Efficacy of a New E. coli-derived Phytase (Expressed in Yeast) for Phosphorus Release in Pigs Nathan R. Augspurger*, Douglas M. Webel** and David H. Baker* *Department of Animal Sciences, University of Illinois **United Feeds Corporation, Sheridan, IN INTRODUCTION The hexaphosphate form of inositol is the prototypical form of the phytate complex. The dephosphorylation of this complex by phytase is initiated at either the 3-position or the 6- position, of which the commercial phytases Natuphos and Ronozyme, respectively, are examples. Years of research have shown the efficacy of phytase, mainly Natuphos, for releasing a portion of the phytate-bound phosphorus (P) in grain-oilseed meal diets fed to monogastric animals. Other supplements known to have phytate P releasing activity are citric acid (Boling et al. 2000) and either 1,25-dihydroxycholecalciferol or 1αhydroxycholecalciferol (Edwards 1993; Biehl et al. 1995). Recently, proteins encoded by genes isolated from different strains of E. coli have been characterized to have phytase activity (Rodriguez et al. 1999a; 2000). These phytase proteins were found to be genetically and structurally unique from the commercial phytase proteins. 1 Previous efficacy work with E. coli phytases has shown that these phytases are as effective as Natuphos when fed at the same dose level to young chicks (Leeson et al. 2000) and young pigs (Stahl et al. 2000). The primary objective of the research presented here was to investigate the P-releasing efficacy of an E. coli phytase () produced from the appa2 gene of an E. coli strain that was obtained from pig intestine (Rodriguez et al. 1999a). Recent chick work with showed that this phytase enzyme was superior in P- releasing efficacy to either Natuphos or Ronozyme (Augspurger et al. 2003). Phytases MATERIALS AND METHODS The experimental E. coli phytase was cloned from E. coli and expressed in a yeast-expression system as described by Rodriguez et al. (1999a; 2000). It is classified as a 6-phytase. The phytase premix was assayed twice for phytase activity prior to inclusion in experimental diets (Han et al. 1999). Phytase activity (FTU/g premix) was based on assay results provided by Dr. X. G. Lei of Cornell University and by the laboratories of Hoffmann-LaRoche. One phytase unit (FTU) was defined as the amount of enzyme required to release 1 µmol inorganic phosphorus (ip) per minute from sodium phytate at 37 C. 1 E. coli phytases differ from the two commercial phytases (Natuphos7 and Ronozyme7) in amino acid sequence, ph optima, susceptibility to pepsin and trypsin, and in kinetic parameters (Rodriguez et al. 1999a,b). The E. coli phytase evaluated herein (from pig intestine) is also somewhat different from other E. coli phytases that have been characterized. 1
General Procedures The University of Illinois Institutional Animal Care and Use Committee approved all housing, handling, and euthanasia procedures. The pig assays were conducted at the United Feeds Research Farm, Frankfort, IN. Ten individually-fed nursery pigs (Pig Assay 1) and 10 individually-fed finishing pigs (Pig Assay 2) were fed each of the experimental diets. Pigs were assigned to blocks or blocks within sex (Pig Assay 2) based on ancestry and body weight. Statistical Analyses Analysis of variance was performed using the GLM procedure of SAS (1990) appropriate for a randomized complete-block design (Pig Assay 1) or a split-plot design (Pig Assay 2). Treatment means were compared using the