purification and characterizations of phytase enzyme form aspergillus niger

MINISTRY OF EDUCATION & TRAINING
CAN THO UNIVERSITY
BIOTECHNOLOGY RESEARCH & DEVELOPMENT INSTITUTE

SUMMARY
BACHELOR OF SCIENCE THESIS
THE ADVANCED PROGRAM IN BIOTECHNOLOGY

PURIFICATION AND CHARACTERIZATIONS OF
PHYTASE ENZYME FORM Aspergillus niger
SIZE 14-15

SUPERVISOR

STUDENT

Dr. DUONG THI HUONG GIANG

NGUYEN CONG DANH
Student code: 4084238
Session: 34

Can Tho, 2013


APPROVAL

SUPERVISOR

Dr. DUONG THI HUONG GIANG

STUDENT

NGUYEN CONG DANH

Can Tho, May 10, 2013
PRESIDENT OF EXAMINATION COMMITTEE


Abstract
The aim of this study was to purify and characterize
an enzyme phytase obtained from Aspergillus niger isolate.
The results showed that extracellular phytase from
Aspergillus niger can be purified by ammonium sulfate
precipitation at 80-90% saturation in combination with
cation-exchange

chromatography

on

SP-streamline

following hydrophobic interaction chromatography on
Phenyl-Sepharose. Phytase purification fold was 12.56 and
activity recovery was 48%. SDS-PAGE revealed that the
purified phytase behaved as a protein with molecular mass
of about 87kDa. Optimum phytase activity was at 65°C in
the presence of 40mM Ca2+.
Key words: Aspergillus niger, hydrophobic interaction,
Cation-exchange

chromatography,

SP-streamline,

Phenyl-sepharose, phytase, phytate.

i


CONTENTS
Abstract

i

Contents

ii

I. Introduction

1

II. Materials and methods

2

2.1 Materials

2

2.2 Methods

2

III. Results and Discussion

8

3.1. Extraction of phytase from A. niger fresh biomass

8

3.2. Purification of phytase

8

3.2.1. Precipitation with ammpnium sulphate saturation

8

3.2.2. Purification of phytase by Cation exchange

9

chromatography
3.2.3. Hydrophobic interaction chromatography

11

3.3. Characterization of phytase
3.3.1. The effect of ion Ca

2+

15
on phytase activity

15

3.3.2. Temperature optimum of phytase activity

16

IV. Conclusions and Suggestions

18

4.1. Conclusions

18

4.2. Suggestions

18

References

19

ii


I. Introduction
Phytate (myo-inositol hexakisphosphate) is the primary storage
form of phosphorus in plants. They occupy about 1-3% of the
seeds of grains and legumes, and 60-80% of the total amount of
plant phosphorus (Nelson, 1967). Phosphorus is essential for
feeding animals, poultry and even for human diet. It is supplied in
the form of phytate or phytic acid. Naturally, phytases are known
as a group of enzyme able to catalyse the hydrolysis of phytate to
free phosphate, a form of phosphorus that is easily to be absorbed
in the animal digestive tract. Unfortunately, monogastric animals
and human are lacking in phytase. As a result, phosphorus from
phytate can not be absorbed and therefore it is excreted in the
feces that cause the environment pollution (Mullaney et al.,
2000). For this reason, supplementing of phytase in the feed or
human diet will help to solve the problem.
Phytase can be obtained from many sources such as bacteria
(Bacillus, Enterobacteria), filamentous fungi (Aspergillus sp.,
Penicillin sp., Mucor sp.). Among them, A. niger is a preferable
species for exploration due to its high phytase production.
Recently, an A. niger strain which was able to give high phytase
production was isolated in the Lab of Enzyme Technology,
Biotechnology R&D Institute (Nguyen Nhat Khoa Tran, 2012).
As the continuation of this research, the thesis on "Purification
and characterizations of phytase enzyme from Aspergillus
niger" has been performed with the aim to obtain purifed phytase
and some its valuable characteristics to aplly in feed or food
industries.

