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Properties of A. ficuum AS3.324 phytase expressed in tobacco
Process Biochemistry 40 (2005) 213–216
Properties of A. ficuum AS3.324 phytase expressed in tobacco Linghua Zhang a,b , Lijia An a , Xiaorong Gao a , Yunji Wang b,∗ a
Department of Bioengineering, School of Chemical Engineering, Dalian University of Technology, Dalian 116012, PR China b College of Bio & Food Technology, Dalian Institute of Light Industry, Dalian 116034, PR China Received 26 June 2003; received in revised form 20 November 2003; accepted 15 December 2003
Abstract Phytase gene phyAI of A. ficuum AS3.324 was transformed into tobacco NC89, resulting in transgenic tobacco and phytase was expressed nonspecifically in tissue of tobacco. The amount of phytase protein expressed accounted for 17.6% of the total soluble protein of tobacco leaves (growing for 8 weeks). The transgenic phytase in tobacco was compared with A. ficuum AS3.324 and was found to have the same specific activity, pH, temperature adaptability and substrate specificity. The differences between the two were as follows: theoretical molecular weight of phytase coded by phyAI was 49.2 kDa, apparent molecular weights of A. ficuum AS3.324 and transgenic tobacco phytase were 68.5 and 76.0 kDa, respectively. Thermal stability of transgenic tobacco phytase was higher than A. ficuum AS3.324 phytase. After 80 ◦ C 15 min heat treatment, the residual enzyme activity of the transgenic tobacco phytase was 4.6 times more than that of the A. ficuum AS3.324 phytase. © 2004 Elsevier Ltd. All rights reserved. Keywords: Phytase; Tobacco; A. ficuum AS3.324; Thermostability; Phytase properties
1. Introduction Sixty to eighty percentage of the total phosphorus presented in crops such as grains, beans, oil-bearing crops is in the form of phytate-phosphorus. The utilization rate of phosphorus in phytate by monogastric animals ranges from 0 to 40% [1]. Phytase EC.3.1.3.8 hydrolyzes phytate into inositol and phosphoric acid in animals both in vivo and in vitro, and therefore, phytase has substituted mineral phosphorus as a supplement to feed [2]. By means of genetic engineering technology, phytase expressed in plants should greatly increase the utilization rate of phosphorus. Thus much attention has been paid to the plant genetic engineering of phytase [3]. Verwoerd et al. [4] transformed the A. niger phytase gene into tobacco and demonstrated its activity in tobacco tissue. The A. niger phytase gene was engineered into soybean (Glycine max) seeds and secretion of active recombinant phytase was detected in the soybean cell-suspension cultures [5]. However, the enzymic properties of microbial phytase expressed in plants have not been reported.
The paper investigates the transformation of A. ficuum AS3.324 phytase into tobacco and the expression level expressed in tobacco, with its attention focused on the enzymatic properties of the recombinant phytase.
2. Materials and methods 2.1. Gene, plasmid, and expression host Cloning vector pMD18-T carrying A. ficuum AS3.324 phytase gene phyAI (pMD18-T/phyAI) was constructed in the laboratory by the author. The gene sequence “phyAI” was shown on the record as GenBank Accession: AY013315. Plasmid pUC19, pMD18-T and E. coli JM109, restriction endonucleases, DNA polymerase were all bought from TaKaRa Corporation Ltd., Dalian Branch. Plasmid pBI121 and Agrobacterium tumefaciens LBA4404 and tobacco (Nicotiana tabacum L. cv.) NC89 were all available in the laboratory. 2.2. Transformation of the phyAI gene into tobacco
∗ Corresponding author. Tel.: +86-411-6307727; fax: +86-411-6323671. E-mail address: [email protected] (Y. Wang).