least significant difference multiple pair-wise comparison procedure (Carmer and Walker 1985). Single degree-of-freedom comparisons were also made to test main effects and interactions. Pen means data for bone ash were regressed on supplemental ip intake to construct the standard curves (Pig Assay 1), and bone ash responses to supplementary phytase were inserted into the standard curve to calculate bioavailable P yield (g). Phosphorus yield was divided by feed intake, and then multiplied by 100 to determine bioavailable P release. Phytase efficacy was based on fibula ash (and metatarsal ash in Pig Assay 2). Pig Assay 1 (Table 2) PROCEDURES, RESULTS AND DISCUSSION Fifty AusGene barrows were weaned at approximately 15 d of age and housed in an environmentally controlled nursery with ad libitum access to a corn-soybean meal-wheyplasma starter diet adequate in all essential nutrients (NRC, 1998). At 1 wk post-weaning, pigs were moved to a nursery facility equipped with individual pens (0.5 x 0.9 m) and allowed a few days to acclimate to the facility. Pigs were then deprived of feed for 12 h, weighed, assigned to uniform blocks based on ancestry and body weight, and then allotted randomly to treatment diets from within blocks. Pigs were penned individually, and given ad libitum access to the experimental diets for a period of 23 d, during which time pigs and feeders were weighed weekly. Ten barrows received each of the five experimental diets. After the 23-d feeding period, pigs were fasted for 12 h and then weighed for determination of weight gain and gain/feed ratio. The five median weight blocks of pigs were killed via CO 2 inhalation and the right fibula bone was removed from each pig for determination of bone ash. Previous research had indicated that, among several bones examined, the fibula was a bone that could be removed quantitatively and processed conveniently for ashing (Biehl and Baker, 1996). Bones were cleaned of adhering tissue, dried for 24 h at 105 C, and ashed in a muffle furnace at 600 C for 24 h. Bone ash was expressed as total mg and % of dry bone weight. The basal diet (Table 1) was formulated to be deficient in P, containing 0.34% total P. Using average P bioavailability estimates of 25% for soybean meal and 15% for corn (Cromwell, 1992), the basal diet contained an estimated 0.075% available P. The diet was also moderately deficient in Ca (0.60%). With the exceptions of Ca and P, the diet was 2
formulated to be adequate to superadequate in all other nutrients, including cholecalciferol (16.5 µg/kg), for 10 to 20 kg pigs (NRC, 1998). The dietary treatments were made by supplementing the basal diet with 0, 0.05, 0.10, and 0.15% ip and 400 of. The weanling pigs in this trial performed well, particularly when considered in light of the fact that a simple corn-soybean meal diet was fed instead of the usual complex diet containing both lactose and blood plasma. All measures of response responded linearly (P < 0.01) to supplemental ip addition, and responses to phytase were also significant (P < 0.05). Total fibula ash was about the same in pigs receiving 400 of as in those receiving 0.10% ip supplementation. Based on the linear regression of total fibula ash on supplemental ip intake (r 2 = 0.87), 400 FTU of was estimated to release 0.108% P from the phytate-bound P present in the corn-soybean meal diet. The linear regression of daily weight gain (g) on supplemental ip intake (g), while significant (P < 0.05), resulted