concentration was 0,5%.
Then 250 µL of enzyme was added into the
tubes. The addition of enzyme from one tube
to another was given some interval. This
interval was meant to synchronize the reaction
time to be exactly 30 minutes for each tube.
After the addition of enzyme, all tubes were
set aside for 30 minutes to react. The 3,5Dinitrosalicylic acid (DNS) solution then
added with the same interval and order. All of
the tube then heated in a boiling water for 20
minutes. All of tubes were then measured on
540 nm wavelength. The result in form of
absorban then converted to U/mL. Unit
protein defined as amount of enzyme that
catalyzes the transformation of 1 micromole
of substrate per minute.
Ammonium
Sulphate
(NH4)2SO4
Precipitation. The crude
enzyme was
precipitated using ammonium sulphate. This
step is to find out the best ammonium sulphate
concentration to precipitate the enzyme.
Different concentrations of ammonium
sulphate tested to enzyme. The analyzed
concentrations were 30%-70% with 10%
interval. Ammonium sulphate poured slowly
into crude enzyme. The solution stirred for 1
hour then centrifugated on 11000 rpm for 15
minutes to obtain its deposit. Finally the
protein deposit was diluted with 0,2 M of
buffer phosphate pH 6,6.
Gel Filtration Chromatography. The
result of ammonium sulphate precipitation
then applied to gel filtration chromatography.
First, the gel was washed with 0,2 M of
buffer phosphate pH 6,6 before applying the
enzyme. 1 ml of enzyme was applied and the
fractions of 1 ml were collected. The purpose
of this process was to gain the protein
gradually based on their size. Enzyme in each
fraction
then
measured
using
spectrophotometer on 280 nm wavelength.
The amylase activity was also measured using
modified Miller method on 540 nm of
wavelength.
Characterization of purified enzyme.
The characterization of purified enzyme
includes optimum pH and temperature. The
temperatures analyzed were 30oC - 80 oC with
10 oC interval. The pH optimization was also
performed with a modification.
The
modification was a change on the buffer that
used to dissolve the substrate and to dilute the
enzyme. The pHs analyzed were pH 6 until 7
with 0,2 interval.
Molecular Weight Determination. The
molecular
weight
determination
was
performed using sodium dodecyl sulfate
polyacrylamide gel electrophoresis (SDS
PAGE) method with 12,5% of polyacrilamide
gel. SDS-PAGE on this research was stained
with silver staining method.
RESULTS
Crude enzyme with the highest activity
was produced by Brevibacterium sp. 96 hours
after cultivation (Figure 1).
Enzyme activity (U/mL)
Production of enzyme. The production
of enzyme was using preculture method in
ASW liquid medium. Preculture medium
volume is 50 mL and the culture medium is
450 mL. Brevibacterium sp. cultured in solid
ASW medium was inoculated to the
preculture medium. Both the preculture and
culture medium were incubated in shaker
incubator with speed 150 rpm and temperature
26oC. The preculture medium was poured into
the culture medium after three days, and then
incubated again with the same condition for 8
days. The crude enzyme from the media was
separated every 24 hours from cell by cold
centrifuge with speed of 11000 rpm for 15
minutes. The amylase activity from each day
isolated crude enzyme was measured using
modified Miller method. The production then
performed once again to harvest the enzyme
with the highest activity.
Figure 1
Hour
Curve of amylase activity from
Brevibacterium sp. on ASW
3
medium with pH 8 at room
temperature.
Enzyme activity (U/mL)
Enzymeactivity
activity(U/mL)
(U/mL)
Enzyme
Enzyme activity (U/mL)
The enzyme activity in a variety of
ammonium sulphate saturation can be seen in
Figure 2. It showed amylase that posses the
highest specific activity was in fraction of
50%. The value of enzyme recovery
concentrated by ammonium sulphate 50%
reached to 41%, the highest among the others.
The temperature and pH effects on
Brevibacterium sp. graphs (Figure 4 & 5)
showed similar pattern. Amylase Activity
keep increasing until reached its peak and then
get lowering. Brevibacterium amylase showed
its activity on temperature 30oC - 80 oC and
high amylase activity only appear on
temperature 50oC. Analysis of pH optimum
showed Brevibacterium sp. has pH optimum
on pH 6,4.