1


II. Materials and Methods
The thesis was done from 12/2012 to 04/2013 in the Laboratory
of Enzyme Technology, Biotechnology R & D Institute, Can Tho
University.
2.1. Materials
The A. niger isolate was supplied by BSc Trần Nguyễn
Nhật Khoa (2012).
Equipments:
- Mini protein II (Bio Rad ).
- Chromatography system (Bio Rad).
- Centrifuge Eppendorf (Germany).
- Spectrophotometer (Japan).
- Refrigerated Centrifuge (Germany)
- Other lab facilities.
Chemicals:
Bovin Serum Albumin (BSA) (Merck), Tris-HCl (Sigma),
Trichloroacetic

acid

(TCA)

(Merck),

Casein

(Prolabo),

Acrylamide (Bio Rad), Sodium hydroxide (NaOH) (Merck),
Sodium Dodecyl Sulfate (SDS) (Sigma), Bromophenol blue
(Merck). Glycine (Merck). SP-Streamline gel (GE Healthcare).
Phenyl Sepharose (GE Healthcare), Folin solution, Bradford
solution, Glucose, Sucrose, Malt extract, KH2PO4, KCl,
MgSO4.7H2O, NaCl, CaCl2.2H2O, MnSO4.4H2O, FeSO4.7H2O
(China).
2.2. Methods

2


2.2.1. Extraction of phytase from A. niger fresh biomass
2.2.1.1. Collection of crude phytase extract
Aim: Extracting crude phytase from A. niger fresh biomass
for purification.
Procedure: A. niger was cultured on the semi-solid medium
containing 30g husk + 60g corn powder + 50ml supplement
solution including Glucose (5g/L), Sucrose (5g/L), Malt extract
(5g/L), KH2PO4 (1g/L), KCl (0.5g/L), MgSO4.7H2O (0.1g/L),
NaCl (0.1g/L), CaCl2.2H2O (5g/L), MnSO4.4H2O(0.01g/L),
FeSO4.7H2O(0.01g/L).
Crude phytase solution was extracted accordingly to the
following scheme.
Semi-solid culture medium
Inoculate 2ml spores (108 CFU/ml)
five days incubation at 30°C

Extraction of phytase from fresh mold biomass by
glycine-HCl 0.2M, pH 3.5

Centrifugation at 7000 rpm, 20 mins, 4oC
Remove particles
Crude phytase extract
Protein concentration was measured by Bradford method
(1976) and phytase activity was determined by the method of
Heinonen and Lahti (1980).

3


2.2.1.2 Studying the effect of Ca2+ ion on phytase activity
Experimental design: Completely random with a factor:
the concentration of Ca2+ varied from 0, 5, 10, 20, 30, 40 to
50mM. The experiment was repeated three times with 7
treatments. Totally, there were 21 experimental units.
Experimental performance: CaCl2 salt was added in
crude phytase extract to achieved different concentrations as
mentioned above. Phytase activity was determined by Heinonen
and Lahti method (1980).
Evaluation criteria: phytase activity (U / mg protein).
2.2.1.3. Purification of phytase by ammonium suphate
precipitation
Crude phytase was precipitated from the extract with
amonium sulfate 80% saturation for 1-2 hour(s) at 4°C. Then, the
precipitated protein was dialyzed against buffer glycine-HCl
0.2M, pH 3.5
Protein concentration was measured by Bradford method

(1976) and phytase activity was determined by the method of
Heinonen and Lahti (1980).
2.2.1.3.

Purification

of

phytase

by

cation

exchange

chromatography
The enzyme solution after dialysis was aplied onto the
cation-exchange column SP-streamline.

4


The chromatography column was equilibrated with the
buffer glycine-HCl 0.2M pH 3.5.
The protein solution was loaded into the column at the rate
of 0.8ml/minute. The column was washed with the same buffer to
remove the unbound protein. Then, bound phytase was eluted
with gradient NaCl from 0 to 0.5M.
Obtained protein fractions were determined protein

concentration by Bradford (1976) and phytase activity by
Heinonen and Lahti method (1980).
Fractions with enzyme activity were checked for purity on
SDS-PAGE. Then they were further dialyzed against glycine-HCl
0.2M containing 30% ammonium sulphate, pH 3.5 and loaded
onto the hydrophobic interaction column.
2.2.1.4 Purification of phytase by hydrophobic interaction
chromatography
Hydrophobic interaction column was equiblirated with
glycine-HCl 0.2M pH 3.5 containing 30% AS saturation. The
enzyme fractions obtained from the ion-exchange column was
loaded into this column at the rate of 0.8ml/minute. Bound
proteins were eluted with gradient ammonium sulfate saturation
from 30% to 0%.
Protein fractions were collected and measured by Bradford
method (1976). Phytase activity was determined by Heinonen and
Lahti method (1980). The purity of the enzyme was checked on
SDS-PAGE.

