purification of phytase from aspergillus fumigatus

The results showed that ammonium sulfate fractionation gave two phytases of different molecular mass Ammonium sulfate precipitation in combination with cation exchange chromatography on

MINISTRY OF EDUCATION & TRAINING

CAN THO UNIVERSITY

BIOTECHNOLOGY RESEARCH & DEVELOPMENT INSTITUTE

SUMMARY BACHELOR OF SCIENCE THESIS

THE ADVANCED PROGRAM IN BIOTECHNOLOGY

PURIFICATION OF PHYTASE

FROM Aspergillus fumigatus

SUPERVISOR STUDENT

Dr DUONG THI HUONG GIANG HUYNH THAO TIEN

Student code: 3083766 Session: 34 (2008-2013)

Can Tho, 2013

APPROVAL

SUPERVISOR STUDENT

Dr DUONG THI HUONG GIANG HUYNH THAO TIEN

Can Tho, May 10, 2013

PRESIDENT OF EXAMINATION COMMITTEE

Abstract

Aspergillus fumigatus, a potential phytase producing isolate, which has recently been found in the Laboratory of Enzymology, Biotechnology R&D Institure, Can Tho University Preliminary studies showed that the phytase enzymes from this fungi strain adopted high thermostability, which

is favorable for animal feed production Since there was little information about the phytase form this species this thesis aimed at purification of phytase from the A fumigatus isolate The results showed that ammonium sulfate fractionation gave two phytases of different molecular mass

Ammonium sulfate precipitation in combination with cation exchange chromatography on SP-Streamline allowed purifying a high molecular

which adopted a molecular mass of 87.7 kDa This phytase of Aspergillus fumigatus was purified to about 2.68-fold and exposed a high specific activity of 4.398 U/mg protein

Key words: Aspergillus fumigatus, ammonium sulfate fractionation,

cation exchange chromatography, phytase, SP-Streamline

CONTENTS

2.2.3.2 Phytase purification by hydrophobic interaction

2.2.3.3 Phytase purification by cation-exchange chromatography

3.2.2 Purification of phytase by hydrophobic interaction

3.2.2 Purification of phytase by cation exchange chromatography on

1 INTRODUCTION

Phosphorus (P) plays major structural and metabolic roles in living cells It is a structural component of important molecules, such as nucleic acid (DNA, RNA), phospholipid membranes, high-energetic molecules

(ATP, NADPH, ) (Jahnke, 2000) Phytases are

myo-inositol-1,2,3,4,5,6-hexakisphosphate phosphohydrolases that catalyze the degradation of

orthophosphate, which is easily to be absorbed in animal digestive tract However, monogastric animals, such as pigs and poultry, are not able to utilize phytate, so phytate in their digestive tracts cannot be absorbed The unutilized phytate is excreted to the environment lead to pollution in areas

of intensive husbandry Besides, phytic acid can act as an anti-nutrient factor by chelating with minerals, such as zinc, iron, calcium and magnesium (Cheryan, 1980) Phytate-degrading enzymes have been studied intensively in recent years because of the interest in such enzymes for reducing the levels of inorganic as well as organic phosphate pollution in livestock areas, and also improving nutritional values of organic phosphate (phytate) (Lei and Porres, 2003)

Phytases are found naturally in plants and microorganisms, particularly fungi Most of the studies focused on phytases produced from

Aspergillus sp., they are high phytase producers (Rao et al., 2009) Among them A fumigatus is a potential fungi strain producing phytase of valuable

properties such as heat-labile, broad pH activity, broad substrate specificity v.v Up until present, very limited information about the purification of

phytase from A fumigatus is available In order to obtain pure enzyme phytase from A fumigatus to apply in feed/food industries, the research on

“Purification of phytase from Aspergillus fumigatus” has been carried

out

Aim of the thesis: Purification of the phytase form A fumigatus by

combination of the methods including ammonium sulfate precipitation, Ion-exchange chromatography and hydrophobic interaction chromatography

