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Supplementation effect of ectoine on thermostability of phytase
JOURNAL OF BIOSCIENCE AND BIOENGINEERING Vol. 102, No. 6, 560–563. 2006 DOI: 10.1263/jbb.102.560
© 2006, The Society for Biotechnology, Japan
Supplementation Effect of Ectoine on Thermostability of Phytase Linghua Zhang,1§* Yue Wang,1,2 Chunyu Zhang,1 Yunji Wang,2 Daochen Zhu,3 Chenxiang Wang,3 and Shinichi Nagata3 College of Environmental Science and Engineering, Dalian Maritime University, Lingshui Road, Dalian 116026, P.R. China,1 College of Bio & Food Technology, Dalian Institute of Light Industry, 1 Qinggong-Yuan, Ganjingzi District, Dalian 116034, P.R. China,2 and Environmental Biochemistry Division, Research Center for Inland Seas, Kobe University, 5-1-1 Fukae, Higashinada-ku, Kobe 658-0022, Japan3 Received 3 July 2006/Accepted 26 September 2006
In this study, we elucidated the supplementation effect of compatible solutes on the thermostability of phytase, designated as PHYA II, which was encoded by the phytase gene phyA I (GeneBank AY013315) from Aspergillus ficuum As3.324 and expressed in Pichia pastoris GS115. When PHYA II in acetate buffer was heated at 90°C for 15 min, more than 80% of the residual activity was retained by adding the cyclic amino acid ectoine, a representative compatible solute. Furthermore, the presence of ectoine led to an increase in the relative hydrolytic rate of sodium phytate by 15.7% with heating at 80°C for 15 min. Among the compatible solutes examined, ectoine was confirmed to be the most efficient thermoprotectant for PHYA II. [Key words: phytase, thermostability, ectoine, hydrolytic rate, denaturation]
Phytase (EC 3.1.3.8) is a phosphomonoesterase that hydrolyzes phytate and releases inorganic phosphate, which can be directly ingested by monogastric animals. Thus, it is important not only to increase the availability of phytate-P in animal feed, but also to decrease the amount of environmental pollution caused by phosphorus contents in manure from nonruminant livestock and poultry (1, 2). Because the activity of phytase usually decreases during feed pelleting (85– 90°C) by 30% or more, it is important to enhance the thermostability of phytase (3). Some studies have been performed for the improvement of phytase thermostability as follows: phytase genes were reconstructed using site-directed mutagenesis (4); a thermostable phytase molecule was newly established by exploiting the consensus technique (Lehmann, M., EP patent 0897985 A2, 1999); phytase was modified through glycosylation (5); and thermostable strains producing phytases were isolated from various environments (6). In addition, protectants or stabilizers such as metals, polyols or other solutes have been applied to increase the thermostability of this enzyme (7–9). The cyclic amino acid ectoine (1,4,5,6-tetrahydro-2-methyl-4-pyrimidine carboxylic acid), one of the representative compatible solutes, accumulates in halophilic and halotolerant bacteria in hypersaline environments, the role of which is to establish a balance between the osmotic pressures inside and outside the cells (10). Previous studies indicated
that ectoine functions on proteins as a stabilizer against some adverse conditions. For example, Lippert and Galinski (11) reported that the thermal stabilities of lactate dehydrogenase and phosphofructokinase in the presence of ectoine increase. Ectoine was also been shown to lower the melting temperature of dsDNA in PCR application (12). In our previous study (13), the phytase gene phyA I (GeneBank AY013315) from Aspergillus ficuum As3.324 was inserted into the plasmid pPIC9K, which was 1515 bp in length, contained an intron of 111 nucleotides and encoded 467 amino acids. The recombinant plasmid was transformed in Pichia pastoris GS115 using an electrical pulse treatment. Phytase, which was encoded by phytase gene from A. ficuum As3.324 and expressed in P. pastoris GS115, was designated as PHYA II (13). The optimum temperature for the purified PHYA II, which showed two peaks of activity at pHs 2.5 and 4.5, was 40°C and the enzyme was unstable during heating. In this study, we