5


The purification process of phytase from A. niger fresh
biomass was performed by the following procedure:
A. niger fresh biomass
homogeniztion with glycin-HCl 0.2M, pH 3.5

Crude phytase extract
Saturated ammonium sulfate 80% precipitation
Ion exchange chromatography

Hydrophobic interaction
chromatography

SDS-PAGE
electrophoresis
SDS-PAGE
electrophoresis

Pure phytase
2.2.2. Studying the optimal temperature of phytase activity
The experimental design: Completely random with a
factor: temperature of the reaction 35°C, 45°C, 55°C, 65°C, 75°C,
85°C, 95°C . The experiment was repeated three times with 7
treatments. In totall there was 21 experimental units.
Experimental performance: The phytase hydrolysis
reactions on synthetic phytate as a substrate were performed at
different temperature as mentioned above. Phytase activity was
determined by Heinonen and Lahti method (1980).
Evaluation criteria: phytase activity (U / mg protein).
2.3. Statistical analysis method

6


Microsoft Excel software version 2003 and Statgraphic
software version 15.0 were used to analyze the experimental
data.

7

III. Results and Discussion
3.1. Extraction of crude phytase from A. niger fresh biomass
Fresh A. niger biomass 900g was homogenized with 1000ml
Glycine-HCl 0.2M pH 3.5 buffer. The mixture was centrifuged to
obtain the crude phytase solution. The volume of crude phytase
extract were obtained about 700ml.
There was 498.71mg protein obtained with specific activity about
0.141U/mg.
3.2. Purification of phytase
3.2.1. Precipitation with ammpnium sulphate saturation
Crude phytase solution were precipitated with 80%
ammonium sulfate saturation (Đỗ Thị Thu Trang, 2011) in 2
hours at 4oC. The protein precipitate was redisolved in GlycineHCl 0.2M pH 3.5 and dialyzed against the same buffer to remove
ammonium sulfate. The specific phytase activity after dialysis
was about 0.770U/mg. It was 5.44 fold higher comparing with the
crude enzyme extract. SDS-PAGE was performed to check for the
purity of this protein fraction.
SDS-PAGE electrophoresis showed that after precipitation
some contaminated proteins were removed, however the enzyme
impurity was still high, so it was needed to purify it further by
other methods.

8


116kDa
66,2kDa
45kDa

35kDa
25kDa

18,4kDa

1

2

3

Figue 5 . SDS-PAGE of ammonium sulfate preparation of
phytase. 1. Standard protein; 2. Crude phytase extract;

3.

Ammonium sulfate protein preparation

3.2.2.

Purification

of

phytase

by

Cation

exchange

chromatography
Phytase from A. niger was rather stable at low pH and its pI
was about 4.7. Thus, the cation exchange chromatography at pH
3.5 were used to purify phytase.

9


The enzyme fraction obtained from ammonium sulphate
precipitation was loaded onto cation exchange column SPStreamline.
4.5

0.6

4

OD 280nm

3

0.4

2.5
0.3
2
1.5

0.2

1

NaCl concentration (M)

0.5
3.5

0.1
0.5
0

0
1

6

11

16

21

26

31

36

41

46

51

56

61

66

71

76

Protein fractions

Figue 6. Chromatogram of ammonium sulphate phytase
preparation on SP-Streamline cation exchange column.
The chromatogram of ammonium sulphate preparation on SPStreamline column (Figure 6) showed that there was only one
protein peak eluted at 0.116M-0.256M NaCL. This protein had
specific phytase activity about 1,154(U/mg), which was 1,5 fold
and 8.16 fold

purity, higher than the specific activity of

ammonium sulphate enzyme preparation and crude enzyme
extract respectively.
SDS-PAGE (Figure 7) showed that after cation exchange
chromatography SP-Streamline, several contaminated proteins in

the phytase fraction have been removed (lane 4). It has been
partially purified in camparison with the ammonium sulphate
preparation (lane 3). However, there were still four protein bands
in this fraction so it should be further purify to get the

10


homogenous form. For this reason, hydrophobic interaction
chromatography on Phenyl Sepharose column was aplied.

116kDa
66,2kDa
45kDa
35kDa
25kDa

18,4kDa

1

2

3

4

Figue 7. SDS-PAGE of enzyme fraction after SP-Streamline
cation exchange chromatography.

1. Standard

protein; 2. Crude phytase extract; 3. Saturated ammonium sulfate
80% precipitation sample; 4. Enzyme fraction eluted at 0.116M0.256M NaCL.
3.2.3. Hydrophobic interaction chromatography
The hydrophobic interaction chromatogram of phytase fraction
after SP-streamline column has been shown on the Figure 8.
There were two main protein peaks. Peak FI had no phytase
activity. Peak FII was the enzyme phytase with specific acticity of
1,778U/mg, it was 12,56 fold purified incomparison with crude
phytase extract.

