• Fisheries and Aquatic Sciences
• /
• v.6 no.1
• /
• pp.20-26
• /
• 2003

A bacterial strain was isolated from seawater to screen for phytase activities. A colony had the highest activity and was identified as an Escherichia coli strain. Using primers derived from E. coli acid phosphatase appA sequence, we cloned a 1,495 bp DNA fragment connected with the pGEM-T vector. It was over-expressed under lac promoter combined with its native promoter in E. coli $DH5\alpha$. The expression of the phytase gene occurred during late exponential growth and the intracellular phytase production was 16.9 units/ml. The yield of recombinant phytate was 412-fold higher than that of wild type E. coli K13.

• Microbiology and Biotechnology Letters
• /
• v.30 no.1
• /
• pp.1-7
• /
• 2002

Phytase from Escherichia coli WC7 was purified from cell extracts and its molecular mass was estimated to be 45 kDa by SDS-PAGE. Its optimum temperature and pH for phytate hydrolysis was 6$0^{\circ}C$ and pH 5.0, respectively. The enzyme was stable up to 6$0^{\circ}C$ and over broad pH range (pH 2-12). The enzyme had higher affinity for sodium phytate than p-nitrophenylphosphate (pNPP). That is, the apparent Km value for sodium phytate and pNPP were $0.15\pm$0.02 mM and 2.82$\pm$0.05 mM, respectively. The gene encoding the phytase was cloned in E. coli XL1-Blue. Sequence analysis showed an open reading frame of 1241 Up encoding a signal peptide (22 aa) and a mature enzyme (410 aa). WC7 phytase was expressed up to 17.5 U/ml in the transformed E. coli XL1-Blue/pUEP, which was 23-fold higher than the activity from wild strain.

• Journal of Animal Science and Technology
• /
• v.50 no.1
• /
• pp.79-88
• /
• 2008

This study was conducted to evaluate effects of dietary Escherichia coli phytase supplementation on growth performance, carcass quality and excretion of copper and zinc concentrations in finishing pigs. The total of seventy two [(Landrace×Yorkshire)×Duroc] pigs(65.43±0.72 kg in average initial body weight) were used in 56 days assay. Dietary treatments included 1) CON (basal diet) 2) P5(basal diet+phytase 0.05%) and 3) P10 (basal diet + phytase 0.1%). There were three dietary treatments with six replicate pens per treatment and four pigs per pen. During the overall periods, ADFI(Average daily feed intake) was increased (P<0.05) in phytase supplementation treatments compared to CON treatment. At the 5th week, dry matter, nitrogen and ash digestibilities were higher in P5 treatment than in CON and P10 treatments(P<0.05) and CON treatment showed the lowest effect on nutrition digestibility(P<0.05). However, phosphorus digestibility was higher in both phytase supplementation treatments than in CON treatment(P<0.05). The pH of M. logissimus dorsi was significantly higher in CON treatment than phytase supplementation treatments(P<0.05). L* value of M. logissimus dorsi muscle color was significantly increased(P<0.05) in P10 treatment compared to CON and P5 treatments. Also, a* value was increased (P<0.05) in CON treatment compared with phytase supplementation treatments. However, excretion of copper and zinc concentrations were no significant difference among the treatments. In conclusion, the effect of Escherichia coli phytase showed in ADFI, digestibilities, pH and color of meat in finishing pigs.

• Journal of Microbiology and Biotechnology
• /
• v.15 no.3
• /
• pp.466-471
• /
• 2005

An Escherichia coli strain with high phytase activity was screened from pig excreta. The phytase gene, appA, was amplified by PCR technique. To obtain large amounts of appA phytase, the appA gene was subcloned into the baculovirus transfer vector pVL1393 under the control of the Polyhedrin promoter. The recombinant baculovirus harboring the appA gene was obtained after co-transfection and screening. The early $5^{th}$ instar larvae of silkworm were infected with the recombinant virus. Using this system, the appA phytase was overproduced up to 7,710 U per ml hemolymph. SDS-PAGE analysis revealed the baculovirus-derived appA phytase to be approximately 47 kDa in size. The optimal temperature and pH of the expressed phytase were $60^{\circ}C$ and pH 4.5, respectively. The enzymatic activity was increased by the presence of 1 mM $Ca^{2+}$, 1 mM $Mn^{2+}$, or $0.02\%$ Triton X-100.

