study on culture conditions of high phytase production from aspergillus fumigatus size 1415

Key words: Aspergillus fumigatus T1, KH2PO4, malt extract, one-day-old mold spore, phosphorus, phytase, phytate... fumigatus 4 fumigatus 4 2.2.3 Studying the effect of nitrogen sources

MINISTRY OF EDUCATION & TRAINING

CAN THO UNIVERSITY

BIOTECHNOLOGY RESEARCH & DEVELOPMENT INSTITUTE

SUMMARY BACHELOR OF SCIENCE THESIS

THE ADVANCED PROGRAM IN BIOTECHNOLOGY

STUDY ON CULTURE CONDITIONS

OF HIGH PHYTASE PRODUCTION

FROM Aspergillus fumigatus

SIZE 14-15

Dr DUONG THI HUONG GIANG NGUYEN THI NGOC DIEP

Student code: 3083916 Session: 34 (2008-2013)

Can Tho, 2013

APPROVAL

SUPERVISOR STUDENT

Dr DUONG THI HUONG GIANG NGUYEN THI NGOC DIEP

Can Tho, May 15, 2013

PRESIDENT OF EXAMINATION COMMITTEE

ABSTRACT

Phytase is a group of enzymes that are able to release phosphorus from phytate so that it can be easily digested Therefore, phytase is largely used in animal feed Recently, in the Laboratory of Enzyme Technology, Biotechonology R&D Institute,

a new isolated strain of Aspergillus fumigatus had high potential for phytase production In this study, the growth conditions to improve the enzyme production of this new A fumigatus T1 were investigated The strain showed maximal productivity on semi-solid state medium, inoculated with one-day-old mold spores, and

glucose and 0.5% nitrogen in the form of malt extract The overall three-fold improvement of phytase activities was achieved The general scheme for high phytase production of A fumigatus was also established

Key words: Aspergillus fumigatus T1, KH2PO4, malt extract, one-day-old mold spore, phosphorus, phytase, phytate

CONTENTS

2.2.1 Studying the effect of the age of spore inoculum

on phytase production of A fumigatus

4

fumigatus

4

2.2.3 Studying the effect of nitrogen sources and

concentrations on phytase production of A fumigatus

5

2.2.4 Studying the effect of carbon sources and

concentrations on phytase production of A fumigatus

5

3.1 The effect of the age of spore inoculum on phytase

production of A fumigatus

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on phytase production of A fumigatus

8 3.3 The effect of nitrogen sources and concentrations on

phytase production of A fumigatus

3.4 The effect of carbon sources and concentrations on

phytase production of A fumigatus

3.5 Suggested procedure for phytase production from

A fumigatus

4 CONCLUSIONS AND SUGGESTIONS

4.1 Conclusions

4.2 Suggestions

REFERENCES

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1 INTRODUCTION

Phosphorus is one of the elements playing an essential role

in living things since it is a structure component of nucleic acid (DNA, RNA), phospholipid membranes, high-energy compounds (such as ATP, NADPH) and connective tissues, etc (Jahnke, 2000) Therefore, inorganic phosphorus as well as organic phosphorus from plants is added into feed to support the animal development in husbandry However, the main storage form of phosphorus in plants is in the form of phytate which can not be utilized by non-ruminant animals Additionally, phytate is considered as an anti-nutritive factor because it is a strong chelator of cations It binds strongly to essential minerals such as

Ca2+, Zn2+, Mg2+, Mn2+, Fe2+/3+, and this leads to the decrease in the mineral-absorbing ability of livestock (Singh, 2008) For these reasons, farmers routinely add inorganic phosphorus to the diet The excess of inorganic phosphorus as well as phytate excreted in the animal faeces can cause environmental pollution

Phytase is a group of enzymes that are able to degrade phytate into easily digested phosphorus In fact, using phytase can help not only to improve the nutritional value of phytate in feed but also to reduce the waste of inorganic phosphorus, and this benefits the reduction of the cost for feed

Phytase enzyme can be found in animals, plants and microorganisms Among them, microorganisms are the main

producers are Aspergillus genus such as A fumigatus, A ficuum,

A oryzae and A niger (Shimizu, 1993; Volfova et al., 1994; Liu

et al., 1999)

In comparison with phytases from other Aspergillus sp., phytase from Aspergillus fumigatus has a special characteristic

with high ability of refolding in heat denaturation process (Pasamontes et al., 1997; Wyss et al., 1998), which is suitable for implementing in feed production