11


AS concentration (%)
4

35

3.5

30

3

25

OD 280nm

2.5
20
2
15
1.5
10

1

5

0.5
0

0
1

6

11

16

21

26

31

36

41

46

51

56

61

66

71

76

81

86

91

96

101

106

111

116

121

126

131

136

Protein fractions

Figure 8.

Hydrophobic interaction chromatogram on Phenyl

Sepharose of Phytase fraction after SP-Streamline column
SDS-PAGE (Figure 9) revealed the homogenous phytase with one
major protein band of 87kDa (lane 5). This result was similar to
results of Greiner et al. (2009), the molecular mass of
extracellular phytase from A. niger 11T53A9 was about 85kDa.
This result is in accordance with the research by Ashok Pandey et
al. (2001) which showed phytase of some Aspergillus strains had
molecular mass from 40 to 100kDa. Sariyska et al. (2005) found
an extracellular phytase from wild strain A. niger with a low
molecular mass 39kDa. In addition, the molecular mass of the
phytases from bacteria is also low. For example, extracellular
phytase from Bacillus sp. DSll was 44 kDa (Young-Ok Kim et al.,
1998) and alkaline phytase from Lilium longiflorum pollen grain

was 52-55 kDa (Barry G. et al., 2006).

12


87kDa
116kDa
66,2kDa
45kDa
35kDa
25kDa
18,4kDa

1

2

3

4

5

Figue 9. SDS-PAGE of phytase fractions after hydrophobic
interaction chromatography on Phenyl Sepharose. 1. Standard
protein; 2. Crude phytase extract; 3. Ammonium sulfate 80%
precipitate; 4. Phytase after SP- Streamline column; 5. Phytase
after Phenyl Sepharose column.
Table 2 showed the purification scheme of the enzyme phytase
from A. niger isolate. The phytase was purified by three steps:

ammonium sulfate precipitation at 80% saturation, cation
exchange chromatography on SP-Streamline and hydrophobic
interaction chromatography on Phenyl sepharose. The enzyme
yield was 3.85% with 48% recovery activity. Specific activity
was 12.56 fold higher than crude phytase extract from the fresh A.
niger biomass.

13


Table 2. Purification scheme

Purification
steps
Crude extract

Protein
Total
(mg)

Yield (%)

Activity

Specific

Purifi-

Total

Recovery

activity

cation

(U)

(%)

(U/mg)

(fold)

498.31

100

58.117

100

0.141

1

47.339

11.53

36.437

63

0.770

5.44

27.598

6.72

31.846

55

1.154

8.16

15.813

3.85

28.112

48

1.778

12.56

Ammonium
sulfate 80%
precipitation
SP-Streamline
Phenyl
Sepharose

In comparison with the other purifcation procedures such as
Sariyska et al. (2005), who purified phytase from

A. niger

wild species by three steps: PS 50 membrane filtration, Sephadex
G-100 gel filtration chromatography column and DEAESepharose CL 6B ion exchange chromatography column, and
Greiner et al. (2009) purified phytase from A. niger 11T53A9
through these steps of ammonium sulfate precipitation from 090% saturation, four ion exchange chromatography column and
gel filtration chromatography (DEAE Sepharose CL 6B, CM
Sepharose CL 6B, Sephacryl S -200 HR and Mono S HR 5/5), the
purification procedure in this thesis was rather simpler and the
enzyme preparation was purer.
In general the purification procedure for phytase from

A.

niger can be established as in the following scheme:

14

Crude phytase extract from fresh A. niger biomass
Saturated ammonium sulfate 80% precipitation
Dialysis against glycine-HCl
0,2M pH3,5 buffer solution

Ion exchange chromatography on
SP-Streamline
Eluted with NaCl 0-0,5M

Eluted enzyme fraction
SDS-PAGE

Hydrophobic interaction on Phenyl Sepharose gel
Eluted with ammonium sulfate 30%-0%

Eluted Phytase faction
SDS-PAGE

Pure phytase
Figure 10. Purification procedure of Phytase from

A.

niger isolate
3.3. Characterization of purified phytase
3.3.1. The effect of ion Ca2+ on crude phytase activity.
Figue 11 demonstrated that crude phytase activity was
affected by Ca2+ ion. The presence of ion Ca2+ from

5- 50mM

enhanced the enzyme activity and the specific activity was
significantly different compared to control (Table 4, Appendix 2).
Adding 40mM Ca2+ increased specific activity of phytase
74,07% comparable to the control (without Ca 2+).