in a fit (r 2 ) value of only 0.55. Thus, fibula ash was confirmed to be a far better criterion of P utilization than weight gain. Pig Assay 2 (Tables 3 and 4) This assay involved 30 barrows and 30 gilts and was designed as a split-plot, with gender being the main plot and dietary treatment being the subplot. Pigs were fed individually in 1.2 x 1.7 m pens from 49 to 118-kg body weight. Each pen was equipped with an individual self feeder. The pigs were exposed to an overnight period of feed removal preceding the initial and final weighings. Pigs were assigned to uniform blocks (within sex) based on ancestry and body weight, after which they were allotted randomly to treatment and pens within sex. When individual block weights reached 118 + 3 kg, all pigs in that block were weighed off. After being weighed off, all pigs were killed, and the right fibula and 4 th metatarsal bone were quantitatively removed for processing and bone-ash determination, as previously described. Two P-deficient basal diets were used (Table 1), one during the 50 to 80-kg early-finishing growth period (0.09% available P, 0.50% Ca) and the other during the 80 to 120-kg latefinishing growth period (0.05% available P, 0.45% Ca). When a given block of pigs reached the target weight of 80 + 3 kg, all pigs in that block were switched to the late-finishing diet. Experimental diets were the basal diet supplemented with 0 or 0.10% ip, and 250, 500, 1,000 and 10,000. Finishing pigs in this 75-d trial responded to supplemental ip and to phytase. Barrows gained faster (P < 0.05) and ate more (P < 0.05) feed than gilts, and in terms of growth performance, 250 of was as efficacious as higher levels, and also as efficacious as 0.10% ip supplementation (Table 3). The sex x ip vs. phytase interaction (P < 0.05) for weight gain suggested that was superior to ip in gilts but was equal to ip in barrows. The bone ash data (Table 4) generally support the conclusion that (a) responses to were equivalent to responses obtained from 0.10% ip supplementation, (b) levels of greater than 250 were slightly more efficacious than the 250 dose level, and c) an excessive dose level of 10,000 of was both safe and efficacious for finishing pigs. 3
SUMMARY Phosphorus-releasing efficacy of a new phytase enzyme derived from an E. coli strain present in pig intestine was evaluated in both weanling and finishing pigs. Bone ash was the principal response criterion. Supplemental E. coli phytase () or inorganic P (ip) from KH 2 PO 4 was added to P-deficient corn-soybean meal diets that contained an estimated 0.05 to 0.09% available P and 0.27 to 0.30% phytate P. The two pig trials confirmed the ip-releasing efficacy of phytase. Weight gain and bone ash responses to doses of 400 to 500 were equal to those resulting from 0.10% ip supplementation. Also, an excessive dose level of (10,000 ) was found to be both efficacious and safe. The E. coli based phytase in a yeast expression system evaluated herein would appear to have excellent potential as a feed additive for poultry and swine. Its efficacy would reside in lowering the amount of supplemental ip needed in diets, and also in lowering the amount of P in swine and poultry excreta. REFERENCES AOAC. 1995. Official Methods of Analysis (16th ed.). Association of Official Analytical Chemists, Washington, DC. Augspurger, N.R., D.M. Webel, X.G. Lei, and D.H. Baker. 2003. Efficacy of an E. coli phytase expressed in yeast for releasing phytate-bound phosphorus in young chicks and pigs. J. Anim. Sci. (In press). Biehl, R.R. and D.H. Baker. 