• Journal of Microbiology and Biotechnology
• /
• v.12 no.3
• /
• pp.490-496
• /
• 2002

The production of E. coli WC7 phytase from a recombinant E. coli strain was optimized using a statistical experimental design approach. Two-level complete factorial designs with seven variables were used for the media optimization. In the first optimization step, the influence of disodium succinate, yeast extract, $K_2HPO_4,\;NH_4H_2PO_4,\;MgSO_4$, NaCl, and trace elements on phytase production was evaluated. As a result, disodium succinate, yeast extract, $NH_4H_2PO_4$, NaCl, and the trace elements were found to have a positive influence on the phytase production, while $K_2HPO_4\;and\;MgSO_4$ had a negative influence. In the second step, the concentrations of disodium succinate and yeast extract were further optimized using central composite designs. The maximum phytase activity obtained was 234 U/ml using 15.9 g/1 disodium succinate, 20 g/1 yeast extract, 5 g/1 K_2HPO_4,\;10 g/1 NH_4H_2PO_4,\;1.5 g/1 MgSO_4$, 4 g/1 NaCl, and 1.5 m1/1 trace elements, which was about a 14-fold increase in comparison with that obtained using the basal medium.

• Journal of Microbiology and Biotechnology
• /
• v.29 no.3
• /
• pp.419-428
• /
• 2019

Phytases are enzymes that can hydrolyze phytate and its salts into inositol and phosphoric acid, and have been utilized to increase the availability of nutrients in animal feed and mitigate environmental pollution. However, the enzymes' low thermostability has limited their application during the feed palletization process. In this study, a combination of B-value calculation and protein surface engineering was applied to rationally evolve the heat stability of Escherichia coli phytase. After systematic alignment and mining for homologs of the original phytase from the histidine acid phosphatase family, the two models 1DKL and 1DKQ were chosen and used to identify the B-values and spatial distribution of key amino acid residues. Consequently, thirteen potential amino acid mutation sites were obtained and categorized into six domains to construct mutant libraries. After five rounds of iterative mutation screening, the thermophilic phytase mutant P56214 was finally yielded. Compared with the wild-type, the residual enzyme activity of the mutant increased from 20% to 75% after incubation at $90^{\circ}C$ for 5 min. Compared with traditional methods, the rational engineering approach used in this study reduces the screening workload and provides a reference for future applications of phytases as green catalysts.

• Asian-Australasian Journal of Animal Sciences
• /
• v.27 no.2
• /
• pp.237-246
• /
• 2014

Two experiments were conducted to evaluate the efficacy of feeding an Escherichia coli (E. coli) derived phytase to pigs fed P deficient, corn-soybean meal diets. In Exp. 1, one hundred and twenty crossbred piglets ($9.53{\pm}0.84$ kg) were allocated to one of five treatments which consisted of four low P diets (0.61% Ca, 0.46% total P and 0.24% non-phytate P) supplemented with 0, 500, 1,000, or 20,000 FTU/kg E. coli phytase as well as a positive control formulated to be adequate in all nutrients (0.77% Ca, 0.62% total P and 0.42% non-phytate P). The treatments were applied to six pens with four pigs per pen for 28 days. In Exp. 2, ten crossbred pigs ($19.66{\pm}1.16$ kg) fitted with ileal T-cannula were used in a nutrient balance study. The pigs were assigned to treatments similar to those used in Exp. 1 in a doubly replicated $5{\pm}4$ incomplete Latin square design (5 diets with 4 periods). Each period consisted of a 5-d adjustment period followed by a 3-d total collection of feces and urine and then a 2-d collection of ileal digesta. Supplementation with phytase linearly increased (p<0.05) weight gain, feed intake, feed efficiency, bone breaking strength and fat-free dry and ash bone weight. There were linear increases (p<0.01) in the apparent ileal digestibility (AID) of DM, GE, CP, Ca, total P, inositol hexaphosphate ($IP_6$) and some AA with increasing dose of E. coli phytase. Pigs fed 20,000 FTU/kg had a greater (p<0.05) AID of IP6 (80% vs 59% or 64%, respectively) than pigs fed diets with 500 or 1,000 FTU/kg phytase. There were linear increases (p<0.05) in the total tract digestibility of Ca, total P, Na, K, Mg, and Zn as well as in the retention of Mg and Zn with increased phytase dose. The retention and utilization of Cu, and the total tract digestibility of CP and Cu quadratic increased (p<0.05) with increased phytase dose. In conclusion, supplementation of 500 FTU of phytase/kg and above effectively hydrolyzed phytate in low-P corn-soybean diets for pigs. In addition, a super dose of phytase (20,000 FTU/kg) hydrolyzed most of the IP6 and consequently further improved mineral use, protein utilization and performance.