2 MATERIALS AND METHODS

2.1 Materials

Laboratory

in Xuan Khanh market, Ninh Kieu District, Can Tho City

Germany, spectrometer (Hitachi – Japan), centrifuge (Rotor – Germany) and other lab facilities

- Chemicals: Sodium phytate (C6H6Na12O24P6H2O), L(+)- Ascorbic acid (C6H8O2) (Sigma), Sodium acetate (Merck), Trichloroacetic acid (TCA) (Merck), Acetone (China),…

 PGA – Potato Glucose Agar: 2% D-glucose, 1.8% (w/v) agar, 20% (w/v) potato

 Semi-solid substrate medium (Arpana et al., 2012): 30g phytate substrate, 15g rice husk, 25 mL mineral solution (Spieck and Lipski, 2011)

pH 5.0

glucose : sucrose (1:1) 1%

2.2 Methods

2.2.1 Preparation of fungi strain and culture medium

Aspergillus fumigatus was maintained in PGA medium and incubated in 45°C for 2 days Determine the centration of A fumigatus

For phytase production, semi-solid substrate medium was used 1 mL A

medium and incubated in 35°C After 2 days incubation, fungi fresh biomass was collected and the crude enzyme was extracted

2.2.2 Extraction of the crude enzyme phytase

Enzyme extraction was performed accordingly to the method of Nguyễn Văn Tính (2012)

Semi-solid substrate medium

Sterilized in 20 minutes, 121°C

Incubated for 2 days, 35°C Homogenization of fungi biomass in 50mL sodium acetate buffer

0.02M, pH 5.5

Centrifugation for 20 minutes, 13000 rpm, 4°C

Take off the pellet Crude phytase extract

Figure 1 Phytase extraction procedure

Protein content of crude phytase extract were measured by Bradford method (1976) and the enzyme activity was determined by the method of Heinonen and Lahti, (1981)

2.2.3 Phytase purification

2.2.3.1 Ammonium sulfate fractionation

Ammonium sulfate (AS) was added into the crude enzyme extract with the concentration from 0, 20, 30, 40, 50, 60, 70, 80 and 90% saturation (Appendix 1) The solution was stirred and kept at 4°C for 1 – 2 hours, then centrifuged at 13000 rpm for 20 minutes The pellet of each ammonium

sulfate fraction was collected and determined the protein content by Bradford method, enzyme activity was determined by the method of Heinonen and Lahti, (1981)

2.2.3.2 Phytase purification by hydrophobic interaction chromatography on Phenyl Sepharose

The crude enzyme extract was precipitated with ammonium sulfate saturation chosen from 2.2.3.1, and centrifuged at 7000 rpm for 20 minutes Dissolving the pellet in the buffer Tris-HCl 0.02M pH 7.6 + AS 30% saturation, and dialyzed against the same buffer in the fridge for 24 hours After dialysis, the enzyme solution was loaded onto the Phenyl Sepharose column Washing the column with buffer Tris-HCl 0,02M pH 7.6 + AS 30% to remove unbound proteins Bound proteins were eluted by decreasing AS concentration gradient from 30% – 0%

Protein content and specific activity of precipitated fractions were determined by Bradford (1976) and Heinonen and Lahti, (1981) methods respectively SDS-PAGE was used to check for phytase purity

2.2.3.3 Phytase purification by cation-exchange chromatography on SP-Streamline column

The crude enzyme extract was precipitated with the ammonium sulfate saturation concentration chosen from 2.2.3.1, centrifuged at 7000 rpm for 20 minutes at 4°C The obtained pellet was dissolved in Tris-HCl 0,02M pH 7.6 and dialyzed against the same buffer in the fridge for 24 hours After dialysis, the enzyme solution was passed through SP-Streamline column with the rate of 0.8 mL/minute The column was washed with buffer Tris-HCl 0.02M, pH 7.6 to remove the unbound proteins The bound proteins were released by increasing NaCl concentration gradient from 0 – 0.5M with the rate of 1 mL/minute

