Phytase 생산 균주의 분리 및 동정

양시용,배경숙,김창원,오태광,박근규 건국대학교 동물자원연구센터 1999 動物資源硏究誌 Vol.20 No.-

본 연구는 산업적으로 유용한 exo-type의 phytase 활성이 높은 균주의 선발 및 동정을 위해 실시하였다. 썩은 나무, 흙 등에서 분리한 1.500여 균주(곰팡이 700여 균주, 방선균 500여 균주, 세균 300여 균주)를 0.5% Ca-phytate를 함유한 phytase screening 배지를 이용하여 1차적으로 phytase 활성을 나타내는 균주를 선발한 결과 300여 균주를 얻었으며, 2차로 Na-phytate를 기질로 하여 exo-type의 phytase 활성을 측정한 결과 0.1 units/ml 이상의 높은 phytase 활성을 나타내는 곰팡이 5균주를 얻었으며, 이중 가장 phytase 활성 및 specific activity가 높은 No. 343 균주를 최종적으로 선발하여 형태학적 특징을 관찰하여 동정한 결과 Penicillium waksmanii로 동정되었다. 5 L jar fermenter(2 vvm, 500 rpm)를 이용하여 YM 배지에서 28℃ 배양조건으로 배양한 결과 96 시간 경과시 가장 높은 phytase 활성을 나타내었으며, 이때 배양액의 pH는 3.2∼3.5로 나타났다. Phytase (myo-inositol hexakisphosphate phosphohydrolase; EC 3.1,3.8) catalyzes the hydrolysis of myo-inositol hexakisphosphate, commonly known as phytic acid, that is pricipal storage form of phosphorus in seeds and pollen. The phytase-producing microorganisms occurring on rotten woods and soils were selectively isolated on the phytase screening medium with 0.5% Ca-phytate. Among more than three-hundred isolated of phytase-producing microorganisms, one fungal isolate was selected, having the highest exo-phytase production. The exo-phytase producer, fungal isolate No. 343 was identified as a strain of Penicillium waksmanii on the basis of morphological characteristics. Maximum production of phytase was reached after 96 hr of cultivation with aeration of 2 vvm in a 5 L jar fermenter.

성장 발육에 따른 흰쥐 장기내 phytase의 분포성

양원진 한국생명과학회 1997 생명과학회지 Vol.7 No.2

Phytase(myo-inositol hexakisphosphate phosphohydro-lase;EC 3.1.38)활성은 소화기관인 소장 점막에서나 나타났고, 그 외 다른 장기에서는 alkaline phosphatase활성만을 측정할 수 있었다. 그리고 anti-90kDa phytase항혈청을 이용한 면역조직학적 조사 결과 소장 이외의 장기에서는 본 효소의 단백질 밴드가 검출되지 않았다. 이와 같은 결과 phytase는 소장에만 특이적으로 존재하며, 소장 특이적 효소의 생리적 역할로서 Phytic acid(inositol=hexakisphos-phate)를 가수분해한다. 흰쥐의 성장 발육과 더불어 phytase의 활성은 증가한다. 출생 후부터 이유기 전까지는 70kDa phytase외에도 90kDa phy-tase가 출현한다. 이 90kDa phy-tase는 이유기에 합성되는 것으로 추정된다. The phytase(myo-inositol hexkisphosphate phosphohydrolase ; EC 3.1.3.8) activity was observed only in the homogenate of intestinal mucosa, though the activity of alkaline phisphatase was measurable in various organs. In addition, no protein bands were detected in any other organs on immunoblotting using the anti-90kDa phytase antiserum. Thses results suggest that phytase is specifically present in small intestinal mucosa, and that hydrolysis of phytic acid(inositol-hexakisphosphate) can be allotted for a physiological role of the intestine-specific enzyme. The activities of phytase was increased during development of rat. The 70kDa phytase appeared just after birth, but the 90kDa phytase was not observed until adult period, suggesting that the 90kDa phytase was synthesized in response to weanling.