examined whether the thermostability of PHYA II is increased by adding compatible solutes, particularly ectoine . MATERIALS AND METHODS Strain GS115/phyA II4 The phytase gene phyA I of A. ficuum As3.324 was inserted into the plasmid pPIC9K. The recombinant plasmid was transformed in P. pastoris GS115 using an electrical pulse treatment, and the recombinant P. pastoris GS115/phyA II4 expressing PHYA II was obtained (13). PHYA II purification GS115/phyA II4 fermentation was carried out as described previously (13). Crude phytase was fractionated with 80%-saturated ammonium sulfate, and then dialysed in 0.1 M acetate buffer (pH 5.0). The dialysate was applied to an anion exchange column (5 ml, Hitrapsp; Pharmacia, Peapack, NJ,
* Corresponding author. e-mail: [email protected] phone: +86-411-86307727 fax: +86-411-86323646 § Present address: College of Bio & Food Technology, Dalian Institute of Light Industry, 1 Qinggong-Yuan, Ganjingzi District, Dalian 116034, P.R. China. 560
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USA) on a protein-preparing machine (Pharmacia Biotech AKTA explorer, 10 s) for the phytase purification. The apparent molecular weight of phytase was determined by SDS–PAGE at 100 V for 0.5 h. The concentration of the upper gel was 4.5% and that of the lower gel was 10%, with bromophenol and ethanol used as a stain and a decolorant, respectively. Measurement of enzymatic activity The reaction mixture consisted of 0.9 ml of 0.1 M acetate buffer (pH 5.0) containing 1.25 mM phytate, and 0.1 ml of the enzyme solution. After incubation for 15 min at 37°C, the reaction was stopped by adding of 1 ml of 10% trichloroacetic acid. Inorganic phosphate was subsequently analyzed using the method of Ullah (14). One enzyme unit (U) was defined as the amount of enzyme required to liberate 1 nmol of inorganic phosphate in 1 min. Measurement of residual activity PHYA II in 0.1 M acetate buffer (pH 5.0) was incubated in a water bath at designated temperatures (45–100°C) for 15 min, and placed at 4°C for 15 min. The residual PHYA II activity was determined at 37°C using the method described above and shown relative to the activity shown without heat treatment (100%). Measurement of hydrolytic rate The hydrolytic rate (nmol/ min) was defined as the amount (nmol) of inorganic phosphate released from sodium phytate in 1 min of incubation under the designated conditions. Various ectoine concentrations were directly added to 3.75 mM sodium phytate in 0.1 M acetate buffer (pH 5.0), in which the reaction was started by adding 0.1 ml of PHYA II. After heating at 80°C for 15 min, the amount of inorganic phosphate liberated was measured, from which hydrolytic rate was calculated. Relative hydrolytic rate was designated as 100% in the absence of ectoine at 37°C for 15 min. Chemicals Ectoine, which was of >97% purity, was provided by Dainippon Sumitomo Pharma, Tokyo. Mannitol, glycerol, trehalose, glycine betaine, proline and glutamate were purchased from Sigma (St. Louis, MO, USA).
RESULTS AND DISCUSSION Thermostability of PHYA II PHYA II was identified using SDS–PAGE (Fig. 1), in which a single bright band was observed. The apparent molecular weight and specific activity of PHYA II were about 80 kDa and 1.14 ×105 U/mg, respectively. Using the method of Ullah (14), the temperature dependence of PHYA II activity was examined from 35°C to 80°C. As a result, the optimal temperature for PHYA II activity was 40°C. The residual PHYA II activity after heat treatment was measured according to the method described in Materials and Methods. As shown in Fig. 2, the thermostability of PHYA II was almost constant at < 55°C,
FIG. 1. SDS–PAGE of phytase (PHYA II). Lane 1, Molecular weight markers; lane 2, sample of phytase protein.
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FIG. 2. Relative thermostability of phytase. The thermostability of phytase (PHYA II) was determined after the incubation in a water bath at 45–100°C for 15 min, and after being placed at 4°C for 15 min. Activity was determined at 37°C and referred to as the residual PHYA II activity. Activity without heat treatment was designated as 100%.
