purification and characterizations of phytase enzyme form aspergillus niger

The results showed that extracellular phytase from Aspergillus niger can be purified by ammonium sulfate precipitation at 80-90% saturation in combination with following hydrophobic int

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 STUDENT

Dr DUONG THI HUONG GIANG 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

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 Ca 2+

Key words: Aspergillus niger, hydrophobic interaction,

Cation-exchange chromatography, SP-streamline, Phenyl-sepharose, phytase, phytate

3.3.1 The effect of ion Ca2 + on phytase activity 15

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

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),

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

2.2.1 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

2.2.1.2 Studying the effect of Ca 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

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

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 SDS-PAGE

electrophoresis

Hydrophobic interaction SDS-PAGE chromatography 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

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

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 Glycine-HCl 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

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

Figue 6 Chromatogram of ammonium sulphate phytase preparation on SP-Streamline cation exchange column

The chromatogram of ammonium sulphate preparation on Streamline 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

SP-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

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

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

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)

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

Table 2 Purification scheme

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 DEAE-Sepharose 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 90% 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

0-In general the purification procedure for phytase from A

niger can be established as in the following scheme:

Purification

steps

activity (U/mg)

cation (fold)

Purifi-Total

Total (U)

Recovery (%)

Crude phytase extract from fresh A niger biomass

Saturated ammonium sulfate 80% precipitation

Ion exchange chromatography on

SP-Streamline

3.3 Characterization of purified phytase

3.3.1 The effect of ion Ca 2+ 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 Ca2+) However, when the concentration of Ca2+ increased more than 40mM, the enzyme activity decreased, possibly high concentration of phytase inhibit the enzyme action

Dialysis against glycine-HCl 0,2M pH3,5 buffer solution

Eluted with NaCl 0-0,5M

Eluted with ammonium sulfate 30%-0%

Pure phytase