Eluted protein fractions were determined protein content by Bradford (1976) and phytase activity by Heinonen and Lahti, (1981) The enzyme purity was checked by SDS-PAGE

3 RESULTS AND DISCUSSION

3.1 Extraction of crude enzyme

The crude enzyme extracts 620 mL were collected from 900 g fresh fungi biomass, the total protein content was 235.743 mg, and the enzyme specific activity was 1.643 U/mg protein Similar result was reported by Đỗ

Thị Thu Trang (2011) studying phytase from A niger PE1, the specific

activity of this fungi was 1.65 U/mg protein This result was lower than

phytase from A niger 11T53A9 (2.6 U/mg protein) (Greiner et al., 2009) Wyss et al., (1999) studied several A fumigatus strains and concluded that

A fumigatus phytase specific activity was lower than A niger strains

3.2 Phytase purification

3.2.1 Ammonium sulfate fractionation

The phytases from A fumigatus were preliminary purified from the

crude enzyme extract with ammonium sulfate concentration varied from

protein) (Figure 2)

SDS-PAGE of protein fractions precipitated by AS of different saturation levels showed that these two protein fractions were different mainly by the protein band of about 87.7 kDa (Figure 3, lane 7,8,9,10) In the 20  50% AS precipitate the other, lower molecular mass phytase of about 66.2 kDa and others (Figure 3, lane 4,5,6,7) The existence of different phytases was reported in the work of Vats and Banerjee (2004), and it could be due to the culture conditions

Figure 2 Preliminary purification of phytases from A fumigatus by

ammonium sulfate fractionation

1 2 3 4 5 6 7 8 9 10 Figure 3 SDS-PAGE of ammonium sulfate precipitated fractions

1 Protein standard 2 Crude enzyme extract 3 AS 20% 4 AS 30%

5 AS 40% 6 AS 50% 7 AS 60% 8 AS 70% 9 AS 80% 10 AS 90%

a

b

b

c

b

phytase

3.2.2 Purification of phytase by hydrophobic interaction chromatography on Phenyl Sepharose

Phenyl Sepharose column It was separated into four peaks– unbound, FI, FII and FIII (Figure 4) The three protein fractions unbound, FII and FIII had no enzyme activity The only FI fraction showed low enzyme activity, about 0.380 U/mg protein

Figure 4 Chromatogram of 60  90% AS phytase fraction on

Phenyl Selpharose column

SDS-PAGE showed very high impurity of the phytase fraction (Figure 7, lane 5) with many protein bands It appeared that hydrophobic interaction chromatography was not an appropriate method for phytase

saturation

3.2.2 Purification of phytase by cation exchange chromatography on SP-Streamline

Figure 5 Chromatogram of phytase fraction precipitated with 60-80% ammonium sufate saturation on cation exchange column

SP-Streamline

The phytase fraction precipitated by AS 60  90% saturation was applied on cation exchange SP-Streamline column The chromatogram revealed that there were three protein fractions (unbound, FI and FII) (Figure 4) Surprisingly, the unbound fraction was the phytase that exposed high specific phytase activity about 4.398 U/mg protein While the bound proteins (fraction I&II) did not have enzyme activity It seemed that this phytase adopted the pI > 7.6, under the chromatography pH condition (pH 7.6), it was generally charged negative, due to this, it could not bind to the

Unbound

SP-streamline column and eluted as the unbound fraction SDS-PAGE analysis showed that the purified phytase was homogenous with molecular mass of about 87.7 kDa (Figure 6, lane 6)