1996. Efficacy of supplemental 1α-hydroxycholecalciferol and microbial phytase for young pigs fed phosphorus- or amino acid-deficient cornsoybean meal diets. J. Anim. Sci. 74:2960-2966. Biehl, R.R., D.H. Baker, and H.F. DeLuca. 1995. 1α-hydroxylated cholecalciferol compounds act addivitely with microbial phytase to improve phosphorus, zinc and manganese utilization in soy-based diets fed to chicks. J. Nutr. 125:2407-2416. Boling, S.D., D.M. Webel, I. Mavromichalis, C.M. Parsons, and D.H. Baker. 2000. The effects of citric acid on phytate phosphorus utilization in young chicks and pigs. J. Anim. Sci. 78:682-689. Carmer, S.G. and W.M. Walker. 1985. Pairwise multiple comparisons of treatment means in agronomic research. J. Agron. Educ. 14:19-26. Cromwell, G.L. 1992. The biological availability of phosphorus in feedstuffs for pigs. Pig News and Inform. 13:75N-78N. Edwards, H.M., Jr. 1993. Dietary 1,25-dihydroxycholecalciferol supplementation increases natural phytate phosphorus utilization in chickens. J. Nutr. 123:567-577. 4
Han, Y., D.B. Wilson, and X.G. Lei. 1999. Expression of an Aspergillus niger phytase gene (phya) in Saccharomyces cerevisiae. Appl. Environ. Microbiol. 65:1915-1918. Leeson, S., H. Namking, M. Cottrill, and C.W. Forsberg. 2000. Efficacy of a new bacterial phytase in poultry diets. Can. J. Anim. Sci. 80:527-528. NRC. 1998. Nutrient Requirements of Swine (10th Ed.). National Academy Press, Washington, DC. Rodriguez, E., Y. Han, and X.G. Lei. 1999a. Cloning, sequencing, and expression of an Escherichia coli acid phosphatase/phytase gene (appa2) isolated from pig colon. Bioch. Biophys. Res. Comm. 257:117-123. Rodriguez, E., J.M. Porres, Y. Han, and X.G. Lei. 1999b. Different sensitivity of recombinant Aspergillus niger phytase (r-phya) and Escherichia coli ph 2.5 acid phosphatase (r-appa) to trypsin and pepsin in vitro. Arch. Bioch. Biophys. 365:262-267. Rodriguez, E., Z.A. Wood, P.A. Karplus, and X.G. Lei. 2000. Site-directed mutagenesis improves catalytic efficiency and thermostability of Escherichia coli ph 2.5 acid phosphatse/phytase expressed in Pichia pastoris. Arch. Biochem. Biophys. 382:105-112. SAS. 1990. SAS/STAT User's Guide (Release 6.08). SAS Inst. Inc., Cary, NC. Stahl, C.H., K.R. Roneker, J.R. Thornton, and X.G. Lei. 2000. A new phytase expressed in yeast effectively improves the bioavailability of phytate phosphorus to weanling pigs. J. Anim. Sci. 78:668-674. 5
Table 1. Percentage composition of diets (as-fed basis). Finishing pig assay Ingredient Young pig assay 50-80 kg 80-120 kg Cornstarch to 100 to 100 to 100 Corn 60.85 78.42 83.85 Soybean meal, dehulled 31.19 18.08 12.65 Soybean oil 3.00 Limestone, ground 1.06 1.06 1.07 Dicalcium phosphate 0.16 Trace-mineral premix a 0.35 0.35 0.35 Vitamin premix b 0.20 0.10 0.10 d Antibacterial premix 1.00 c 0.75 d 0.75 d Copper sulfate 0.08 L-Lysine HCl, feed grade 0.17 0.16 0.11 L-Threonine, feed grade 0.02 DL-Methionine, feed grade 0.05 Chemical composition Crude protein, % e 20.8 15.1 13.0 Total phosphorus, % e 0.35 0.38 0.32 Available phosphorus, % f 0.075 0.09 0.05 Calcium, % f 0.60 0.50 0.45 ME, kcal/kg f 3420 3310 3315 a Supplied the following per kilogram of complete diet: Fe, 90 mg (FeSO 4 H 2 O); Zn, 100 mg (ZnO); Mn, 20 mg (MnO); Cu, 8 mg (CuSO 4 H 2 O); I, 0.35 mg (CaI 2 ); Se, 0.3 mg (Na 2 SeO 3 ); NaCl, 3 g. b Supplied the following per kilogram of complete diet: retinyl acetate, 2,273 µg; cholecalciferol, 16.5 µg; DL-α-tocopheryl acetate, 88 mg; menadione, 4.4 mg (menadione sodium bisulfite complex); niacin, 33 mg; D-Ca-pantothenate, 24.2 mg; riboflavin, 8.8 mg; vitamin B 12, 35 µg; choline chloride, 319 mg. c Provided 55 mg of mecadox per kilogram of complete diet. d Provided 38 mg of roxarsone per kilogram of complete diet. e Analyzed (AOAC, 1999) f Calculated (NRC, 1998) 6
