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

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 INTRODUCTION 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 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 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 CONCLUSIONS AND SUGGESTIONS 4.1 Conclusions 4.2 Suggestions REFERENCES ii i ii 3 4 5 7 10 11 12 12 12 13 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, 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 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 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 days of cultivation Mold spore inoculum were used from one-day to seven-day old The experiment was done in triplicate with treatments Totally, there were 21 experimental units Experimental performance: 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 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 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 days (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 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 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 factors: (i) four different carbon sources (glucose, fructose, maltose and sucrose) and (ii) four levels of carbon concentration with 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 above, spore density about 108 spores/mL, pH 4, incubation temperature 35°C in 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 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 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 medium with different concentrations of KH2PO4 (Figure 8) Figure 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 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 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% 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 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 for 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 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 Production of phytase by acido-thermophilic strain of Klebsiella sp DB-3 FJ711774.1 using orange peel flour under submerged fermentation Inno Romanian Food Biotechnol, 10:18-27 Gargova, S and M Sariyska 2003 Effect of culture conditions on the biosynthesis of Aspergillus niger phytase and acid phosphatase Enzyme Microbial Technol, 32(2):231-235 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 In Earth System Science, ed R.C.M Jacobson: Academic Press, pp.360376 Liu, B.L., C.H Jong and Y.M Tzeng 1999 Effect of immobilization on pH and thermal stability of Aspergillus ficuum phytase Enzyme Microbial Technol, 25(6):517-521 Nair, V.C., J Laflamme and Z Duvnjak 1991 Production of phytase by Aspergillus ficuum and reduction of phytic acid 13 content in canola meal J Sci Food and Agri, 54(3):355365 Pasamontes, L., M Haiker, M Wyss, M Tessier and A.P van Loon 1997 Gene cloning, purification, and characterization of a heat-stable phytase from the fungus Aspergillus fumigatus Appl Environ Microbiol, 63(5):1696-1700 Sano, K., H Fukuhara and Y Nakamura 1999 Phytase of the yeast Arxula adeninivorans Biotechnol Letters, 21(1):3338 Shieh, T.R and J.H Ware 1968 Survey of Microorganisms for the Production of Extracellular Phytase Appl Microbiol, 16(9):1348-1351 Shieh, T.R., R.J Wodzinski and J.H Ware 1969 Regulation of the formation of acid phosphatases by inorganic phosphate in Aspergillus ficuum J Bacteriol, 100(3):1161-1165 Shimizu, M 1993 Purification and Characterization of Phytase and Acid Phosphatase Produced by Aspergillus oryzae K1 Biosci Biotechnol Biochem, 57(8):1364-1365 Singh, B and T Satyanarayana 2012 Production of phytatehydrolyzing enzymes by thermophilic moulds African J Biotechnol, 11(59):12314-12324 Singh, P.K 2008 Significance of phytic acid and supplemental phytase in chicken nutrition: a review World's Poultry Sci J, 64(04):553-580 Vats, P and U.C Banerjee 2002 Studies on the production of phytase by a newly isolated strain of Aspergillus niger var 14 teigham obtained from rotten wood-logs Pro Biochem, 38(2):211-217 Vohra, A and T Satyanarayana 2001 Phytase production by the yeast, Pichia anomala Biotechnol Letters, 23(7):551-554 Volfova, O., J Dvorakova, A Hanzlikova and A Jandera 1994 Phytase from Aspergillus niger Folia Microbiol (Praha), 39(6):481-484 Wyss, M., L Pasamontes, R Remy, J Kohler, E Kusznir, M Gadient, F Muller and A van Loon 1998 Comparison of the thermostability properties of three acid phosphatases from molds: Aspergillus fumigatus phytase, A niger phytase, and A niger PH 2.5 acid phosphatase Appl Environ Microbiol, 64(11):4446-4451 15 ... 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. .. production of A fumigatus 3.2 The effect of phosphorus concentrations (KH PO4) on phytase production of A fumigatus 3.3 The effect of nitrogen sources and concentrations on phytase production of A fumigatus. .. effect of carbon sources and concentrations on phytase production of A fumigatus 3.5 Suggested procedure for phytase production from A fumigatus CONCLUSIONS AND SUGGESTIONS 4.1 Conclusions 4.2