• Journal of Microbiology and Biotechnology
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• 제20권11호
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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.

• 한국유기농업학회지
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• 제16권3호
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• pp.331-339
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• 2008

본 연구는 반추가축에 있어서 단백질 Bypass처리가 광물질의 체내이용에 미치는 영향을 조사하기 위하여 실시하였다. 반추위 Cannula를 장착한 면양 3두에 나이론백 실험방법을 이용하여 사양시험을 실시하였다. 시험사료로는 대두박과 채종박을 사용하였으며, 본 시험사료에 가열($133^{\circ}C$, $143^{\circ}C$) 처리와 포름알데이드(0.3%, 0.5%)처리를 하고 나이론 백을 사용하여 반추위내에서 배양한 후 Phytate와 P, Ca, Mg, Cu, Fe, Cu에 대해 각 항목의 반추위내의 소실율을 구하였다. 시험결과에 의하면 대두박과 채종박에 대한 단백질 Bypass처리는 Phytate를 포함한 모든 광물질에서 단백질 Bypass 처리의 강도가 높아짐에 따라 반추위내의 각 광물질의 소실율이 유의하게 감소하였다(P<0.05). 그리고 각 광물질간의 비교에서 Cu, Zn, Ca의 소실율이 타광물질에 비하여 낮은 경향을 보여 특히 본 광물질의 이용성 감소가 예상된다. 이상의 결과로 반추가축에 있어서 이용률이 매우 높은 것으로 알려진 Phytate는 단백질 Bypass처리를 함으로써 Phytate와 결합되어 사료내에 존재하는 광물질의 반추위내의 분해 율을 감소시켜 반추가축에 있어서의 광물질 부족 및 분으로의 배설 증가로 인한 토양의 오염증가가 예상된다.

• 한국식품과학회지
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• 제32권2호
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• pp.231-237
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• 2000

한국산 소맥 고유의 무기질에 관한 특성을 조사하기 위하여, 한국산 소맥 5품종과 외국산 소맥 3가지 품종의 회분함량과 밀가루 및 반죽의 색깔, 무기질 조성과 phytate 함량을 조사하였다. 한국산 밀 품종은 수입 소맥보다 회분 함량이 많으며 통밀의 회분 함량이 많을수록 밀가루의 회분 함량도 증가되었다. 또한 통밀의 회분 함량이 중가하면 밀가루의 적색도가 높아지며 따라서 백색도가 저하되는 상관관계를 볼 수 있었다. 한국산 통밀의 무기질 조성은 Mg, Fe과 P 등이 수입소맥들과 비교하여 많이 함유되어 있으며, 제분된 밀가루에서는 Fe의 함량이 수입소맥들과 비교하여 특히 많이 함유되어 있었다. Fe은 통밀과 밀가루 모두에서 한국산 소맥에 월등히 많은 양 함유되어 있는 것으로 미루어 Fe은 제분과정에서 제거되지 않으며, 겨와 호분층 이외에 배유에도 분포되어 있기 때문에 제분된 뒤에도 Fe은 밀가루에서 많은 양 검출되는 것으로 생각된다. 무기질의 흡수를 제해하는 phytate는 한국산 소맥이 수입소맥들과 비교하여 많은 양 함유되어 있었으나, 제분된 밀가루에서는 함량에 있어서 큰 차이를 나타내지 않고, 밀가루와 통밀의 phytate 함량간에 유의적 상관관계가 없는 것으로 보아 많은 양의 phytate가 제분과정에서 제거되는 것으로 생각된다. 또한 통밀의 phytate 함량과 통밀의 P 함량간에는 유의적 상관관계가 확인되며 밀가루의 phytate 함량과 밀가루의 P의 함량간에도 유의적 상관관계가 확인되어 한국산 소맥의 P는 phytate 내에 존재하는 P가 상당부분 차지하며, phytate가 제분과정에서 제거되기 때문에 많은 양의 P도 제거되는 것으로 생각된다.

• Asian-Australasian Journal of Animal Sciences
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• 제14권2호
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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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• 제10권6호
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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.