육계의 인 이용율 향상을 위한 식물성 Phytase의 이용

김병한,남궁환,백인기 한국동물자원과학회 2002 한국축산학회지 Vol.44 No.4

Phytase 공급원으로 밀과 밀기울의 첨가가 육계 생산성 및 P이용률에 미치는 영향을 측정하기 위해 5주간의 사양 실험을 실시하였다. 갓 부화한 1,000수의 병아리(Ross^R)를 20pen에 50수씩(암수 각 25수) 공시하여 5처리 4반복으로 완전임의 배치하였다. T1은 대조구로 정상수준의 nonphytate-P(NPP)를 함유하였고, T2는 T1-0.1% NPP의 低 NPP구, T3는 T2 + 사료 ㎏당 600IU microbial phytase(NOVO^R구, T4는 T2 + 밀과 밀기울로 사료 ㎏당 600IU plant phytase 공급구, T5는 T2 + 밀과 수침 후 건조 처리한 밀기울로 사료 ㎏당 600IU plant phytase 공급구 였다. NPP 수준을 0.1% 감소시킨(T2) 처리에서 생산성과 사료효율이 감소되었고 식물성 phytase 처리시(T4와 T5)이 microbial phytase 처리구(T3)보다 더 나은 결과를 보였다. 정상 밀기울구(T4)와 수침 후 건조 처리한 밀기울구(T5)와는 차이를 보이지 않았다. 폐사율은 低 NPP(T2)에서 가장 높았다. 육성 사료에서 조지방과 조회분 이용률에 일반 밀기울 처리구(T4)가 가장 높았다. Ca과 P의 이용률 또한 T4가 가장 높고 T3와 T5가 다음으로 높았다. Phytase 처리구(T3, T4 와 T5) 들은 MG, Fe와 Zn의 이용률을 크게 증가시켰다. Ca, P, Mg, Fe 와 Zn의 배설량은 microbial phytase가 가장 적었다. 혈청내 Ca과 Mg의 함량은 低 NPP에서 가장 높았고, 경골내 조회분 함량은 T2와 T3가 T1, T4와 T5보다 낮았다. 그러나 경골내 Ca 함량은 T1과 T2가 다른 처리구들보다 높았다. 경골내 P와 Mg의 함량은 T1구가 가장 높았다. 결론적으로 밀기울을 통한 식물성 phytase의 공급은 P 이용률 향상에 효과적이었다. 수침 후 건조처리하는 것은 이용률 향상에 도움이 되지 않았다. This study was conducted to evaluate the efficacy of wheat and wheat bran as the source of phytase in a 5 week broiler feeding trial. One thousand day-old broiler chickens(Ross^R) were divided into 20 pens of 50 broilers(25 male and 25 female) each. Four pens were randomly arranged to one of the five dietary treatment; T1, control diet containing normal nonphytate P(NPP) ; T2, T1 - 0.1% NPP; T3, T2 + 600IU microbial phytase(NOVO^R) per ㎏ diet; T4, T2 + 600IU plant phytase from wheat and wheat bran; T5, T2 + 600IU plant phytase from wheat and hydrothermally treated wheat bran. Reduction of NPP level by 0.1%(T2) reduced weight gain and feed intake but plant phytase treatments(T4 and T5) recovered the lost performance. Plant phytase treatments showed better(p<0.05) weight gain and intake than the microbial phytase treatment(T3). There was no difference between regular wheat bran treatment(T4) and hydrothermally treated wheat bran treatment(T5). Mortality was the highest by low NPP diet(T2). Availability of ether extract and crude ash of grower diet was the highest(p<0.05) in normal wheat bran diet(T4). Availability of Ca and P of grower diet was the highest(p<0.05) in T4 followed by T3 and T5. Availability of Mg, Fe and Zn was drastically improved by phytase treatments(T3, T4 and T5). Excretion of Ca, P, Mg, Fe and Zn was the lowest(p<0.05) with microbial phytase treatment(T3). Serum level of Ca and Mg was the highest(p<0.05) with the low NPP treatment(T2). Tibial ash content of T2 and T3 was lower(p<0.05) than that of T1, T4 and T5. However, tibial Ca content was higher(p<0.05) in T1 and T2 than other treatments. Tibial P and Mg contents were the highest(p<0.05) in T1. It was concluded that plant phytase from wheat bran can be effectively used to improve P utilization. Hydrothermal treatment of wheat bran prior to inclusion in the diet had no benefical effects.

