• Journal of Microbiology and Biotechnology
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• v.20 no.11
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• pp.1491-1499
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• 2010

Rhizosphere microorganisms possessing phytase activity are considered important for rendering phytate-phosphorus (P) available to plants. In the present study, the Citrobacter braakii phytase gene (appA) was overexpressed in rhizobacteria possessing plant growth promoting (PGP) traits, for increasing their potential as bioinoculants. AppA was cloned under the lac promoter in the broadhost-range expression vector pBBR1MCS-2. Transformation of the recombinant construct pCBappA resulted in high constitutive phytase activity in all of the eight rhizobacterial strains belonging to genera Pantoea, Citrobacter, Enterobacter, Pseudomonas (two strains), Rhizobium (two strains), and Ensifer that were studied. Transgenic rhizobacterial strains were found to display varying levels of phytase activity, ranging from 10-folds to 538-folds higher than the corresponding control strains. The transgenic derivative of Pseudomonas fluorescens CHA0, a well-characterized plant growth promoting rhizobacterium, showed the highest expression of phytase (~8 U/mg) activity in crude extracts. Although all transformants showed high phytase activity, rhizobacteria having the ability to secrete organic acid showed significantly higher release of P from Ca-phytate in buffered minimal media. AppA overexpressing rhizobacteria showed increased P content, and dry weight (shoot) or shoot/ root ratio of mung bean (Vigna radiata) plants, to different extents, when grown in semisolid agar (SSA) medium containing Na-phytate or Ca-phytate as the P sources. This is the first report of the overexpression of phytase in rhizobacterial strains and its exploitation for plant growth enhancement.

• Korean Journal of Organic Agriculture
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• v.16 no.3
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• pp.331-339
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• 2008

The effects of heat and formaldehyde treatments of soybean meal and rapeseed meal on ruminal release of minerals from the meals were studied on three sheep fitted with rumen cannula. Oilseed meals were treated at 133, $143^{\circ}C$ for 3h or added with formaldehyde at a level of 3, 5g/kg. The ruminal release of P, Ca, Mg, Cu, Fe and Zn from the oilseed meals was examined using the nylon bag technique. Effective degradabilities of minerals in the oilseed meals were reduced by both treatments. The results suggest that rumen bypass treatments of oilseed meals prevent the release of minerals from the meals during rumen digestion and suppress the availability of mineals for ruminants. Eventually, the increase of heavy metal pollution in soil is anticipated due to the increase of mineral content in the feces of ruminants by ruminal protein bypass treatments.

• Korean Journal of Food Science and Technology
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• v.32 no.2
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• pp.231-237
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• 2000

A Comparative study was performed among five Korean wheat(Greu, Eunpa, Tapdong, Allgreu, Woori) and three imported wheat(Dark Northern Spring, Austrian Standard Wheat, Western White) to investigate the characteristics of minerals and phytate from Korean wheat varieties. Ash contents of kernel was higher in Korean wheat than in imported wheat and related to ash contents and darkness of flour. More contents of Fe, P, and Mg from Korean wheat grains and Fe from Korean wheat flours were observed in comparison to imported wheat varieties. Fe concentration in Korean flour show high, indicating Fe was distributed at endosperm layer of kernels and not eliminated with milling. In addition, phytate affecting availability of minerals was determined from kernels and flours. Even though Korean wheat kernels had more phytate than that of imported wheat kernels, it is believed that most of phytate was removed with milling and there was no major difference between Korean and imported wheat flours. We also observed that the content of phosphate closely parallels the content of phytate from both the flours and kernels.

• Asian-Australasian Journal of Animal Sciences
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• v.14 no.2
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• pp.250-257
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• 2001

An experiment was conducted to determine the effects of phytase supplementation to the diets containing different levels of non-phytate phosphorus (NPP). A $3{\times}2$ factorial arrangement of treatments was employed. There were three dietary NPP levels of control (C) (0.45% for starter diet and 0.35% for grower diet), C-0.1% NPP (0.35% for starter diet and 0.25% for grower diet), and C-0.2% NPP (0.25% for starter diet and 0.15% for grower diet) and two phytase levels (0 and 500 U/kg). Reduced dietary NPP decreased feed intake and weight gain and increased mortality whereas dietary phytase increased feed intake and weight gain and decreased mortality. Supplemental phytase improved availabilities of dry matter, crude fat, ash, P, Zn, Mg, and Cu whereas dietary NPP level did not affect availabilities of nutrients except decreased Zn availability and increased Cu availability in reduced NPP diets. Nutrient retention of N, ash, Ca, P, Mg, and Zn were linearly decreased as dietary NPP levels reduced but dietary phytase increased their retention. Reduced dietary NPP increased ash excretion but decreased P and Cu excretion while dietary phytase decreased N excretion. Weight, length, girth and contents of ash, Ca, P and Mg of tibia linearly decreased as dietary NPP levels reduced. Dietary phytase increased length and ash content of tibia. It is concluded that dietary phytase can reduce P excretion and alleviate adverse affects caused by feeding low dietary NPP. Effects of phytase were greater in the lower NPP diets.