Table 2. Efficacy of phytase for young pigs. (Pig assay 1). Weight Gain/feed, Fibula ash b Bioavailable P Diet gain, g/d a g/kg a % mg release, % c 1. Basal diet 369 f 533 f 29.3 g 666 g 2. As 1 + 0.05% ip (KH 2 PO 4 ) 435 e 576 ef 32.8 f 766 g 3. As 1 + 0.10% ip (KH 2 PO 4 ) 476 de 618 de 36.6 d 972 ef 4. As 1 + 0.15% ip (KH 2 PO 4 ) 509 d 660 d 36.6 d 1123 d 5. As 1 + 400 443 e 583 ef 35.0 def 968 ef 0.108 Pooled SEM 17 21 0.8 38 a Data are means of 10 individually-fed pigs over a 23-d feeding period; average initial weight was 8.4 kg. b Data are means of five individually-fed pigs that were chosen from the median-weight blocks at the end of the 23-d feeding period. c The linear regression of fibula ash (mg) for Diets 1 to 4 as a function of supplemental ip intake (g/23 d) was Y = 664.5 ± 25.5 + 15.3 ± 1.4X (r 2 = 0.87). Bioavailable P release (equivalent P yields) for Diet 5 was determined by calculating equivalent bioavailable P intake (g) from the standard curve, dividing that by the total feed intake (g/23 d), and multiplying by 100. d,e,f,g Means within a column with different superscripts are different, P < 0.05. 7
Table 3. Effect of phytase on growth performance of finishing pigs (Pig assay 2) a. Dietary treatment Response variable P-deficient basal diet As 1 + 0.10% ip As 1 + 250 As 1 + 500 As 1 + 1,000 As 1 + 10,000 Pooled SEM Daily gain, g b Barrows 935 1023 1023 929 993 974 Gilts 752 790 872 909 828 902 Mean 844 907 947 919 910 938 38 Daily feed, g c Barrows 2837 2861 3028 2712 2873 2684 Gilts 2347 2197 2571 2507 2378 2562 Gain/feed, g/kg d Mean 2592 2529 2800 2610 2625 2623 81 Barrows 331 358 338 343 346 365 Gilts 320 363 341 363 349 352 Mean 325 361 339 353 347 359 8 a Data are means of five individually-fed pigs of each sex fed their experimental diets from 48.9 to 117.6 kg body weight. b Sex diet interaction, P < 0.10; Sex ip vs -supplemented diets, P < 0.05. c Barrows vs gilts, P < 0.01. d P-deficient vs ip- and -supplemented diets, P < 0.01. 8
Table 4. Effect of phytase on bone characteristics of finishing pigs (Pig assay 2) a. Dietary treatment As 1 + 250 As 1 + 500 P-deficient As 1 + basal diet 0.10% ip Response variable Fibula ash, % b As 1 + 1,000 As 1 + 10,000 Barrows 52.9 58.8 57.8 58.9 58.5 58.5 Gilts 52.4 59.2 58.9 55.8 58.3 58.8 Pooled SEM Fibula ash, g bcdef Metatarsal ash, % b Metatarsal ash, g bd Mean 52.6 59.0 58.3 57.3 58.4 58.7 0.7 Barrows 4.57 6.30 5.69 6.56 6.37 7.04 Gilts 4.19 6.06 5.90 5.99 6.24 6.86 Mean 4.38 6.18 5.80 6.28 6.31 6.95 0.17 Barrows 40.3 46.9 48.8 47.5 47.3 47.6 Gilts 43.1 48.5 48.3 45.4 49.0 49.0 Mean 41.7 47.7 48.5 46.5 48.1 48.3 1.1 Barrows 5.4 6.6 6.8 7.0 7.2 7.4 Gilts 4.9 6.6 6.5 7.0 6.8 7.3 Mean 5.1 6.6 6.6 7.0 7.0 7.3 0.2 a Data are means of five individually-fed pigs of each sex fed their experimental diets from 48.9 to 117.6 kg body weight. b P-deficient vs ip- and phytase-supplemented diets, P < 0.01. c Barrows vs gilts, P < 0.10. d 250 U/kg vs higher activity levels, P < 0.01. e 500 U/kg vs 1,000 and 10,000 U/kg, P < 0.10. f 1,000 U/kg vs 10,000 U/kg, P < 0. 9
10