• Journal of Microbiology and Biotechnology
• /
• v.25 no.6
• /
• pp.930-935
• /
• 2015

A phytase gene was identified in a publicly available metagenome derived from subsurface groundwater, which was deduced to encode for a protein of the histidine acid phosphatase (HAP) family. The nucleotide sequence of the phytase gene was chemically synthesized and cloned, in order to further overexpress the phytase in Escherichia coli. Purified protein of the recombinant phytase demonstrated an activity for phytic acid of 298 ± 17 µmol P/min/mg, at the pH optimum of 2.0 with the temperature of 37℃. Interestingly, the pH optimum of this phytase is much lower in comparison with most HAP phytases known to date. It suggests that the phytase could possess improved adaptability to the low pH condition caused by the gastric acid in livestock and poultry stomachs.

• Animal Bioscience
• /
• v.34 no.8
• /
• pp.1365-1374
• /
• 2021

Objective: This study was conducted to investigate the synergistic effect of exogenous multienzyme and phytase on growth performance, nutrients digestibility, blood metabolites, intestinal microflora, and morphology in broilers fed corn-wheat-soybean meal diets. Methods: A 2×2 factorial design was used in this study. Four dietary treatments consisted of i) basal diets (corn-wheat-soybean meal based diets without multi-enzyme and phytase), ii) basal diets with phytase (0.05%), iii) basal diets with exogenous multi-enzyme (0.05%), and iv) basal diets with exogenous multi-enzyme including phytase (0.05%). A total of 480 broiler chickens (Ross 308 - one day old) were weighed and allotted to thirty-two cages (15 birds per cage), and chicks were randomly allocated to four dietary treatments. Results: The body weight gain and feed conversion rate were improved by supplementation of exogenous multi-enzyme containing phytase during the finisher period (p<0.05). The birds fed diets with exogenous multi-enzyme containing phytase had a significantly greater digestibility of dry matter, gross energy, crude protein, calcium, and phosphorus compared with birds fed non-supplemented diets (p<0.05). The chickens fed diets with exogenous multi-enzyme containing phytase showed a higher concentration of Ca and P in the serum (p<0.05). The population of Lactobacillus spp., Escherichia coli, and Clostridium were not affected in the ileum and cecum of chickens fed enzyme-supplemented diets. The dietary supplemental exogenous multi-enzyme containing phytase showed a significant improvement in villus height, crypt depth, and villus height and crypt depth ratio, compared to basal diets or dietary supplemental phytase (p<0.05). Conclusion: The supplementation of the exogenous multi-enzyme containing phytase synergistically improved the growth performance, nutrients digestibility, and villus height of the small intestine of broiler chickens fed a corn-wheat-soybean meal based diets.

• Asian-Australasian Journal of Animal Sciences
• /
• v.22 no.10
• /
• pp.1391-1399
• /
• 2009

Two hundred and sixteen 68-week-old Hy-Line brown laying hens were used in a 6-week feeding trial to compare the efficacy of phytases Optiphos (OPT) and Natuphos (NAT), which were isolated from Escherichia coli and Aspergillus niger, respectively. Hens were randomly allotted into six treatments with six replications (six layers in three adjacent cages) per treatment according to their initial BW. The hens were then subjected to one of the following dietary treatments: i) Positive Control (PC; available phosphorus (AP) 0.4%); ii) Negative control (NC; AP 0.2%); iii) NAT1 (NC+250 FTU/kg NAT); iv) NAT2 (NC+500 FTU/kg NAT); v) OPT1 (NC+250 FTU/kg OPT); vi) OPT2 (NC+500 FTU/kg OPT). Feed intake, egg production, egg quality, apparent nutrient digestibility and serum P and Ca concentration were evaluated to compare the effect of the two phytases. Feed intake and eggshell thickness were not affected by the treatments. Superior effects (p<0.05) of OPT were only observed in egg production and egg weight compared with NAT. Characteristics such as eggshell breaking strength, apparent digestibility of N, Ca and P and serum P concentration were equally increased with the supplementation of both phytases (p<0.05), where no significant difference was observed in those characteristics between PC and phytase supplementation at 500 FTU/kg. Equally effective improvements (p<0.05) were also observed in egg production and DM digestibility, where no improvements were observed (p<0.05) between the PC group and the groups with phytase supplementation at 500 FTU/kg. Equal increases in the serum Ca level were observed when the groups with phytase supplementation were compared to the PC group. Overall, the results of this study suggest that NAT and OPT are equally effective at liberating phytate-bound complexes when included in 0.2% available phosphorus diets for 68-week laying hens; either source of phytase can be fed to commercial 68-week laying hens at 500 FTU/kg to correct the negative effects associated with a 0.2% available phosphorus diet. In conclusion, either source of phytase can be fed to commercial first cycle laying hens at 500 FTU/kg to effectively replace inorganic phosphorus when economically justified.