• Asian-Australasian Journal of Animal Sciences
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• v.10 no.6
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• pp.551-566
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• 1997

Phosphorus has been known as an essential component of animal body. However, the requirement has not been determined precisely because of the variable bioavailabilities of feedstuffs from plant origin. The bioavailability of P in various feedstuffs of plant origin varies from 10 to 60%. Digestibility and availability of the P differed considerably depending on the feed. The lowest values were found for maize (under 20%), the highest for wheat and triticale (over 50%). This is due to the proportion of phytate and the presence of intrinsic phytase. And the digestive tract of monogastric animals does not contain sufficient amounts of phytase, an enzyme that hydrolyses the unavailable phytate complexes to available, inorganic orthophosphates. Microbial phytase supplementation improves the P availability, and both intrinsic plant and microbial phytase improves the availability of P in feedstuffs of plant origin. In a mixture of feeds with low and high activity of intrinsic phytase and/or supplemented by commercial phytase, the P availability is additive. However, in the light of current results it seems that exceeding the P availability equal to 60-70% is unrealizable even at large microbial phytase doses.

• Korean Journal of Poultry Science
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• v.27 no.3
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• pp.169-179
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• 2000

An experiment was conducted to measure the effect of crude phytase supplementation on the growing performance, blood concentrations of some minerals and tibia characteristics of broiler chickens. Day-old 240 male broiler chickens (Avian) were randomly allotted to four treatments. There were six replicates per treatment, and ten chicks per replicate. Treatments consisted of two levels of crude phytase (0 and 600U/kg) made from Aspergillus ficuum and two levels of non-phytate P (0.45 and 0.35% NPP for the starter period, and 0.35 and 0.25% NPP for the grower period), making the experiment 2$\times$2 factorial. The starter period was from hatch to 21 d of age, and grower period was from 22 to 35 d of age. Feed intake and weight gain of chicks fed diet containing phytase were higher(P〈0.05) than those of chicks fed diets without phytase, however, no differences was found in feed/gain. mortality, and nutrient availabilities regarding the phytase supplementation. Chickens fed diets with low NPP and phytase excreted lower P than did birds fed diets containing normal NPP without phytase. The level of NPP and phytase did not affect N excretion. The Ca availability was increased by feeding low NPP diet. Dietary phytase increased the availabilities of P and Mg, but decreased those of Fe and Zn. There was interactions between dietary NPP level and phytase addition on mineral availability. Tibia was lighter and shorter in low NPP groups, and heavier in phytase treated groups. The tibial contents of Ca, P and Mg decreased in low NPP treated groups, but increased in phytase treated groups. The ash content of tibia of chickens fed diet with phytase was higher than that of birds fed diets without phytase. These data suggest that the crude phytase supplementation to broiler diets containing low NPP level improves growth performance and mineral availability and, reduces fecal P excretion.

• Asian-Australasian Journal of Animal Sciences
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• v.13 no.5
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• pp.673-680
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• 2000

An experiment was conducted with day-old 300 commercial male broiler chicks (Arbor Acres$^{(R)}$) to evaluate the efficacy of crude phytase preparerations produced from a culture of Aspergillus ficcum. The experiment consisted of five dietary treatments; T1, com-soy control diet with 0.45% non-phytate phosphorus (NPP) for starter period and 0.35% NPP for grower period; T2, control - 0.1% NPP; T3, control 0.2% NPP; T4, T3+600 U of crude phytase (broth+cell); and T5, T3+600 U of crude phytase (broth). The body weight gain, feed intake, and feed/gain of chickens fed T1 diet was highest (p<0.01) among treatments. BW gain and feed intake of T4 and T5 were greater than those of T3 but were less than those of T1 and T2. T3 was highest in mortality among treatments. Decreasing the NPP level lowered availability of DM, crude ash, ether extract, crude fiber, Zn, and Fe but supplementation of crude phytase preparations improved the availability of these nutrients as well as those of Ca, P and Cu. Excretion of P and Cu significantly decreased as the NPP level in the diet decreased. Further reduction of P and Cu excretion and reduction of Ca, Mg and Fe excretion were achieved by supplementation of crude phytase preparations. The serum concentrations of Ca, P, Mg, Zn, Fe, and Cu were significantly increased by crude phytase supplementation. The weight and length of tibia, and contents of crude ash, Ca, P, Mg, and Zn were adversely affected by lowering NPP level but partially recovered by supplementation of crude phytase preparations. In conclusion, lowering NPP level in the broiler diet significantly depressed the performance. Supplementation of crude phytase preparations produced from Aspergillus ficuum could partially recover the depression.