but it rapidly decreased at temperature > 60°C and was only 11.3% at 90°C. From these findings, PHYA II is regarded as a heat-sensitive enzyme. Supplementation effect of ectoine on the thermostability of PHYA II The supplementation effect of ectoine on the thermostability of PHYA II was determined for heating at 90°C for 15 min, taking the actual heat treatment of phytase during feed pelleting into consideration. As shown in Table 1, the addition of ectoine led to an increase in PHYA II thermostability, i.e., the residual activity reached 83.4% in the presence of 0.5 mM ectoine, which was about 72% higher than that reached in the absence of ectoine when PHYA II was treated at 90°C. Because the temperature of feed pelletTABLE 1. Residual PHYA II activities (%) after heat treatment in the presence of compatible solutesa Ectoine Proline Mannitol Glycerol Trehalose (mM) (M) (M) (mM) (M) 0 0 0 0 0 (11.3 ± 1.3) (11.3 ± 1.3) (11.3 ± 1.3） (11.3 ± 1.3） (11.3 ± 1.3） 0.25 0.3 0.3 0.1 0.4 (70.8 ± 4.4) (77.4 ± 2.7) (21.5± 0.9） (45.6 ±0.8） (13.1 ±0.8） 0.5 0.5 0.5 0.5 0.8 (83.4 ± 3.5) (84.3 ± 0.1) (62.0± 0.3） (53.1 ±2.2） (29.8 ±0.9） 0.75 0.7 0.7 1.0 1.2 (79.1 ± 0.7) (86.2 ± 0.6) (54.8± 0.7） (47.3 ±0.5） (40.3 ±2.0） 1.5 0.9 0.9 5.0 1.6 (46.1 ± 1.7) (81.8 ± 0.5) (51.3± 2.3） (39.1 ±3.5） (20.0 ±1.3） a To examine the supplementation effects of compatible solutes on the thermostability of phytase (PHYA II), proline, mannitol, glycerol, trehalose, glycine betaine, glutamate and ectoine were incubated with PHYA II at 90°C for 15 min, and placed at 4°C for 15 min. PHYA II activity was determined at 37°C and designated as 100% without heat treatment. The values in parentheses are residual PHYA II activities (%). They are the averages ± SD from three separate experiments.
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FIG. 3. Effect of ectoine concentration on hydrolysis using phytase. Hydrolysis using phytase (PHYA II) with different concentrations of ectoine was carried out at 80°C. The reaction was initiated by adding PHYA II and heating the mixture at 80°C for 15 min, and the hydrolytic rate for sodium phytate was measured. The relative hydrolytic rate was designated as 100% and measured without ectoine at 37°C for 15 min.
ing is usually in the range of 85–90°C, the residual phytase activity is quite useful and important for monogastric animals. This result showed that supplemented ectoine efficiently protected and maintained the catalytic activity of phytase during heating, which is a favorable method of processing animal feed. Supplementation effect of ectoine on hydrolysis During the processing of some foods, hydrolyzing phytate with phytase is quite important (15). Thus, the supplementation effect of ectoine on PHYA II during phytate hydrolysis at high temperatures was determined, taking the concomitancy of phytase and phytate in processing into consideration. We tried to examine the supplementation effect of ectoine on the relative hydrolytic rate of PHYA II for sodium phytate at 80°C in the presence of ectoine as an example of phytate hydrolysis at a high temperature. As shown in Fig. 3, the relative hydrolytic rate increased with an increase in ectoine concentration. The rates were 1.6% and 17.3% under conditions without and with 4.5 mM ectoine, respectively. Thus, it can be concluded that adding an appropriate amount of ectoine leads to the maintenance of the activity of PHYA II exposed to a high temperature, i.e., adding an appropriate ectoine concentration results in thermoprotection for the hydrolysis of PHYA II at a high temperature (Fig. 3). According to the results in Table 1 and Fig. 3, the optimal ectoine concentration was 0.5 mM for maintaining the residual PHYA II activity during heating in the absence of sodium phytate, whereas it was 4.5 mM for the direct hydrolysis of
J. BIOSCI. BIOENG.,
FIG. 4. Protective effect of ectoine on thermostability of phytase. Sample I: activity was measured immediately after heat treatment at 90°C for 15 min without ectoine. Sample II: activity was measured after heat treatment at 90°C for 15 min and renaturation at 4°C for 15 min without ectoine. Sample III: before heat treatment at 90°C for 15 min, 0.5 mM ectoine was added to the solution of PHYA II, and then activity was measured immediately. Sample IV: before heat treatment at 90°C for 15 min, 0.5 mM ectoine was added to the solution of PHYA II following renaturation at 4°C for 15 min, and then activity was measured. Sample V: after heat treatment at 90 ℃ for 15 min, 0.5 mM ectoine was added the solution of PHYA II following renaturation at 4°C for 15 min, and then activity was measured. The activity without heat treatment was designated as 100%.