Phytase를 생산하는 Enterobacter cloacae의 분리 및 효소 생산의 배지 최적화

김영훈,양시용,김대영,김창원,정원형,권문남,송민동 한국산업미생물학회 2001 한국미생물·생명공학회지 Vol.29 No.2

본 연구는 phytic acid를 myo-inositol과 무기태인으로 분해시키는 효소인 phytase 생산균주의 분리 및 효소생산의 배지최적화에 관한 것이다. 1차로 calcium phytate를 기질로 함유한 phytase screening 배지를 이용하여 phytase 생산을 나타내는 균주 35가지를 이용하여 분리한 후, sodium phytate를 기질로 하여 재현성 있는 phytase 활성을 나타내는 균주 12가지를 선발하였다. 12개의 균주 중 BHI broth에서 배양한 조효소액의 phytase 활성이 가장 우수한 것으로 나타난 YH100을 선발하여 주사현미경 관찰, 16S rRNA sequence 분석, GC content(mol%) 조성, 지방산 분석, API 20E kit를 이용한 test 결과 Enterobacter cloacae로 동정되어, 이 균주를 Enterobacter cloacae YH100이라 명명하였다. Enterobacter cloacae YH100에 의한 phytase생산을 위한 최적 배지 조성을 파악한 결과 glucose 2.0%(w/v), peptone 1.0%(w/v), beef extract 1.0%(w/v), KCl 0.1%(w/v), sodium phytate 0.1%(w/v)로 나타났다. Phytase(myo-inositol hexakisphosphate phosphohydrolase: EC 3.1.3.8) hydrolyzes phytic acid(myo-inositol hexakisphosphate) to myo-inositol and monophosphates. In order to obtain phytase producing bacteria, many samples were collected from various soils. Among thirty-five phytase-producing strains, YH100 showed the highest phytase activity. In order to identify the selected YH100 strain, the morphological and physiological characteristics were examined according to the method of Bergey's manual by 16S rRNA sequence, cellular fatty acids profile, G+C contents and physiological test using API 20E kit. The strain YH100 identified to be a genus of Enterobacter cloacae and was named as Enterobacter cloacae YH100. Optimum medium for the phytase production by the Enterobacter cloacae YH100 was composed of 2.0%(w/v) glucose, 1.0%(w/v) peptone, 1.0%(w/v) beef extract, 0.1%(w/v) KCl, and O.1%(w/v) sodium phytate.

Acid Phytase를 생산하는 Pseudomonas fragi의 분리와 phytase의 생산조건

김영진,장은석,인만진,오남순,Kim, Young-Jin,Jang, Eun-Seok,In, Man-Jin,Oh, Nam-Soon 한국응용생명화학회 2003 한국농화학회지 Vol.46 No.4

축산폐수가 방류되는 단위동물 밀집사육장 인근의 하천수로부터 phytase를 생산하는 세균들 중 활성이 가장 우수한 균주를 분리하여, Pseudomonas fragi Y9451로 동정, 명명하였다. 탄소원으로 fructose를 첨가할 경우에 phytase의 생산능이 가장 우수하였고, 배지에 7% 첨가할 경우에 72시간 배양 후 효소활성이 1,078 mU/ml로 최대의 phytase 생산능을 보였다. 또한, 질소원으로는 nutrient broth(NB)가 적합하였으며, NB를 3%의 농도로 첨가할 경우에 96시간 배양 후 효소활성이 1,425 mU/ml까지 향상되었다. 인산원인 $KH_2PO_4$, phytate는 첨가 농도가 높을수록 phytase 생산은 억제되었으며, 무기염으로 $MgSO_4$와 $CaCl_2$를 첨가하는 경우는 첨가농도와 상관없이 phytase의 생산을 현저히 억제하였다. 산업생산을 목적으로 하는 scale-up에 있어서의 통기교반 효과는 phytase의 생산량에 큰 영향을 미치는 것으로 나타났다. A bacterial strain producing a high level of an extracellular phytase was isolated from livestock waste water, identified as a strain of Pseudomonas fragi and designated as Pseudomonas fragi Y9451. Under the phytase production medium, the activity of phytase reached the highest level after 120 hours of incubation. On the effect of carbon sources on the phytase production, the most favorable carbon source for phytase production was fructose. As for the effect of nitrogen sources, high levels of phytase activity were detected in the medium containing nutrient broth as the nitrogen source. Free $PO_4^{3-}$ inhibited phytase production with increasing concentration of $KE_2PO_4$ and phytate in the media. The addition of $CaCl_2$ and $MgSO_4$ also resulted in the inhibition of phytase production. To investigate the effect of aeration on the phytase production, different volumes of culture broth in Erlenmeyer flasks were incubated in rotary shaker at the speed of 200 rpm. As a result, a high level of phytase activity was detected at small volume of culture broth as compared to larger volume because of its more aerobic condition.