However, the research of phytase on this mold in Vietnam

is still limited Recently, a new strain A fumigatus T1 producing

phytase has been isolated and studied in the Enzyme Technology Laboratory, Biotechnology R&D Institute As the continuation of this work, the thesis “Study on culture conditions of high phytase

production from Aspergillus fumigatus” has been performed to

have more complete figure about this strain, with the hope to

produce high phytase from this new A fumigatus isolate

Thesis objectives: This study aimed at studying culture

conditions of the high phytase yield from A fumigatus

2 MATERIALS AND METHODS

2.1 Materials

An A fumigatus T 1 strain was isolated by Nguyen Van

Tinh (2012)

Media: PGA (Potato Glucose Agar), semi-solid substrate medium (Arpana et al., 2012)

Wheat flour (organic phytase source) was purchased in Xuan Khanh market, Ninh Kieu District, Cantho City

Nitrogen sources: Malt extract, yeast extract, ammonium sulfate

Carbon sources: Glucose, fructose, maltose, sucrose

Equipment: Eppendorf - Germany, spectrometer (Hitachi - Japan), centrifuge (Rotor - Germany) and other lab facilities

tricloro-acetic (TCA) (Merck), acetone (China), glucose, fructose, maltose, sucrose (China), malt extract, yeast extract (Merck)

2.2 Methods

Studying culture conditions of high phytase production

from A fumigatus isolate

Purpose: Choosing the appropriate factors such as the

nitrogen source and nitrogen concentration, carbon source and carbon concentration to obtain high yield of phytase from

A fumigatus

The semi-solid state culture medium: 45 g of wheat flour and rice husk with the ratio of 2:1 w/w; 25 mL of mineral

g/L); MnSO4 (0.01 g/L); NaCl (0.1 g/L); CaCl2 (5 g/L) and pH 5.5 (Arpana et al., 2012)

2.2.1 Studying the effect of the age of spore inoculum on

phytase production of A fumigatus

Experimental design: Completely random with a following

factor: spore age within 7 days of cultivation

Mold spore inoculum were used from one-day to seven-day old The experiment was done in triplicate with 7 treatments Totally, there were 21 experimental units

Experimental performance: 7 samples of the mold spores at

different ages as mentioned above was inoculated into semi-solid state culture medium with wheat flour as a phytate substrate,

35°C for 2 days (Nguyen Van Tinh, 2012) The enzyme activity was determined by the method of Heinonen and Lahti (1981)

Evaluation criteria: Phytase activity (U/g fresh biomass)

2.2.2 Studying the effect of phosphorus concentrations (KH2PO4) on phytase production of A fumigatus

Experimental design: Completely random with a factor:

concentration such as: 0%, 0.5%, 1%, 1.5%, 2% and 2.5% The experiment was done in triplicate with 6 treatments Totally, there were 18 experimental units

Experimental performance: A fumigatus was grown on the

culture medium with wheat flour (phytate substrate) at different concentrations of phosphorus as mentioned above, spore density

(Nguyen Van Tinh, 2012), the appropriate spore age was chosen

from 2.2.1 The enzyme activity was determined by the method

of Heinonen and Lahti (1981)

Evaluation criteria: Phytase activity (U/g fresh biomass)

2.2.3 Studying the effect of nitrogen sources and

concentrations on phytase production of A fumigatus

Experimental design: Completely random with 2 factors: (i)

three nitrogen sources (malt extract, yeast extract and ammonium sulfate) and (ii) four different levels of nitrogen concentration (0%, 0.25%, 0.5% and 0.75%) The experiment was done in triplicate with 12 treatments Totally, there were 36 experimental units

Experimental performance: A fumigatus was grown on

semi-solid state medium with wheat flour (phytate substrate) of different nitrogen sources at different concentration levels as

incubation temperature 35°C in 2 days (Nguyen Van Tinh, 2012), the appropriate spore age and phosphorus concentration were chosen from 2.2.1 and 2.2.2 The enzyme activity was determined by the method of Heinonen and Lahti (1981)

Evaluation criteria: Phytase activity (U/g fresh biomass)

2.2.4 Studying the effect of carbon sources and

concentrations on phytase production of A fumigatus

Experimental design: Completely random with 2 factors: (i)

four different carbon sources (glucose, fructose, maltose and sucrose) and (ii) four levels of carbon concentration with 4 concentrations (0%, 0.5%, 1% and 1.5%) The experiment was done in triplicate with 16 treatments Totally, there were 48 experimental units