Similarly, Wang et al., (2007) studied on phytases from A fumigatus

WY-2 also showed that the molecular mass of the phytase is about 88 kDa

Other research on phytase from A fumigatus isolate found another phytase

of 60 kDa (Pasamontes et al., 1997) Wyss et al., (1999) studied on phytases from six fungi strains revealed that molecular mass of phytases

from the two different A fumigatus batches were 72.3 kDa and 60.7 kDa

Figure 6 also revealed that cation exchange chromatography by SP-Streamline was an appropriate method for purification of a high molecular

1 2 3 4 5 6 7

Figure 6 SDS-PAGE analysis of phyase fractions from Phenyl

sepharose and SP-Streamline column

1 Protein standard 2 Crude phytase extract

3 60-80% AS preciptated phytase

4 Unbound fraction from Phenyl Sepharose column

5 Bound fraction from phenyl sepharose column

6 Unbound fraction from SP-streamline column

7 Bound fraction from SP-Streamline column

Based on the purification scheme of the high molecular phytase

(87,7kDa) from A fumigatus (Table 1), it can be concluded that this

phytase can be successfully purified by combination of the two methods ammonium sulfate fractionation and cation exchange chromatography The phytase was purified 2.68 fold in comparison with the crude extract

Phytase 87.7 kDa

Table 1 Purification scheme of a high molecular mass phytase

from A fumigatus by hydrophobic interaction chromatography

Step

Total protein (mg)

Total activity (U)

Specific activity (U/mg protein)

Purification (folds)

60  90% AS

Hydrophobic

interaction

chromatography

Table 2 Purification scheme of a high molecular mass phytase

from A fumigatus by ion exchange chromatography

Step

Total protein (mg)

Total activity (U)

Specific activity (U/mg protein)

Purification (folds)

Crude enzyme

60  90% AS

Ion exchange

4 CONCLUSIONS AND SUGGESTIONS

4.1 Conclusions

- Ammonium sulfate fractionation allowed separating the two

phytases from the crude enzyme extract of fresh A fumigatus biomass

- A high molecular mass phytase (87.7 kDa) was completely purified by AS fractionation following cation exchange chromatography The purification factor of the enzyme phytase was about 2.68-fold with high specific activity (4.398 U/mg protein)

4.2 Suggestions

precipitate

- Optimizing the purification procedure to get higher phytase yield

- Characterizing phytases from A fumigatus such as optimum pH and

temperature, and the effect of metal ions on the enzyme activity

- Application of phytase in animal feed

REFERENCES Vietnamese

Đỗ Thị Thu Trang, 2011 Tinh sạch và khảo sát một số đặc điểm của

enzyme phytase từ nấm Aspergillus niger Luận văn Thạc sĩ Đại học

Cần Thơ 27-36

Dương Thị Hương Giang 2010 Bài giảng hóa protein Viện nghiên cứu và phát triển công nghệ sinh học, Đại học Cần Thơ

English

Arpana, M., S Gulab, V.G., A.Y., N.K and N.K Aggarwal 2012 Production of phytase by acido-thermophilic strain of Klebsiella sp DB-3FJ711774.1 using orange peel flour under submerged

fermentation Inovative Romanian Food Biotechnology 10:18-27 Cheryan, M 1980 Phytic acid interactions in food systems Crit Rev Food Sci Nutr 13(4):297-335

Greiner, Ralf, Lucineia Gomes da Silva and Sonia Couri 2009 Purification and characterisation of an extracellular phytase from Aspergillus

niger 11T53A9 Brazilian Jf Microbiol 40:795-807

Heinonen, J K and R J Lahti 1981 A new and convenient colorimetric determination of inorganic orthophosphate and its application to the

assay of inorganic pyrophosphatase Anal Biochem 113(2):313-317 Jahnke, R.A (2000) The phosphorus cycle, Earth System Science,

pp.360-376

Lei, X G and J M Porres 2003 Phytase enzymology, applications, and

biotechnology Biotechnol Lett 25(21):1787-1794

Pasamontes, L., M Haiker, M Wyss, M Tessier and A P van Loon 1997 Gene cloning, purification, and characterization of a heat-stable