However,

2+

when the concentration of Ca increased more than 40mM, the
enzyme activity decreased, possibly high concentration of phytase
inhibit the enzyme action.

15


Specific activity (U/µg)

160
b

140
120
100

a

c

d

d

5

10

b

e

80
60
40
20
0
0

20

30

40

50

Concentration of Ca2+ (mM)

Figue 11. The effect of Ca2+ion on phytase activity

Also, Sariyska et al. (2005) reported that crude phytase
from A. niger wild type was active in the presence of 0.1mM
Ca2+. Phytase from Bacillus sp. DS11 active in the presence of
5mM Ca2+ (Young-Ok Kim et al., 1998).
3.3.2. Temperature optimum of phytase activity
Temperature is an important factor directly affecting the
catalytic activity of the enzyme. Almost all enzymes were
denatured under high temperature due to the disruption of enzyme
structure, except some thermophylic enzymes.
The results in Figue 12 showed that the phytase from A.
niger isolate was active in a range of 45 oC to 95 oC, and the
optimum temperature was 65oC.

It seemed this phytase is

thermotolerant. Similarly, Kim et al., 1998 reported that the active
temperature of different phytase was in a range of 40 oC - 77 oC.
Also, Sariyska et al. (2005) showed that the extracellular phytase
from wild Aspergillus niger exposed the optimum temperature at
55°C.

16


Specific activity (U/µg)

1600
a

1400

1200

b

b

1000
800

400

c

d

600
e

200
f

0
35°C

45°C

55°C

65°C

75°C

85°C

95°C

Temperature (°C)

Figure 12. Optimum temperature of Phytase from A. niger
isolate

17


IV. Conclusions and Suggestions
4.1. Conclusions
Phytase from A. niger isolate could be purified through
three steps including ammonium sulfate saturation precipitation
cation

exchange

chromatography

on

SP-Streamline

and

hydrophobic interaction chromatography on Phenyl-Sepharose.
Purified phytase has the molecular mass of bout 87KDa. The ion
Ca2+ is necessary for enzyme activity. It appeared that Phytase
from A. niger isolate in this study was thermotolerant.
4.2. Suggestions
Optimizing the purification procedure to get higher
enzyme yield and recovery activity.
Studying the preservation conditions for phytase activity
Applying the obtained phytase in

feed and food

industries.
Exploring gene cloning to produce Phytase in larger
scale.

18


Reference
Vietnamese
Đỗ Thị Thu Trang. 2011. Tinh sạch và khảo sát một số đặc điểm
của enzyme phytase từ nấm A. niger PE1. Luận văn tốt
nghiệp cao học khóa 16 chuyên ngành công nghệ sinh học.
Đại học Cần Thơ
Trần Nguyễn Nhật Khoa. 2012. Phân lập một số chủng nấm mốc
sinh phytase cao và khảo sát các điều kiện bảo quản chế
phẩm phytase từ Aspergillus niger. Luận văn tốt nghiệp đại
học chuyên ngành công nghệ sinh học. Đại học Cần Thơ.
Phan Thị Bích Trâm. 2010. Nghiên cứu hệ protease trùn quế

(Perionyx excavatus) trong quá trình tự phân và khả nămg
ứng dụng. Luận án tiến sĩ sinh học. Đại học khoa học tự
nhiên thành phố Hồ Chí Minh.
English
Ashok, P., George, S., Carlos, R.S., Jose, A.R.L., Vanete, T.S.
2001. Production, purification and properties of microbial
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Barry G. Garchow, Sonali P. Jog, Bakul Dhagat Mehta, Jodi M.
Monosso, Pushpalatha P.N. Murthy. 2006. Alkaline
phytase from Lilium longiflorum: PuriWcation and
structural characterization. Protein Expression and
Purification 46, pp.221–232.
Bradford, M.M. 1976. A rapid and sensitive method for
quantitation of microgram quantities of protein utilizing the

19


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254.
Dvorakova, J., Volfova, O. and Kopecky, J. 1997.
Characterization of phytase produced by Aspergillus niger.
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Greiner R., Lucineia Gomes da Silva, Sonia Couri. 2009.
Purification and characterisation of an extracellular phytase
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Heinonen, J., Lahti, R. 1980. A new and convenient colorimetric
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20


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A. niger phytase, and A. niger pH 2.5 acid phosphatase.
Appl. Environ. Microbiol 64, pp.4446-4451.
Young-Ok Kim, Hyung-Kwoun Kim , Kyung-Sook Bae, Ju-Hyun
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21