Experimental performance: A fumigatus was grown on the

culture medium with wheat flour (phytate substrate) of different carbon sources at different concentration levels as mentioned

temperature 35°C in 2 days (Nguyen Van Tinh, 2012), the appropriate inoculum spore age, phosphorus concentration, nitrogen source and concentration were taken from previous experiments The enzyme activity was determined by the method

of Heinonen and Lahti (1981)

Evaluation criteria: Phytase activity (U/g fresh biomass)

2.2.5 Statistical analysis methods

Microsoft Exel software version 2010 and Statgraphic software version 15.0 were used to analyze the experimental data

3 RESULTS AND DISCUSSION

3.1 The effect of the age of spore inoculum on phytase

production of A fumigatus

Inoculum spore age was known as an important factor affecting the microbial growth as well as enzyme production of microorganisms (Singh and Satyanarayana, 2012) In this

experiment, the A fumigatus spores at different ages (from

one-day old to seven-one-day old) were inoculated into the semi-solid state culture medium with wheat flour as a phytate substrate The enzyme production was measured by phytase activity per gram fresh biomass

Figure 7 Effect of the age of spore inoculum on phytase

production of A fumigatus

Based on the results in the Figure 7, it was found that the productivity of the enzyme was highest (1.347 U/g) in case of using one-day-old spore inoculum, and it was significantly different from the other older spore ages The spore of seven day old gave lowest enzyme yield (0.647 U/g) It appeared that this

A fumigatus isolate has an ability to develop in a very short time

The one-day-old fungus was readily adapted to the culture conditions to produce high level phytase for development While the older spores seemed to quickly enter into the dormant phase,

so they took a longer time to germinate and this lead to low enzyme production (Singh and Satyanarayana, 2012)

Therefore, the mold spores of one-day-old was applied for the next experiments

3.2 The effect of phosphorus concentrations (KH2PO4) on

phytase production of A fumigatus

It was reported that the phosphorus concentration plays an important role in phytase production (Shieh and Ware, 1968; Shieh et al., 1969) To determine the effect of phosphorus on

phytase production, A fumigatus was cultivated in the growth

Figure 8 Effect of KH2PO4 concentrations on phytase

production of A fumigatus

concentration of 1% (1.861 U/g) Similarly, Vats and Banerjee (2002) and Garvova and Sariyska (2003) also showed that

phytase synthesis was inhibited at high phosphorus content

experiments

3.3 The effect of nitrogen sources and concentrations on

phytase production of A fumigatus

Figure 9 Effect of nitrogen sources and concentrations on

phytase production of A fumigatus

As it was shown in the Figure 9, the nitrogen source and nitrogen content significantly affected phytase production of

A fumigatus Yeast extract had the most effect at 0.25%

(3.945 U/g) However, the maximum yield of phytase was obtained at the concentration of 0.5% (4.490 U/g), it was remarkably higher than other malt extract concentration tested (0.25 and 0.75%) and also higher at 0.25% yeast extract Among two organic nitrogen sources explored, malt extract gave much higher phytase yield (4.490 U/g) than yeast extract (3.945 U/g),

it appeared that the plant nitrogen is a suitable source for phytase

production of A fumigatus Besides, the organic nitrogen sources

(malt extract and yeast extract) gave higher phytase production

than inorganic one (ammonium sulphate) Similarly, Sano et al (1999) and Vohra and Satyanarayana (2001) also reported that organic nitrogen sources were generally better for protein production than inorganic nitrogen sources Furthermore, the nitrogen supplementation >0.5% seemed to inhibit phytase production, the phytase yield was dropped remarkably

In sum, malt extract of 0.5% could be used for further

experiment

3.4 The effect of carbon sources and concentrations on

phytase production of A fumigatus

Similar to nitrogen sources, the carbon sources and carbon content also seemed to have significant effect on the phytase

production of A fumigatus (Figure 10)

ê

Figure 10 Effect of carbon sources and concentrations on

phytase production of A fumigatus

Among four different sugars tested (glucose, fructose, maltose and sucrose), the glucose of 0.5% gave highest enzyme yield of 4.673 U/g (Figure 10) Glucose concentration >5%

appeared to inhibit the enzyme production (3.523 U/g at 1% and

2.984 U/g at 1.5%) A similar result was reported by Nair et al

(1991) that phytase production of A ficuum was also inhibited by

high glucose concentrations

In general, 0.5% glucose was chosen for high phytase

production of A fumigatus

3.5 Suggested procedure for phytase production of

A fumigatus

Based on the above obtained results, the general scheme for

high phytase production from A fumigatus could be established

as in Figure 11

Figure 11 General scheme for phytase production from

A fumigatus