0032-9592/$ – see front matter © 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.procbio.2003.12.005
pMD18-T/phyAI was cleaved with SmaI and XbaI and then blunt-ended. Plant expression vector pBI121 was cleaved by SmaI. The digested pMD18-T/phyAI and pBI121
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L. Zhang et al. / Process Biochemistry 40 (2005) 213–216
are connected by T4 DNA ligase. The plasmid pBI121 carrying phyAI (pBI121/phyAI) with correct orientation were selected by PCR and introduced into E. coli JM109 competent cells. The pBI121/phyAI was extracted from E. coli JM109 and was transformed into A. tumefaciens LBA4404, thus obtaining A. tumefaciens LBA4404 with phyAI(LBA4404/phyAI). The tobacco NC89 leaf discs were transformed using LBA4404/phyAI according to the method described by Wang [6]. Transformants was confirmed by PCR and analyzed by Southern blotting [7]. 2.3. Purification of phytase NC89/phyAI leaf discs were ground in 0.1 M HAC–NaAC buffer at pH 5.5. Supernatant was obtained by centrifugation. The crude phytase preparation was fractionated with 80% saturated ammonium sulfate. The pellet collected after a second centrifugation was dialyzed against 0.1 M HAC–NaAC at pH 5.0 by using an ultrafiltration membrane (Amicon YM-10). The concentration was applied to an anion exchange column (5 ml Hitrapsp, Pharmacia) on a protein preparing machine (Pharmacia Biotech AKTA explorer 10s) to purify phytase protein. Phytase apparent molecular weight was monitored by SDS-polyacrylamide gel electrophoresis. 2.4. Enzyme activity and properties One unit (U, mol/mg) of phytase activity was defined as the amount of phytase required to hydrolyze sodium phytate and to produce 1 mol of phosphorus per min at 37 ◦ C and pH 5.5. Phytase activity was measured as described by Ullah [8]. pH activity profiles were determined at different levels of pH (pH 1.0–8.0) of a sodium acetate buffer at 37 ◦ C for 10 min. To obtain the optimum temperature, the enzyme activity was measured at different temperature (35–70 ◦ C) at pH 5.5 for 10 min. For thermal stability assays, phytase was incubated for 15 min in a water bath at temperatures 55–80 ◦ C. After the heat treatment, the samples were stored at 4 ◦ C for 15 min or until activity assays were performed. And the standard activity assay was performed as described previously. To determine Km and Vmax values, 2.7 × 10−5 mM phytase was incubated with different substrate concentrations ranging from 8 × 10−6 to 800 × 10−6 M. The Km and Vmax values were determined from a double reciprocal plot of the data.
Fig. 1. Identification of the transgenic tobacco by PCR. 1: marker DL-2000; 2: negative control, nontransgenic NC89; 3–13: NC89 transformed by phyAI; 14: positive control phyAI.
cef (500 mg/l) + Km (300 mg/l)), calli were formed after 15 days, and were differentiated into buds that could be regenerated and moved to rooting medium (MS + NAA (0.2 mg/l) + Km (100 mg/l)) to form plantlets containing km (Kanamycin) resistance. For confirmation of transgenic tobacco by PCR, genomic DNA was isolated from transformed tobacco leaves [9]. Agarose gel electrophoresis revealed a specific band at 1.4 kb (Fig. 1). Two transformed plants, which were tested as positive by PCR, were selected and were used to perform Southern blot analysis. The results showed that the phytase gene was integrated into the genome of NC89 plants (Fig. 2). 3.2. Expression of phyAI in tobacco Phytase protein from transgenic tobacco leaf discs were separated and purified with the method shown in Section 2.3. Phytase protein was separated by SDS-polyacrylamide gel electrophoresis (Fig. 3). The apparent molecular weight of phytase was 76 kDa, and the specific activity was approximately 109 U/mg. The amount of expressed phytase increased with extension of time (data not shown). Until the eighth week, phytase protein accounted for 17.6% of tobacco
3. Results 3.1. Transgenic tobacco The phyAI gene was transformed into tobacco (NC89) leaf discs (as shown in Section 2.2). At 26 ◦ C under screen medium (MS + 6BA (1 mg/l) + NAA (0.1 mg/l) +
Fig. 2. Southern blotting analysis of the transgenic tobacco. 1: marker DL-2000; 2: positive control phyAI; 3: transgenic tobacco genomic DNA digested by EcoRI/PstI; 4: transgenic tobacco genomic DNA digested by EcoRI/PstI; 5: NC89 genomic DNA digested by EcoRI/PstI.
L. Zhang et al. / Process Biochemistry 40 (2005) 213–216
215
Fig. 3. SDS-polyacrylamide gel electrophoresis of transgenic tobacco phytase. 1: marker protein molecular weight; 2: sample of phytase protein. Fig. 5. The optimum temperature of enzyme.