sodium phytate at 80°C. This result indicates that the optimal thermoprotective concentration of ectoine for the direct hydrolysis of sodium phytate for maintainting the residual PHYA II activity is markedly higher than that of ectoine for maintainting the residual PHYA II activity. Thus, we can infer that the optimal thermoprotective concentration of ectoine is influenced by the substrates and products, their concentrations, and temperature. Protective effect of ectoine on thermostability of PHYA II The protective effect of ectoine on the thermal stability of PHYA II was examined. As shown in Fig. 4, the relative activity for sample I remained at 11.3%, which was almost the same as that for sample II (12.0%). In the presence of ectoine supplemented to the reaction mixture prior to the heat treatment, the residual activities were 83.9% and 84.3% for samples III and IV, respectively. These results indicate that ectoine markedly stabilizes the catalytic capacity of the enzyme, showing a seven fold increase compared with that in the case without ectoine. For sample V, where ectoine was added after heating, ectoine was ineffective for maintaining PHYA II activity. Thus, supplementation with ectoine exhibits a stabilizing effect on enzyme catalysis during heat treatment; however, we could not recover the denatured PHYA II. Moreover, renaturation had no effect on the recovery of the activity of the enzyme treated at a high temperature.
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Effect of other compatible solutes on the thermostability of PHYA II The supplementation effects of representative compatible solutes such as proline, mannitol, glycerol, trehalose, glycine betaine, and glutamate were examined at concentrations different from those of ectoine. As shown in Table 1, ectoine and proline were more effective than mannitol, glycerol and trehalose as thermoprotectants of PHYA II, whereas both glycine betaine and glutamate had no thermal protection effect on PHYA II activity (data not shown). When determining thermoprotective efficiencies of additives examined, the addition of 0.7 M proline resulted in the highest residual PHYA II activity (86.2%), but 83.4% was retained at a far lower concentration of ectoine, 0.5 mM (Table 1). In this regard, it is interesting to note that ectoine had a higher thermoprotection efficiency for PHYA II than proline from a comparison of their optimal concentrations. The additions of mannitol, glycerol, and trehalose brought about lower protection efficiencies than those of ectoine and proline. In general, the ideal protectants for enhancing the thermal stability of an enzyme should have the following characteristics: nontoxicity, self-stabilization, and high protective ability at low concentrations. The present data indicates that ectoine possesses such characteristics and is a more suitable thermally stable protectant for PHYA II than the other compounds. Further study should focus in detail on correlation factors and the mechanisms underlying ectoine’s improvement in the thermal stability of phytase and the increase in this enzyme’s catalytic capability at a high temperature. REFERENCES 1. Keshavarz, K.: Nonphytate phosphorus requirement of laying hens with and without phytase on a phase feeding program. Poult. Sci., 79, 748–763 (2000). 2. Stahl, C. H., Roneker, K. R., Thornton, J. R., and Lei, X. G.: A new phytase expressed in yeast effectively improves the bioavailability of phytate phosphorus to weanling pigs. J. Anim. Sci., 78, 668–674 (2000). 3. Ullah, A. H.: Production, rapid purification and catalytic characterization of extracellular phytase from Aspergillus ficuum.
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Prep. Biochem., 18, 43–58 (1988). 4. Rodriguez, E., Wood, Z. A., Karplus, P. A., and Lei, X. G.: Site-directed mutagenesis improves catalytic efficiency and themostability of Escherichia coli pH 2.5 acid phosphatase/ phytase expressed in Pichia pastoris. Arch. Biochem. Biophys, 382, 105–112 (2000). 5. Yanming, H. and Gen, L. X.: Role of glycosylation in the functional expression of an Aspergillus niger phytase (phyA) in Pichia pastoris. Arch. Biochem. Biophys, 364, 83–90 (1999). 6. Randy, M. B., Michael, W. R., Kimberly, M. B., Tony, B., and Alan, V. K.: Molecular characterization and expression of a phytase gene from the thermophilic fungus Thermomyces lanuginosus. Appl. Environ. Microbiol., 64, 4423–4427 (1998). 