• 한국가금학회지
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• 제27권3호
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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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• 제13권5호
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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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• 제45권2호
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• pp.229-240
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• 2003

밀기울의 수침처리가 밀기울 내 phytase에 의한 피틴태 인의 분해에 미치는 영향을 연구하기 위하여 in vitro 실험을 실시하였고, 육계사료에 밀기울을 첨가 하였을 때 밀기울에 들어 있는 phytase가 육계 생산성 및 P 이용률에 미치는 영향을 구명하기 위해 5주간의 사양실험을 실시하였다. 수침처리는 배양온도 55$^{\circ}C$와pH 5.5 완충용액에서 배양액과 밀기울의 비율 및 배양 시간을 달리하고, 배양 후 55, 65와 75$^{\circ}C$에서 건조 시켰다. 피틴태 인 함량 감소(phytic acid 분해)에 가장 큰 영향을 미치는 요인으로는 밀기울과 완충용액의 비율로 밀기울:완충용액 비율이 증가할수록 곡선적(quadratic)으로 피틴태 인 함량이 감소 하였으며 1:5 전후가 효율적이었고 배양시간은 10분 이상에서는 큰 차이가 없었다. 건조온도(55$^{\circ}C$, 65$^{\circ}C$와 75$^{\circ}C$)와 완충용액의 pH(5.5 및 7.0)는 큰 영향을 미치지 않았다. 사양 실험은 갓 부화한 240수의 병아리(Ross®)를 24 cage에 10수씩 공시하여 4처리 6반복(처리별 암수 각 3반복)으로 완전임의 배치하였다. 처리구 들은 대조구; 정상수준의 nonphytate-P(NPP)구, LP구; 저 NPP구로 대조구보다 0.1% 낮음, LPWB구; 저 NPP구(LP)에+밀기울로 475IU phytase 공급, LPHWB구; LP+수침 후 건조 처리한 밀기울을 LPWB와 동량 공급한 구 등이었다. 실험 결과 증체량에 있어서 전기(1~21일)의 경우 타 처리구들에 비해 LP구가 유의하게 낮았으며 육성기(22-5일)와 전 기간에 있어서는 LP구의 웅추만이 대조구에 비해 유의하게 낮았다. 전 기간동안 LPWB구와 LPHWB구는 대조구와 차이가 없었다. 전 기간동안 사료 섭취량은 LPWB는 대조구와 유의한 차이가 없었으나 LP구는 LPHWB보다 그리고 LPHWB구는 대조구보다 유의하게 낮았다. 사료 요구률은 LPHWB와 LP구가 대조구와 LPWB보다 유의하게 낮았다. 폐사율은 LPHWB구에서 가장 높았다. 영양소 이용률에 있어서 LP구가 조지방, 조회분과 Ca 이용률이 타처리구에 비해 유의하게 낮았으나 Fe이용률 만은 가장 높았다. P, Mg 및 Zn의 이용률은 LPWB 및 LPHWB구가 대조구나 LP구 보다 높았다. P 배설량은 대조구에 비해 저 NPP구들이 낮았으나 저 NPP구들 간에는 차이가 없었다. 혈청 내 Ca 함량은 LP구가 가장 높았고 P 함량은 LP구가 가장 낮았다. 경골 내 조회분 함량은 밀기울 처리들에서 높았지만 Ca 함량은 대조와 LP구가 높았다. P 함량에선 LP가 LPWB보다 낮았다. Fe 함량은 LP구가 가장 높았다. 결론적으로 밀기울의 피틴태 인 감소에 수침처리 조건으로 밀기울과 완충용액의 비율이 가장 큰 영향을 미치는데 밀기울에 대한 완충용액의 비율이 증가함으로써 피틴태 인 함량이 감소하였으며 1:5 전후가 효율적이었고 저 NPP사료에 밀기울을 phytase 공급원으로 사용 시 육계의 생산성 감소를 방지하고 P의 배설량을 줄일 수 있다. 밀기울의 수침처리는 광물질 이용률 향상에 다소 도움이 되나 생산성 향상에는 도움이 되지 않았다.

• Journal of Microbiology and Biotechnology
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• 제34권5호
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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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• 제29권3호
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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.