Fig. 6. The most ability of phytase. Fig. 4. The optimum pH of enzyme.
total soluble protein. Phytase expressed in wild type strain A. ficuum AS3.324 accounted for 0.2% of fermentation total liquid protein only. While the amount of phytase expressed in tobacco increased by a large margin. The phenomenon of accumulating phytase expressed by tobacco indicates that phytase was stable in the tissues of tobacco. 3.3. Properties of transgenic tobacco phytase Properties of transgenic tobacco phytase were determined according to the method described in Section 2.4. Test result showed the peak of phytase activity appeared at pH 2.0 and 5.5 (Fig. 4). The optimum temperature was 50 ◦ C (Fig. 5). Thermal stability of phytase is shown in Fig. 6. The optimum pH and the optimum temperature of transgenic tobacco phytase were the same as wild type strain A. ficuum AS3.324 on the whole, but thermal stability of phytase was different from each other. When phytase expressed by tobacco was treated under different temperatures, the residual enzyme activity improved in different degrees comparing with that of
wild type strain A. ficuum AS3.324. For instance, after 80 ◦ C 15 min heat treatment, residual enzyme activity was 25.1%, and under the same condition, residual phytase activity of A. ficuum AS3.324 was 5.5%. The Km and Kcat values of transgenic tobacco phytase were determined with sodium phytate and calcium phytate as substrate separately and according to the method described in Section 2.4. The results were shown in Table 1. As shown in Table 1, phytase was expressed by two hosts (A. ficuum AS3.324 and tobacco NC89), the Km and Kcat values shown no significant differences under the same substrate, while for the substrate of sodium phytate and calcium phytate, there were significant differences in order of magnitude. The result shows both A. ficuum AS3.324 phytase and transgenic tobacco phytase had higher substrate specificity and catalytic efficiency with sodium phytate as substrate than with calcium phytate. Both phytases had different substrate specificity and catalytic efficiency under all kinds of phytate as substrate, while they had no hydrolytic ability on other compounds with a phosphoester bond (data not shown).
Table 1 The kinetic parameters of phytase Sample
Sodium phytate Km (mol/l)
AS3.324 Tobacco
10−4
7.5 × 7.3 × 10−4
Calcium phytate Kcat
(s−1 )
9.4 × 1.2 × 104 103
Kcat /Km (l/mol s) 1.3 × 1.6 × 107 107
Km (mol/l)
Kcat (s−1 )
Kcat /Km (l/mol s)
10−3
3.4 × 5.1 × 103
2.8 × 106 3.9 × 106
1.2 × 1.3 × 10−3
103
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L. Zhang et al. / Process Biochemistry 40 (2005) 213–216
4. Discussions The phytase gene phyAI of A. ficuum AS3.324 was transformed into tobacco NC89 by way of A. tumefaciens introduction, resulting in transgenic tobacco. Phytase was expressed nonspecifically in the tissues of tobacco. When the transgenic tobacco phytase with A. ficuum AS3.324 were compared, they had the same specific activity, pH, temperature adaptability and substrate specificity. The differences between the two are as follows: The amount of phyAI expressed in host A. ficuum AS3.324 was very low, and enzyme activity in the fermentation supernatant was only 8.2 U (nmol/ml), accounting for 0.2% of total secreted protein. The amount of phytase expressed in tobacco increased by a large margin. Phytase protein accounted for 17.6% of total soluble protein in the growing tobacco leaves in 8 weeks. Phytase had various degrees of expression in leaf, stem, root and seed of tobacco. The expression amount in the blade was the highest. In addition, phytase was stable in tobacco and was in the state of accumulation. Thermal stability of transgenic tobacco phytase was higher than A. ficuum AS3.324 phytase. Among the heat treatment range of 55–80 ◦ C, the average residual enzyme activity of the former was 2.9 times more than that of the latter. The difference between molecular weight determined by SDS-PAGE (apparent molecular weight) and the molecular weight calculated by using the amino acid sequence (theoretical molecular weight) is an indication of the extent of glycosylation of a protein [10]. Theoretical molecular weight of phytase coded by phyAI was 49.2 kDa. Apparent molecular weights of A. ficuum AS3.324 and transgenic tobacco phytase were 68.5 and 76.0 kDa, respectively. There are 10 potential N-glycosylation sites in the phyAI gene. The difference at thermal stability between the two kinds of phytase expressed by two hosts separately, might be related
to the extent and patterns of glycosylation [10,11]. But the thermal stability of the recombinant phytase cannot totally meet the demand of industry processes yet. Researchers hope to improve its thermal stability through protein engineering.
