Tài liệu Study on culture conditions of high phytase production from aspergillus fumigatus

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 SUPERVISOR STUDENT Dr. DUONG THI HUONG GIANG NGUYEN THI NGOC DIEP Student code: 3083916 Session: 34 (2008-2013) Can Tho, 2013 APPROVAL SUPERVISOR Dr. DUONG THI HUONG GIANG STUDENT 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 semisolid state medium, inoculated with one-day-old mold spores, and supplemented with 1% phosphorus in the form of KH2PO4, 0.5% 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. i CONTENTS ABSTRACT CONTENTS 1. INTRODUCTION 2. MATERIALS AND METHODS 2.1 Materials 2.2 Methods 2.2.1 Studying the effect of the age of spore inoculum on phytase production of A. fumigatus 2.2.2 Studying the effect of phosphorus concentrations (KH2PO4) on phytase production of A. fumigatus 2.2.3 Studying the effect of nitrogen sources and concentrations on phytase production of A. fumigatus 2.2.4 Studying the effect of carbon sources and concentrations on phytase production of A. fumigatus 2.2.5 Statistical analysis methods 3. RESULTS AND DISCUSSION 3.1 The effect of the age of spore inoculum on phytase production of A. fumigatus 3.2 The effect of phosphorus concentrations (KH 2 PO4) on phytase production of A. fumigatus 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 ii i ii 1 3 3 3 4 4 5 5 6 7 7 8 9 10 11 12 12 12 13 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 sources of phytase production. Especially, the most active producers are Aspergillus genus such as A. fumigatus, A. ficuum, 1 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 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. Chemicals: Sodium phytate (C6H6Na12O24P6H2O), L(+)ascorbic acid (C6H8O2) (Sigma), sodium acetate (Merck), tricloroacetic (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 inoculum mold spore age, phosphorus (KH2 PO4) concentration, 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 solution (MgSO4.7H2O (0.1 g/L); KCl (0.5 g/L); FeSO4 (0.01 3 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, spore density of 108 spores/mL, pH 4, incubation temperature 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 (KH 2PO4) on phytase production of A. fumigatus Experimental design: Completely random with a factor: phosphorus in the form of KH2 PO4 with six levels of 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 of 108 spores/mL, pH 4, incubation temperature 35°C in 2 days (Nguyen Van Tinh, 2012), the appropriate spore age was chosen 4 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 mentioned above, spore density of 108 spores/mL, pH 4, 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. 5 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 above, spore density about 108 spores/mL, pH 4, incubation 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. 6 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 oneday old to seven-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. 7 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 medium with different concentrations of KH2PO4 (Figure 8). Figure 8. Effect of KH2PO4 concentrations on phytase production of A. fumigatus The enzyme activity reached a peak at the KH2PO4 concentration of 1% (1.861 U/g). Similarly, Vats and Banerjee (2002) and Garvova and Sariyska (2003) also showed that 8 phytase synthesis was inhibited at high phosphorus content. Therefore, KH2PO4 of 1% was used for the next 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 9 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% 10 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 11 4. CONCLUSIONS AND SUGGESTIONS 4.1 Conclusions The optimum conditions for phytase production from A. fumigatus T1 were determined. A. fumigatus T1 exhibited maximum enzyme yield on the medium with wheat flour as a phytate substrate, spore density of 108 spores/mL, incubation temperature 35°C, pH 4 for 2 days (Nguyen Van Tinh, 2012); and mineral supplement such as malt extract with nitrogen content of 0.5%, 1% KH2PO4, 0.5% glucose, and one-day-old spore inoculum. The general scheme for high phytase production from A. fumigatus was also established. An overall three-fold improvement in the phytase production was achieved. 4.2 Suggestions - Studying submerged incubation conditions for phytase production since that would be easier for extraction of crude phytase from the A. fumigatus biomass than on semi-solid culture media. - Studying storage conditions such as time and preservative agents for crude phytase extract from A. fumigatus to apply efficiently in animal feed. - Purification and characterization of the phytase enzyme from this A. fumigatus isolate. - Studying application of this phytase enzyme product in aquaculture and husbandry. 12 REFERENCES Vietnamese Nguyễn Văn Tính. 2012. Phân lập và khảo sát môi trường nuôi cấy nấm Aspergillus fumigatus sinh tổng hợp phytase cao. Luận văn tốt nghiệp Đại học chuyên ngành Công nghệ Sinh học. Đại học Cần Thơ. English Arpana, M., S. Gulab, G. Varsha, Y. Anita and N.K. Aggarwal. 2012. 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