Ammonium sulphate concentration
Figure 2 Protein precipitation using various
concentration
of
ammonium
sulphate.
Figure
4
Temperature
effects
on
Brevibacterium sp. amylase
activity using 0,5% substrate
at pH 6,6.
Enzyme activity (U/mL)
U/mL
OD 280
The protein pattern (measured on 280 nm
of wavelength) and activity of amylase
obtained from gel filtration chromatography
can be seen on Figure 4. The result showed
the absorban on 280 nm and 540 nm of
wavelength shared a same pattern. The pattern
showed that protein and amylase activity were
at peak on fraction number 48-52. The
enzyme on fraction number 48-52 were
collected to be characterized.
Temperature (oC)
pH
Fraction number
Figure 5 pH effects on Brevibacterium sp.
amylase activity using 0,5%
substrate at room temperature.
Figure 3 Amylase activity and protein profile
of fraction from gel filtration
chromatography.
Purification process using ammonium
sulphate precipitation and gel filtration
chromatography method (Table 1) indicated
the decreasing of crude enzyme total activity.
4
Table 1 Purification of Brevibacterium sp. amylase
Total
Total Protein
Purification Step
Activity
(mg)
(Unit)
Crude enzyme
629.5
55
Ammonium sulphate
257.46
7.28
precipitation
Sephadex G-75
31.12
0.35
Purification
(fold)
Yield
(%)
1
100
35.37
3.1
40.9
88.91
7.8
4.9
was made with Rf as vertical axis and log
MW as horizontal axis.
Rf
Protein precipitation using ammonium
sulphate made specific enzyme activity
reduced by 59,1% and caused total protein
decreased sharply from 55 mg to 7,28 mg.
After all the precipitation and purification
process, the overall yield of 4,9% was
obtained with the improvement of specific
enzyme activity 7,8 times.
Specific
Activity
(U/mg)
11.4
(1)
(2)
(3)
260
260 kDa
kDa
140 kDa
238 kDa
100 kDa
79 kDa
Log MW
Figure 7 Protein marker linear curve.
70 kDa
40 kDa
55,7 kDa
35 kDa
33,6 kDa
25 kDa
Figure 6 SDS PAGE profile (1) marker, (2)
crude enzyme, (3) purified enzyme.
The SDS PAGE with silver staining
showed there were four bands of protein. To
determine the enzyme molecular weight, the
first step was to find the Rf (Retardation
factor) value of the protein marker. The
migration distance of substance and the
migration distance of solven front must be
measured in order to find the Rf value.
Rf =
migration distance of substance
migration distance of solven front
After obtaining Rf and log MW value of
protein marker, the standard curve was made
to find thelinier equation. The standard curve
According to the analysis of molecular
weight and retardation factor of marker, the
linear equation was: y = -1.049x + 2.251.
Molecular weight of the enzyme was
determined with this equation. X is Rf, and Y
is the logaritma value of the enzyme’s
molecular weight. From the calculation using
this equation, the molecular weight of the
purified proteins were 238 kDa, 79 kDa, 55,7
kDa and 33,6 kDa.
DISCUSSION
Brevibacterium sp. crude enzyme with the
highest activity was produced 96 hours after
incubation. Different incubation time can be
seen on a research conducted by Nouadri
(2010). In his research he found that
Penicillium camemberti PL21 produced crude
enzyme with the highest activity on 168 hours
after incubation. Each bacteria produced crude
enzyme with the highest activity on different
time, depends on many factors, such as the
adaptability of bacteria in their growth
medium.
Purification was very important to study a
particular enzyme because in analysis there
5
might be interference from other enzymes in
the extract that use the same substrate or
cofactor. Purified enzyme also very important
for industrial use, especially in pharmaceutical
and clinical sectors. The crude enzyme usually
purified using chromatography methods.
Among the other precipitation methods,
precipitation by ammonium sulphate and
aseton was the most common method
(Seftiono 2001). Ammonium sulphate used
oftenly because of its high solubility, low cost,
and the stability of protein in ammonium
sulphate solution can last for years. The salt in
the solution was then removed by dialysis.