References [1] Keshavarz K. Nonphytate phosphorus requirement of laying hens with and without phytase on a phase feeding program. Poult Sci 2000;79(5):748–63. [2] Stahl CH, Roneker KR, Thornton JR, Lei XG. A new phytase expressed in yeast effectively improves the bilavailability of phytate phosphorus to weanling pigs. J Anim Sci 2000;78(3):668–74. [3] Yao B, Zhang Y-C, Wang J-H, et al. Isolation of Aspergillus niger 963 with phytase activity and cloing its phytase gene phyA2 [J]. J Agric Biotechnol 1998;6(1):1–6. [4] Verwoerd TC, van Paridon PA, Ooyen AJ, et al. Stable accumulation of Aspergillus niger phase transgenic tobacco leaves. Plant Physiol 1995;109:1199–205. [5] Li J, Carla E. Secretion of active recombinant phytase from soybean cell-suspension cultures [J]. Plant Physiol 1997;114:1103–11. [6] Wang G. Plant gene engineering principle and technology. Scientific Publishing Company, Beijing, 1998. p. 552–5. [7] Sambrook J, Fritsch EF, Maniatis T. Molecular cloning: a laboratory manual, vols. 323–324. 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, 1989. p. 602–14. [8] Ullah AHJ. Aspergillus ficuum phytase: partial primary structure, substrate selectivity and kinetic characterization. Prep Biochem 1988;18(4):459–71. [9] Zhang L-L, Zhang L-H, Wang Y-J. Isolation and molecular biological analyses of genomic DNAs from fungi using benzyl chloride. J Dalian Inst Light Ind 2000;19:36–9 [in Chinese with English abstract]. [10] Wyss M, Pasamontes L, Friedlein A, Remy R, Tessier M, Kroneberger A, et al. Biophysical characterization of fungal phytases (myo-inositol hexakisphosphate phosphohytrolases): molecular size glycosylation pattern, and engineering of proteolytic resistance. Appl Environ Microbiol 1999;65(2):359–66. [11] Han Y, Lei X-G. Role of glycosylation in the functional expression of an Aspergillus niger phytase(phyA) in Pichia Pastiris. Arch Biochem Biophys 1999;364(1):83–90.
Properties of A. ficuum AS3.324 phytase expressed in tobacco Linghua Zhang a,b , Lijia An a , Xiaorong Gao a , Yunji Wang b,∗ a
Department of Bioengineering, School of Chemical Engineering, Dalian University of Technology, Dalian 116012, PR China b College of Bio & Food Technology, Dalian Institute of Light Industry, Dalian 116034, PR China Received 26 June 2003; received in revised form 20 November 2003; accepted 15 December 2003
Abstract Phytase gene phyAI of A. ficuum AS3.324 was transformed into tobacco NC89, resulting in transgenic tobacco and phytase was expressed nonspecifically in tissue of tobacco. The amount of phytase protein expressed accounted for 17.6% of the total soluble protein of tobacco leaves (growing for 8 weeks). The transgenic phytase in tobacco was compared with A. ficuum AS3.324 and was found to have the same specific activity, pH, temperature adaptability and substrate specificity. The differences between the two were as follows: theoretical molecular weight of phytase coded by phyAI was 49.2 kDa, apparent molecular weights of A. ficuum AS3.324 and transgenic tobacco phytase were 68.5 and 76.0 kDa, respectively. Thermal stability of transgenic tobacco phytase was higher than A. ficuum AS3.324 phytase. After 80 ◦ C 15 min heat treatment, the residual enzyme activity of the transgenic tobacco phytase was 4.6 times more than that of the A. ficuum AS3.324 phytase. © 2004 Elsevier Ltd. All rights reserved. Keywords: Phytase; Tobacco; A. ficuum AS3.324; Thermostability; Phytase properties
1. Introduction Sixty to eighty percentage of the total phosphorus presented in crops such as grains, beans, oil-bearing crops is in the form of phytate-phosphorus. The utilization rate of phosphorus in phytate by monogastric animals ranges from 0 to 40% [1]. Phytase EC.3.1.3.8 hydrolyzes phytate into inositol and phosphoric acid in animals both in vivo and in vitro, and therefore, phytase has substituted mineral phosphorus as a supplement to feed [2]. By means of genetic engineering technology, phytase expressed in plants should greatly increase the utilization rate of phosphorus. Thus much attention has been paid to the plant genetic engineering of phytase [3]. Verwoerd et al. [4] transformed the A. niger phytase gene into tobacco and demonstrated its activity in tobacco tissue. The A. niger phytase gene was engineered into soybean (Glycine max) seeds and secretion of active recombinant phytase was detected in the soybean cell-suspension cultures [5]. However, the enzymic properties of microbial phytase expressed in plants have not been reported.