7. Qu, Y., Bolen, C. L., and Bolen, D. W.: Osmolyte-driven contraction of a random coil protein. Proc. Natl. Acad. Sci. USA, 95, 9268–9273 (1998). 8. Kaushik, J. K. and Bhat, R.: Thermal stability of proteins in aqueous polyol solutions. J. Phys. Chem. B, 102, 7058–7066 (1998). 9. Anjum, F., Rishi, V., and Ahmad, F.: Compatibility of osmolytes with Gibbs energy of stabilization of proteins. Biochim. Biophys. Acta, 1476, 75–84 (2000). 10. Galinski, E. A., Pfeiffer, H. P., and Trüper, H. G.: 1,4,5,6Tetrahydro-2-methyl-4-pyrimi-dine carboxylic acid. A novel cyclic amino acid from halophilic phototrophic bacteria of the genus Ectothiorhodospira. Eur. J. Biochem., 149, 135–139 (1985). 11. Lippert, K. and Galinski, E. A.: Enzyme stabilization be ectoine-type compatible solutes:protection against heating, freezing and drying. Appl. Microbiol. Biotechnol., 37, 61–65 (1992). 12. Schnoor, M., Voss, P., Cullen, P., Boking, T., Galla, H. J., Galinski, E. A., and Lorkowski, S.: Characterization of the synthetic compatible solute homoectoine as a potent PCR enhancer. Biochem. Biophys. Res. Commun., 322, 867–872 (2004). 13. Zhang, L. H., An, L. J., Yuan, X. D., Gao, X. R., and Wang, Y. J.: Expression of gene (phyA I) encoding phytase in Pichia pastorisis and the study on the properties of phytase. J. Dalian Univ. Technol., 42, 294–300 (2002). 14. Ullah, A. H. J.: Aspergillus ficuum phytase: partial primary structure, substrate selectivity, and kinetic characterization. Prep. Biochem., 18, 459–471 (1988). 15. Han, Y. W. and Wilfred, A. G.: Phytate hydrolysis in soybean and cottonseed meals by Aspergillus ficuum phytase. J. Agric. Food Chem., 36, 259–262 (1988).
© 2006, The Society for Biotechnology, Japan
Supplementation Effect of Ectoine on Thermostability of Phytase Linghua Zhang,1§* Yue Wang,1,2 Chunyu Zhang,1 Yunji Wang,2 Daochen Zhu,3 Chenxiang Wang,3 and Shinichi Nagata3 College of Environmental Science and Engineering, Dalian Maritime University, Lingshui Road, Dalian 116026, P.R. China,1 College of Bio & Food Technology, Dalian Institute of Light Industry, 1 Qinggong-Yuan, Ganjingzi District, Dalian 116034, P.R. China,2 and Environmental Biochemistry Division, Research Center for Inland Seas, Kobe University, 5-1-1 Fukae, Higashinada-ku, Kobe 658-0022, Japan3 Received 3 July 2006/Accepted 26 September 2006
In this study, we elucidated the supplementation effect of compatible solutes on the thermostability of phytase, designated as PHYA II, which was encoded by the phytase gene phyA I (GeneBank AY013315) from Aspergillus ficuum As3.324 and expressed in Pichia pastoris GS115. When PHYA II in acetate buffer was heated at 90°C for 15 min, more than 80% of the residual activity was retained by adding the cyclic amino acid ectoine, a representative compatible solute. Furthermore, the presence of ectoine led to an increase in the relative hydrolytic rate of sodium phytate by 15.7% with heating at 80°C for 15 min. Among the compatible solutes examined, ectoine was confirmed to be the most efficient thermoprotectant for PHYA II. [Key words: phytase, thermostability, ectoine, hydrolytic rate, denaturation]
Phytase (EC 3.1.3.8) is a phosphomonoesterase that hydrolyzes phytate and releases inorganic phosphate, which can be directly ingested by monogastric animals. Thus, it is important not only to increase the availability of phytate-P in animal feed, but also to decrease the amount of environmental pollution caused by phosphorus contents in manure from nonruminant livestock and poultry (1, 2). Because the activity of phytase usually decreases during feed pelleting (85– 90°C) by 30% or more, it is important to enhance the thermostability of phytase (3). Some studies have been performed for the improvement of phytase thermostability as follows: phytase genes were reconstructed using site-directed mutagenesis (4); a thermostable phytase molecule was newly established by exploiting the consensus technique (Lehmann, M., EP patent 0897985 A2, 1999); phytase was modified through glycosylation (5); and thermostable strains producing phytases were isolated from various environments (6). In addition, protectants or stabilizers such as metals, polyols or other solutes have been applied to increase the thermostability of this enzyme (7–9). The cyclic amino acid ectoine (1,4,5,6-tetrahydro-2-methyl-4-pyrimidine carboxylic acid), one of the representative compatible solutes, accumulates in halophilic and halotolerant bacteria in hypersaline environments, the role of which is to establish a balance between the osmotic pressures inside and outside the cells (10). Previous studies indicated