Salt precipitation does not usually lead to a
highly purified protein, but can assist in
eliminating some unwanted proteins in a
mixture and concentrating the sample. That is
why this precipitation was followed by gel
filtration chromatography method.
The enzyme was purified using gel
filtration chromatography. Gel filtration
chromatography separates biomolecules based
on their molecular size differences (Stanton
2004). Gel-filtration chromatography is a
popular and versatile technique that permits
the effective separation of proteins and other
biological molecules in high yield. Gel on gel
filtration consisted series of three dimension
molecule that crosslinked each other in form
of beads (Harsono 2001). In the column, pores
that formed inside the gel selected the
molecules based on their size. Small
molecules diffuse freely into the pores and
their movement through the column is
retarded, whereas large molecules are unable
to enter the pores and are therefore eluted
earlier. This selection makes the larger
molecule will pass through the gel faster than
the small one. The separating media used in
this research was Sephadex G-75, which is
effectively separate proteins with 3.00080.000 Da size.
Purification process made total protein
decrease because each process eliminates
unwanted substances from enzyme solution
and this eliminated substances also could be in
form of protein. Specific activity is enzyme
activity value per milligram protein.
Purification factor is the comparation of
specific activity value of every purification
process with the crude enzyme specific
activity. The purification factor indicated the
purity of enzyme as the result of purification
process compared to crude enzyme.
Yield value is total amount of enzyme
after purification compared to total amount of
enzyme before purification. Compared to
another bacterias amylase purified with same
method, yield value of Brevibacterium sp.
amylase is very low. Purification of
Penicillium camemberti (Nouadri et al. 2010)
and Pseudomonas sp. (Liu et al. 2011)
resulting enzyme with yield value 34,6% and
44%, while purified Brevibacterium sp.
amylase only 4,9%.
Characterization of enzyme conducted to
find out the enzyme’s characteristics. The test
of temperature effects on amylase activity
showed that amylase activity increasing until
the optimum temperature on 50 oC. Low
activity on lower temperature was caused by
the lack of activation energy available.
Amylase activity was then decreased
significantly on 60 oC, and keep decreasing on
higher temperature. Higher temperature
affected the conformation of substrate. It
makes the active site of substrate inhibited to
enter the enzyme’s active site which makes
the enzyme activity low. Beside that, the
higher temperature was also caused enzyme
denaturation. Denaturation makes the folding
structure of the enzyme opened on the surface
and change the active site and causing low
enzyme activity (Hames & Hooper 2000).
The effects of pH showed the same pattern
as temperature effects on amylase activity.
Amylase activity increasing until its optimum
pH on 6,4. An extreme pH change could make
the enzyme denaturated. pH change can also
disturb non kovalen interaction that keep the
stability of enzyme’s three dimension
structure (Hames & Hooper 2000). The
purified amylase has optimum activity on pH
6,4 and temperature 50oC.
Each bacteria has unique characteristic
based on their living environment. Therefore,
each of them produce enzyme with different
characteristic as well. In his research, Ashwini
et al. (2011) found that the amylase extracted
from Bacillus sp. marini has optimum activity
on 40 oC and pH 7. This result indicated a
similarity with the characterization result of
Brevibacterium sp. amylase. Both bacteria
have a similar type of living environment.
Both bacteria live on marine environment,
near the land.
In another research, amylase purified and
characterized from Pyrococcus furious has
optimum activity on 100 oC (Laderman et al.
1993). This various result were also caused by
the differences of the bacterias living
environment. Pyrococcus furious which is
living on high temperature environment,
secreted enzymes that able to work optimum
on high temperature.
6
Various result of amylase optimum pH can
be seen in many researches as well. The
differences of the amylase optimum pH of
each bacteria was caused by the differences of
its living environment. pH of living
environment known to affect the synthesis and
secretion of amylase just like its stability
(Sivaramakrishnan et al. 2006). The research
about amylase production from Aspergillus
sp. JGI 12 (Alva et al. 2007) showed that the
optimum pH of medium growth for the
bacteria was same to the optimum pH of its
crude enzyme. The optimum pH of medium
growth and crude enzyme were 5,8 ; 7,5 and
9. In another research,
Penicillium
camemberti has optimum pH on pH 5, which
is an acidic environment (Nouadri et al.