The paper investigates the transformation of A. ficuum AS3.324 phytase into tobacco and the expression level expressed in tobacco, with its attention focused on the enzymatic properties of the recombinant phytase.
2. Materials and methods 2.1. Gene, plasmid, and expression host Cloning vector pMD18-T carrying A. ficuum AS3.324 phytase gene phyAI (pMD18-T/phyAI) was constructed in the laboratory by the author. The gene sequence “phyAI” was shown on the record as GenBank Accession: AY013315. Plasmid pUC19, pMD18-T and E. coli JM109, restriction endonucleases, DNA polymerase were all bought from TaKaRa Corporation Ltd., Dalian Branch. Plasmid pBI121 and Agrobacterium tumefaciens LBA4404 and tobacco (Nicotiana tabacum L. cv.) NC89 were all available in the laboratory. 2.2. Transformation of the phyAI gene into tobacco
∗ Corresponding author. Tel.: +86-411-6307727; fax: +86-411-6323671. E-mail address: [email protected] (Y. Wang).
0032-9592/$ – see front matter © 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.procbio.2003.12.005
pMD18-T/phyAI was cleaved with SmaI and XbaI and then blunt-ended. Plant expression vector pBI121 was cleaved by SmaI. The digested pMD18-T/phyAI and pBI121
214
L. Zhang et al. / Process Biochemistry 40 (2005) 213–216
are connected by T4 DNA ligase. The plasmid pBI121 carrying phyAI (pBI121/phyAI) with correct orientation were selected by PCR and introduced into E. coli JM109 competent cells. The pBI121/phyAI was extracted from E. coli JM109 and was transformed into A. tumefaciens LBA4404, thus obtaining A. tumefaciens LBA4404 with phyAI(LBA4404/phyAI). The tobacco NC89 leaf discs were transformed using LBA4404/phyAI according to the method described by Wang [6]. Transformants was confirmed by PCR and analyzed by Southern blotting [7]. 2.3. Purification of phytase NC89/phyAI leaf discs were ground in 0.1 M HAC–NaAC buffer at pH 5.5. Supernatant was obtained by centrifugation. The crude phytase preparation was fractionated with 80% saturated ammonium sulfate. The pellet collected after a second centrifugation was dialyzed against 0.1 M HAC–NaAC at pH 5.0 by using an ultrafiltration membrane (Amicon YM-10). The concentration was applied to an anion exchange column (5 ml Hitrapsp, Pharmacia) on a protein preparing machine (Pharmacia Biotech AKTA explorer 10s) to purify phytase protein. Phytase apparent molecular weight was monitored by SDS-polyacrylamide gel electrophoresis. 2.4. Enzyme activity and properties One unit (U, mol/mg) of phytase activity was defined as the amount of phytase required to hydrolyze sodium phytate and to produce 1 mol of phosphorus per min at 37 ◦ C and pH 5.5. Phytase activity was measured as described by Ullah [8]. pH activity profiles were determined at different levels of pH (pH 1.0–8.0) of a sodium acetate buffer at 37 ◦ C for 10 min. To obtain the optimum temperature, the enzyme activity was measured at different temperature (35–70 ◦ C) at pH 5.5 for 10 min. For thermal stability assays, phytase was incubated for 15 min in a water bath at temperatures 55–80 ◦ C. After the heat treatment, the samples were stored at 4 ◦ C for 15 min or until activity assays were performed. And the standard activity assay was performed as described previously. To determine Km and Vmax values, 2.7 × 10−5 mM phytase was incubated with different substrate concentrations ranging from 8 × 10−6 to 800 × 10−6 M. The Km and Vmax values were determined from a double reciprocal plot of the data.