• Journal of Animal Science and Technology
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• v.45 no.2
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• pp.229-240
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• 2003

An in vitro test and a broiler feeding trial were conducted to test the effect of hydrothermal treatment of wheat bran on phytate-P degradation and it’s feeding effect on performance of broilers. Hydrothermal treatment of wheat bran was carried out at 55$^{\circ}C$ with pH 5.5 buffer solution. Phytate-P content of wheat bran decreased quadrically as the wheat bran: buffer solution ratio increased from 1:0.5 to 1:5. Phytate-P degradation was not significantly affected by incubation times above 10 min., drying temperature (55$^{\circ}C$, 65$^{\circ}C$ and 75$^{\circ}C$) or pH of the buffer solution (5.5 and 7.0). A feeding trial was conducted with 240 sex separated d-old broiler chickens (Ross$^{\circledR}$). Broilers were randomly housed to 24 cages of 10 birds each. Six cages (3 of each sex) were assigned to 4 treatments: Control-normal level of non-phytate-P (NPP); LP-low NPP treatment which had 0.1% lower NPP than Control; LPWB-LP with wheat bran which provided 475 IU of plant phytase per kg diet; LPHWB-LP with hydrothermally treated wheat bran. Results of the feeding trial showed that broilers in the LP treatment gained significantly less than other treatments in starter period (1-21d) but only male broilers for growing LP gained significantly less than Control in the grower (22-35d) and overall period. There were no significant differences in weight gain among the birds of LPWB, LPHWB and Control. Feed intake during the overall period was not significantly different between LPWB and Control but that of LP was lower than LPHWB and that of LPHWB was lower than Control. Feed/gain ratio was significantly lower in LPHWB and LP than in Control and LPWP. Mortality was highest in LPHWB. Availability of crude fat, crude ash and Ca was significantly lower in LP than other treatments. Availability of P and Zn was higher in LPWB and LPHWB than in Control and LP. Availability of P, Mg and Zn was highest in LPHWP treatment. Excretion of P was significantly lower in low NPP treatments than in Control. Serum Ca level was highest whereas serum P level was lowest in LP. Tibial crude ash content was higher in wheat bran treatments, but lower in LP than Control. However, tibial Ca content was higher in Control and LP than wheat bran treatments. Tibial P content of LP and LPWB was lower than Control. However, tibial content of Fe was highest in LP. It was concluded that wheat bran, a source of plant phytase, could be used in low NPP broiler diets to prevent the depression of performance. Reduction of P excretion can be achieved concomitantly. Hydrothermal treatment of wheat bran was effective in improving utilizability of some minerals but was not effective in improving performance of broilers.

• Journal of Microbiology and Biotechnology
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• v.34 no.5
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• pp.1119-1125
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• 2024

Phytase increases the availability of phosphate and trace elements by hydrolyzing the phosphomonoester bond in phytate present in animal feed. It is also an important enzyme from an environmental perspective because it not only promotes the growth of livestocks but also prevents phosphorus contamination released into the environment. Here we present a novel phytase derived from Turicimonas muris, TmPhy, which has distinctive structure and properties compared to other previously known phytases. TmPhy gene expressed in the Pichia system was confirmed to be 41 kDa in size and was used in purified form to evaluate optimal conditions for maximum activity. TmPhy has a dual optimum pH at pH3 and pH6.8 and exhibited the highest activity at 70℃. However, the heat tolerance of the wildtype was not satisfactory for feed application. Therefore, random mutation, disulfide bond introduction, and N-terminal mutation were performed to improve the thermostability of the TmPhy. Random mutation resulted in TmPhyM with about 45% improvement in stability at 60℃. Through further improvements, a total of three mutants were screened and their heat tolerance was evaluated. As a result, we obtained TmPhyMD1 with 46.5% residual activity, TmPhyMD2 with 74.1%, and TmPhyMD3 with 66.8% at 80℃ heat treatment without significant loss of or with increased activity.

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