2010). This variety of amylase characteristic
is the reason why amylase has been widely
used in various industrial fields.
Molecular weight of the enzyme was
determined using SDS PAGE method. It is an
electrophoresis
technique
that
use
polyacrilamide as its separation material. The
SDS PAGE result showed that there were four
bands obtained. The appearance of four bands
occured possibly because the purified enzyme
was an isozyme. Isozyme was enzymes that
have different structure but catalyze the same
reaction. Another possibility is because
another proteins with different molecular
weight that entered the same fraction with the
enzyme fraction. Among the others, the
second band was the thickest. According the
analysis and calculation, this band has
molecular weight 79 kDa, while the other
three have 238 kDa, 55,7 kDa and 33,6 kDa of
molecular weight.
CONCLUSION
The purification and characterization
process of Brevibacterium sp. amylase were
performed
using
amonium
sulphate
precipitation
and
gel
filtration
chromatography method. The purification
process resulting an enzyme with optimum
activity on temperature 50oC and pH 6,4.
SUGGESTION
Further purification and characterization
need to be conducted to get more about
Brevibacterium sp. amylase character.
Zymogram is also necessary to know which
protein band that possess the catalytic feature.
BIBLIOGRAPHY
Alva
S et al. 2007. Production and
characterization of fungal amylase
enzyme isolated from Aspergillus sp.
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Ashwini K, Gaurav K, Karthik L, Bhaskara
RKV. 2011. Optimization, production
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Kandra L. 2003. α-Amylases of medical and
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Laderman et al 1993. The purification and
characterization of an extremely
thermostable α-amylase from the
hyperthermophilic
Archaebacterium
Pyrococcus furiosus. The Journal of
Biological Chemistry
268:2439424401.
Liu
J et al. 2011. Isolation and
characterization of α-amylase from
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2002.
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2010
Purification
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8
APPENDIX
9
Appendix 1 Composition of bacteria growth medium :
1. Solid Artificial Sea Water (ASW)
- 3.8 g ASW
- 0.1 g yeast extract
- 0.5 g pepton
- 1.5 g agar
- 1.5 g starch
- 100 ml akuades
2. Liquid ASW
- 3.8 g ASW
- 0.1 g yeast extract
- 0.5 g pepton
- 1.5 g starch
- 100 ml akuades
Appendix 2 Composition of DNS solution :
- 1 g DNS
- 30 g Na K tartat
- 1.6 g NaOH
- 100 ml akuades
Appendix 3 Amylase activity on ASW medium with pH 8 on room temperature
Hour
Amylase
Activity (U/ml)
24
48
72
96
120
144
168
0,44
0,5
0,61
1,26
1,01
0,62
0,16
Appendix 4 Precipitation using Ammonium Sulphate
Ammonium suphate
concentration
Amylase
Activity (U/ml)
30%
0,2
40%
1,07
50%
3,36
60%
1,02
70%
0,63
Appendix 5 Temperature effects on amylase activity
Temperature
30 oC
Amylase Acivity
(U/ml)
0,77
10
40 oC
0,87
o
4,43
o
1,67
o
1,69
o
1,54
50 C
60 C
70 C
80 C
Appendix 6 pH effects on amylase activity
pH
Amylase Activity
(U/ml)
6
0,46
6,2
1,03
6,4
1,27
6,6
0,63
6,8
0,71
7
0,69
Appendix 7 Protein marker standard data
Marker
No.
1
2
3
4
5
6
7
8
9
10
Marker Tracking Distance
B
Rf
Log BM
BM
1,5
5,5
9
18,5
21
27
34
41
54
63
68
68
68
68
68
68
68
68
68
68
0,02
0,08
0,13
0,27
0,31
0,4
0,5
0,60
0,79
0,93
2,41
2,15
2
1,85
1,7
1,60
1,54
1,4
1,18
1
260
140
100
70
50
40
35
25
15
10
Appendix 8 Sediment of protein after precipitation with 50% concentration of ammonium sulphate