that ectoine functions on proteins as a stabilizer against some adverse conditions. For example, Lippert and Galinski (11) reported that the thermal stabilities of lactate dehydrogenase and phosphofructokinase in the presence of ectoine increase. Ectoine was also been shown to lower the melting temperature of dsDNA in PCR application (12). In our previous study (13), the phytase gene phyA I (GeneBank AY013315) from Aspergillus ficuum As3.324 was inserted into the plasmid pPIC9K, which was 1515 bp in length, contained an intron of 111 nucleotides and encoded 467 amino acids. The recombinant plasmid was transformed in Pichia pastoris GS115 using an electrical pulse treatment. Phytase, which was encoded by phytase gene from A. ficuum As3.324 and expressed in P. pastoris GS115, was designated as PHYA II (13). The optimum temperature for the purified PHYA II, which showed two peaks of activity at pHs 2.5 and 4.5, was 40°C and the enzyme was unstable during heating. In this study, we examined whether the thermostability of PHYA II is increased by adding compatible solutes, particularly ectoine . MATERIALS AND METHODS Strain GS115/phyA II4 The phytase gene phyA I of A. ficuum As3.324 was inserted into the plasmid pPIC9K. The recombinant plasmid was transformed in P. pastoris GS115 using an electrical pulse treatment, and the recombinant P. pastoris GS115/phyA II4 expressing PHYA II was obtained (13). PHYA II purification GS115/phyA II4 fermentation was carried out as described previously (13). Crude phytase was fractionated with 80%-saturated ammonium sulfate, and then dialysed in 0.1 M acetate buffer (pH 5.0). The dialysate was applied to an anion exchange column (5 ml, Hitrapsp; Pharmacia, Peapack, NJ,
* Corresponding author. e-mail: [email protected] phone: +86-411-86307727 fax: +86-411-86323646 § Present address: College of Bio & Food Technology, Dalian Institute of Light Industry, 1 Qinggong-Yuan, Ganjingzi District, Dalian 116034, P.R. China. 560
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USA) on a protein-preparing machine (Pharmacia Biotech AKTA explorer, 10 s) for the phytase purification. The apparent molecular weight of phytase was determined by SDS–PAGE at 100 V for 0.5 h. The concentration of the upper gel was 4.5% and that of the lower gel was 10%, with bromophenol and ethanol used as a stain and a decolorant, respectively. Measurement of enzymatic activity The reaction mixture consisted of 0.9 ml of 0.1 M acetate buffer (pH 5.0) containing 1.25 mM phytate, and 0.1 ml of the enzyme solution. After incubation for 15 min at 37°C, the reaction was stopped by adding of 1 ml of 10% trichloroacetic acid. Inorganic phosphate was subsequently analyzed using the method of Ullah (14). One enzyme unit (U) was defined as the amount of enzyme required to liberate 1 nmol of inorganic phosphate in 1 min. Measurement of residual activity PHYA II in 0.1 M acetate buffer (pH 5.0) was incubated in a water bath at designated temperatures (45–100°C) for 15 min, and placed at 4°C for 15 min. The residual PHYA II activity was determined at 37°C using the method described above and shown relative to the activity shown without heat treatment (100%). Measurement of hydrolytic rate The hydrolytic rate (nmol/ min) was defined as the amount (nmol) of inorganic phosphate released from sodium phytate in 1 min of incubation under the designated conditions. Various ectoine concentrations were directly added to 3.75 mM sodium phytate in 0.1 M acetate buffer (pH 5.0), in which the reaction was started by adding 0.1 ml of PHYA II. After heating at 80°C for 15 min, the amount of inorganic phosphate liberated was measured, from which hydrolytic rate was calculated. Relative hydrolytic rate was designated as 100% in the absence of ectoine at 37°C for 15 min. Chemicals Ectoine, which was of >97% purity, was provided by Dainippon Sumitomo Pharma, Tokyo. Mannitol, glycerol, trehalose, glycine betaine, proline and glutamate were purchased from Sigma (St. Louis, MO, USA).