Fig. 1. Identification of the transgenic tobacco by PCR. 1: marker DL-2000; 2: negative control, nontransgenic NC89; 3–13: NC89 transformed by phyAI; 14: positive control phyAI.
cef (500 mg/l) + Km (300 mg/l)), calli were formed after 15 days, and were differentiated into buds that could be regenerated and moved to rooting medium (MS + NAA (0.2 mg/l) + Km (100 mg/l)) to form plantlets containing km (Kanamycin) resistance. For confirmation of transgenic tobacco by PCR, genomic DNA was isolated from transformed tobacco leaves [9]. Agarose gel electrophoresis revealed a specific band at 1.4 kb (Fig. 1). Two transformed plants, which were tested as positive by PCR, were selected and were used to perform Southern blot analysis. The results showed that the phytase gene was integrated into the genome of NC89 plants (Fig. 2). 3.2. Expression of phyAI in tobacco Phytase protein from transgenic tobacco leaf discs were separated and purified with the method shown in Section 2.3. Phytase protein was separated by SDS-polyacrylamide gel electrophoresis (Fig. 3). The apparent molecular weight of phytase was 76 kDa, and the specific activity was approximately 109 U/mg. The amount of expressed phytase increased with extension of time (data not shown). Until the eighth week, phytase protein accounted for 17.6% of tobacco
3. Results 3.1. Transgenic tobacco The phyAI gene was transformed into tobacco (NC89) leaf discs (as shown in Section 2.2). At 26 ◦ C under screen medium (MS + 6BA (1 mg/l) + NAA (0.1 mg/l) +
Fig. 2. Southern blotting analysis of the transgenic tobacco. 1: marker DL-2000; 2: positive control phyAI; 3: transgenic tobacco genomic DNA digested by EcoRI/PstI; 4: transgenic tobacco genomic DNA digested by EcoRI/PstI; 5: NC89 genomic DNA digested by EcoRI/PstI.
L. Zhang et al. / Process Biochemistry 40 (2005) 213–216
215
Fig. 3. SDS-polyacrylamide gel electrophoresis of transgenic tobacco phytase. 1: marker protein molecular weight; 2: sample of phytase protein. Fig. 5. The optimum temperature of enzyme.
Fig. 6. The most ability of phytase. Fig. 4. The optimum pH of enzyme.
total soluble protein. Phytase expressed in wild type strain A. ficuum AS3.324 accounted for 0.2% of fermentation total liquid protein only. While the amount of phytase expressed in tobacco increased by a large margin. The phenomenon of accumulating phytase expressed by tobacco indicates that phytase was stable in the tissues of tobacco. 3.3. Properties of transgenic tobacco phytase Properties of transgenic tobacco phytase were determined according to the method described in Section 2.4. Test result showed the peak of phytase activity appeared at pH 2.0 and 5.5 (Fig. 4). The optimum temperature was 50 ◦ C (Fig. 5). Thermal stability of phytase is shown in Fig. 6. The optimum pH and the optimum temperature of transgenic tobacco phytase were the same as wild type strain A. ficuum AS3.324 on the whole, but thermal stability of phytase was different from each other. When phytase expressed by tobacco was treated under different temperatures, the residual enzyme activity improved in different degrees comparing with that of
wild type strain A. ficuum AS3.324. For instance, after 80 ◦ C 15 min heat treatment, residual enzyme activity was 25.1%, and under the same condition, residual phytase activity of A. ficuum AS3.324 was 5.5%. The Km and Kcat values of transgenic tobacco phytase were determined with sodium phytate and calcium phytate as substrate separately and according to the method described in Section 2.4. The results were shown in Table 1. As shown in Table 1, phytase was expressed by two hosts (A. ficuum AS3.324 and tobacco NC89), the Km and Kcat values shown no significant differences under the same substrate, while for the substrate of sodium phytate and calcium phytate, there were significant differences in order of magnitude. The result shows both A. ficuum AS3.324 phytase and transgenic tobacco phytase had higher substrate specificity and catalytic efficiency with sodium phytate as substrate than with calcium phytate. Both phytases had different substrate specificity and catalytic efficiency under all kinds of phytate as substrate, while they had no hydrolytic ability on other compounds with a phosphoester bond (data not shown).