RESULTS AND DISCUSSION Thermostability of PHYA II PHYA II was identified using SDS–PAGE (Fig. 1), in which a single bright band was observed. The apparent molecular weight and specific activity of PHYA II were about 80 kDa and 1.14 ×105 U/mg, respectively. Using the method of Ullah (14), the temperature dependence of PHYA II activity was examined from 35°C to 80°C. As a result, the optimal temperature for PHYA II activity was 40°C. The residual PHYA II activity after heat treatment was measured according to the method described in Materials and Methods. As shown in Fig. 2, the thermostability of PHYA II was almost constant at < 55°C,
FIG. 1. SDS–PAGE of phytase (PHYA II). Lane 1, Molecular weight markers; lane 2, sample of phytase protein.
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FIG. 2. Relative thermostability of phytase. The thermostability of phytase (PHYA II) was determined after the incubation in a water bath at 45–100°C for 15 min, and after being placed at 4°C for 15 min. Activity was determined at 37°C and referred to as the residual PHYA II activity. Activity without heat treatment was designated as 100%.
but it rapidly decreased at temperature > 60°C and was only 11.3% at 90°C. From these findings, PHYA II is regarded as a heat-sensitive enzyme. Supplementation effect of ectoine on the thermostability of PHYA II The supplementation effect of ectoine on the thermostability of PHYA II was determined for heating at 90°C for 15 min, taking the actual heat treatment of phytase during feed pelleting into consideration. As shown in Table 1, the addition of ectoine led to an increase in PHYA II thermostability, i.e., the residual activity reached 83.4% in the presence of 0.5 mM ectoine, which was about 72% higher than that reached in the absence of ectoine when PHYA II was treated at 90°C. Because the temperature of feed pelletTABLE 1. Residual PHYA II activities (%) after heat treatment in the presence of compatible solutesa Ectoine Proline Mannitol Glycerol Trehalose (mM) (M) (M) (mM) (M) 0 0 0 0 0 (11.3 ± 1.3) (11.3 ± 1.3) (11.3 ± 1.3） (11.3 ± 1.3） (11.3 ± 1.3） 0.25 0.3 0.3 0.1 0.4 (70.8 ± 4.4) (77.4 ± 2.7) (21.5± 0.9） (45.6 ±0.8） (13.1 ±0.8） 0.5 0.5 0.5 0.5 0.8 (83.4 ± 3.5) (84.3 ± 0.1) (62.0± 0.3） (53.1 ±2.2） (29.8 ±0.9） 0.75 0.7 0.7 1.0 1.2 (79.1 ± 0.7) (86.2 ± 0.6) (54.8± 0.7） (47.3 ±0.5） (40.3 ±2.0） 1.5 0.9 0.9 5.0 1.6 (46.1 ± 1.7) (81.8 ± 0.5) (51.3± 2.3） (39.1 ±3.5） (20.0 ±1.3） a To examine the supplementation effects of compatible solutes on the thermostability of phytase (PHYA II), proline, mannitol, glycerol, trehalose, glycine betaine, glutamate and ectoine were incubated with PHYA II at 90°C for 15 min, and placed at 4°C for 15 min. PHYA II activity was determined at 37°C and designated as 100% without heat treatment. The values in parentheses are residual PHYA II activities (%). They are the averages ± SD from three separate experiments.
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FIG. 3. Effect of ectoine concentration on hydrolysis using phytase. Hydrolysis using phytase (PHYA II) with different concentrations of ectoine was carried out at 80°C. The reaction was initiated by adding PHYA II and heating the mixture at 80°C for 15 min, and the hydrolytic rate for sodium phytate was measured. The relative hydrolytic rate was designated as 100% and measured without ectoine at 37°C for 15 min.