Table 1 The kinetic parameters of phytase Sample
Sodium phytate Km (mol/l)
AS3.324 Tobacco
10−4
7.5 × 7.3 × 10−4
Calcium phytate Kcat
(s−1 )
9.4 × 1.2 × 104 103
Kcat /Km (l/mol s) 1.3 × 1.6 × 107 107
Km (mol/l)
Kcat (s−1 )
Kcat /Km (l/mol s)
10−3
3.4 × 5.1 × 103
2.8 × 106 3.9 × 106
1.2 × 1.3 × 10−3
103
216
L. Zhang et al. / Process Biochemistry 40 (2005) 213–216
4. Discussions The phytase gene phyAI of A. ficuum AS3.324 was transformed into tobacco NC89 by way of A. tumefaciens introduction, resulting in transgenic tobacco. Phytase was expressed nonspecifically in the tissues of tobacco. When the transgenic tobacco phytase with A. ficuum AS3.324 were compared, they had the same specific activity, pH, temperature adaptability and substrate specificity. The differences between the two are as follows: The amount of phyAI expressed in host A. ficuum AS3.324 was very low, and enzyme activity in the fermentation supernatant was only 8.2 U (nmol/ml), accounting for 0.2% of total secreted protein. The amount of phytase expressed in tobacco increased by a large margin. Phytase protein accounted for 17.6% of total soluble protein in the growing tobacco leaves in 8 weeks. Phytase had various degrees of expression in leaf, stem, root and seed of tobacco. The expression amount in the blade was the highest. In addition, phytase was stable in tobacco and was in the state of accumulation. Thermal stability of transgenic tobacco phytase was higher than A. ficuum AS3.324 phytase. Among the heat treatment range of 55–80 ◦ C, the average residual enzyme activity of the former was 2.9 times more than that of the latter. The difference between molecular weight determined by SDS-PAGE (apparent molecular weight) and the molecular weight calculated by using the amino acid sequence (theoretical molecular weight) is an indication of the extent of glycosylation of a protein [10]. Theoretical molecular weight of phytase coded by phyAI was 49.2 kDa. Apparent molecular weights of A. ficuum AS3.324 and transgenic tobacco phytase were 68.5 and 76.0 kDa, respectively. There are 10 potential N-glycosylation sites in the phyAI gene. The difference at thermal stability between the two kinds of phytase expressed by two hosts separately, might be related
to the extent and patterns of glycosylation [10,11]. But the thermal stability of the recombinant phytase cannot totally meet the demand of industry processes yet. Researchers hope to improve its thermal stability through protein engineering.
References [1] Keshavarz K. Nonphytate phosphorus requirement of laying hens with and without phytase on a phase feeding program. Poult Sci 2000;79(5):748–63. [2] Stahl CH, Roneker KR, Thornton JR, Lei XG. A new phytase expressed in yeast effectively improves the bilavailability of phytate phosphorus to weanling pigs. J Anim Sci 2000;78(3):668–74. [3] Yao B, Zhang Y-C, Wang J-H, et al. Isolation of Aspergillus niger 963 with phytase activity and cloing its phytase gene phyA2 [J]. J Agric Biotechnol 1998;6(1):1–6. [4] Verwoerd TC, van Paridon PA, Ooyen AJ, et al. Stable accumulation of Aspergillus niger phase transgenic tobacco leaves. Plant Physiol 1995;109:1199–205. [5] Li J, Carla E. Secretion of active recombinant phytase from soybean cell-suspension cultures [J]. Plant Physiol 1997;114:1103–11. [6] Wang G. Plant gene engineering principle and technology. Scientific Publishing Company, Beijing, 1998. p. 552–5. [7] Sambrook J, Fritsch EF, Maniatis T. Molecular cloning: a laboratory manual, vols. 323–324. 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, 1989. p. 602–14. [8] Ullah AHJ. Aspergillus ficuum phytase: partial primary structure, substrate selectivity and kinetic characterization. Prep Biochem 1988;18(4):459–71. [9] Zhang L-L, Zhang L-H, Wang Y-J. Isolation and molecular biological analyses of genomic DNAs from fungi using benzyl chloride. J Dalian Inst Light Ind 2000;19:36–9 [in Chinese with English abstract]. [10] Wyss M, Pasamontes L, Friedlein A, Remy R, Tessier M, Kroneberger A, et al. Biophysical characterization of fungal phytases (myo-inositol hexakisphosphate phosphohytrolases): molecular size glycosylation pattern, and engineering of proteolytic resistance. Appl Environ Microbiol 1999;65(2):359–66. [11] Han Y, Lei X-G. Role of glycosylation in the functional expression of an Aspergillus niger phytase(phyA) in Pichia Pastiris. Arch Biochem Biophys 1999;364(1):83–90.