ing is usually in the range of 85–90°C, the residual phytase activity is quite useful and important for monogastric animals. This result showed that supplemented ectoine efficiently protected and maintained the catalytic activity of phytase during heating, which is a favorable method of processing animal feed. Supplementation effect of ectoine on hydrolysis During the processing of some foods, hydrolyzing phytate with phytase is quite important (15). Thus, the supplementation effect of ectoine on PHYA II during phytate hydrolysis at high temperatures was determined, taking the concomitancy of phytase and phytate in processing into consideration. We tried to examine the supplementation effect of ectoine on the relative hydrolytic rate of PHYA II for sodium phytate at 80°C in the presence of ectoine as an example of phytate hydrolysis at a high temperature. As shown in Fig. 3, the relative hydrolytic rate increased with an increase in ectoine concentration. The rates were 1.6% and 17.3% under conditions without and with 4.5 mM ectoine, respectively. Thus, it can be concluded that adding an appropriate amount of ectoine leads to the maintenance of the activity of PHYA II exposed to a high temperature, i.e., adding an appropriate ectoine concentration results in thermoprotection for the hydrolysis of PHYA II at a high temperature (Fig. 3). According to the results in Table 1 and Fig. 3, the optimal ectoine concentration was 0.5 mM for maintaining the residual PHYA II activity during heating in the absence of sodium phytate, whereas it was 4.5 mM for the direct hydrolysis of
J. BIOSCI. BIOENG.,
FIG. 4. Protective effect of ectoine on thermostability of phytase. Sample I: activity was measured immediately after heat treatment at 90°C for 15 min without ectoine. Sample II: activity was measured after heat treatment at 90°C for 15 min and renaturation at 4°C for 15 min without ectoine. Sample III: before heat treatment at 90°C for 15 min, 0.5 mM ectoine was added to the solution of PHYA II, and then activity was measured immediately. Sample IV: before heat treatment at 90°C for 15 min, 0.5 mM ectoine was added to the solution of PHYA II following renaturation at 4°C for 15 min, and then activity was measured. Sample V: after heat treatment at 90 ℃ for 15 min, 0.5 mM ectoine was added the solution of PHYA II following renaturation at 4°C for 15 min, and then activity was measured. The activity without heat treatment was designated as 100%.
sodium phytate at 80°C. This result indicates that the optimal thermoprotective concentration of ectoine for the direct hydrolysis of sodium phytate for maintainting the residual PHYA II activity is markedly higher than that of ectoine for maintainting the residual PHYA II activity. Thus, we can infer that the optimal thermoprotective concentration of ectoine is influenced by the substrates and products, their concentrations, and temperature. Protective effect of ectoine on thermostability of PHYA II The protective effect of ectoine on the thermal stability of PHYA II was examined. As shown in Fig. 4, the relative activity for sample I remained at 11.3%, which was almost the same as that for sample II (12.0%). In the presence of ectoine supplemented to the reaction mixture prior to the heat treatment, the residual activities were 83.9% and 84.3% for samples III and IV, respectively. These results indicate that ectoine markedly stabilizes the catalytic capacity of the enzyme, showing a seven fold increase compared with that in the case without ectoine. For sample V, where ectoine was added after heating, ectoine was ineffective for maintaining PHYA II activity. Thus, supplementation with ectoine exhibits a stabilizing effect on enzyme catalysis during heat treatment; however, we could not recover the denatured PHYA II. Moreover, renaturation had no effect on the recovery of the activity of the enzyme treated at a high temperature.
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EFFECT OF ECTOINE ON PHYTASE THERMOSTABILITY
Effect of other compatible solutes on the thermostability of PHYA II The supplementation effects of representative compatible solutes such as proline, mannitol, glycerol, trehalose, glycine betaine, and glutamate were examined at concentrations different from those of ectoine. As shown in Table 1, ectoine and proline were more effective than mannitol, glycerol and trehalose as thermoprotectants of PHYA II, whereas both glycine betaine and glutamate had no thermal protection effect on PHYA II activity (data not shown). When determining thermoprotective efficiencies of additives examined, the addition of 0.7 M proline resulted in the highest residual PHYA II activity (86.2%), but 83.4% was retained at a far lower concentration of ectoine, 0.5 mM (Table 1). In this regard, it is interesting to note that ectoine had a higher thermoprotection efficiency for PHYA II than proline from a comparison of their optimal concentrations. The additions of mannitol, glycerol, and trehalose brought about lower protection efficiencies than those of ectoine and proline. In general, the ideal protectants for enhancing the thermal stability of an enzyme should have the following characteristics: nontoxicity, self-stabilization, and high protective ability at low concentrations. The present data indicates that ectoine possesses such characteristics and is a more suitable thermally stable protectant for PHYA II than the other compounds. Further study should focus in detail on correlation factors and the mechanisms underlying ectoine’s improvement in the thermal stability of phytase and the increase in this enzyme’s catalytic capability at a high temperature. REFERENCES 1. Keshavarz, K.: Nonphytate phosphorus requirement of laying hens with and without phytase on a phase feeding program. Poult. Sci., 79, 748–763 (2000). 2. Stahl, C. H., Roneker, K. R., Thornton, J. R., and Lei, X. G.: A new phytase expressed in yeast effectively improves the bioavailability of phytate phosphorus to weanling pigs. J. Anim. Sci., 78, 668–674 (2000). 3. Ullah, A. H.: Production, rapid purification and catalytic characterization of extracellular phytase from Aspergillus ficuum.
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