Wheat phytase can alleviate the cellular toxic and inflammatory effects of lipopolysaccharide

( Jeongmin An ),( Jaiesoon Cho ) 한국축산학회(구 한국동물자원과학회) 2021 한국축산학회지 Vol.63 No.1

The objective of this study was to characterize the enzymatic hydrolysis of lipopolysaccharide (LPS) by wheat phytase and to investigate the effects of wheat phytase-treated LPS on in vitro toxicity, cell viability and release of a pro-inflammatory cytokine, interleukin (IL)-8 by target cells compared with the intact LPS. The phosphatase activity of wheat phytase towards LPS was investigated in the presence or absence of inhibitors such as L-phenylalanine and L-homoarginine. In vitro toxicity of LPS hydrolyzed with wheat phytase in comparison to intact LPS was assessed. Cell viability in human aortic endothelial (HAE) cells exposed to LPS treated with wheat phytase in comparison to intact LPS was measured. The release of IL-8 in human intestinal epithelial cell line, HT-29 cells applied to LPS treated with wheat phytase in comparison to intact LPS was assayed. Wheat phytase hydrolyzed LPS, resulting in a significant release of inorganic phosphate for 1 h (p < 0.05). Furthermore, the degradation of LPS by wheat phytase was nearly unaffected by the addition of L-phenylalanine, the inhibitor of tissue-specific alkaline phosphatase or L-homoarginine, the inhibitor of tissue-non-specific alkaline phosphatase. Wheat phytase effectively reduced the in vitro toxicity of LPS, resulting in a retention of 63% and 54% of its initial toxicity after 1-3 h of the enzyme reaction, respectively (p < 0.05). Intact LPS decreased the cell viability of HAE cells. However, LPS dephosphorylated by wheat phytase counteracted the inhibitory effect on cell viability. LPS treated with wheat phytase decreased IL-8 secretion from intestinal epithelial cell line, HT-29 cell to 14% (p < 0.05) when compared with intact LPS. In conclusion, wheat phytase is a potential therapeutic candidate and prophylactic agent for control of infections induced by pathogenic Gram-negative bacteria and associated LPS-mediated inflammatory diseases in animal husbandry.

Acid Phytase를 생산하는 Pseudomonas fragi의 분리와 phytase의 생산조건

김영진,장은석,인만진,오남순 한국응용생명화학회 2003 Applied Biological Chemistry (Appl Biol Chem) Vol.46 No.4

cnrtkscP수가 방류되는 단위동물 밀집사육장 인근의 하천수로부터 phytase를 생산하는 세균들 중 활성이 가장 우수한 균주를 분리하여 Paseudomonas fragi Y9451로 동정, 명명하였다. 탄소원으로 fructose를 첨가할 경우에 phytase의 생산능이 가장 우수하였고, 배지에 7% 첨가할 경우에 72시간 배양 후 효소활성이 1,078mU/m로 최대의 phytase생산능을 보였다. 또한, 질소원으로는 nutrient broth(NB)가 적합하였으며, NB를 3%의 농도로 첨가할 경우에 96시간 배양 후 효소활성이 1,425mU/m까지 향상되었다. 인산원인 KH₂PO₄ phytate는 첨가 농도가 높을수록 phytase생산은 억제되었으며, 무기염으로 MgSO₄와 CaCI₂를 첨가하는 경우는 첨가농도와 상관없이 phytase의 생산을 현저히 억제하였다. 산업생산을 목적으로 하는 scale-up에 있어서의 통기교반 효과는 phytase의 생산량에 큰 영향을 미치는 것으로 나타났다. A bacterial strain producing a high level of an extracellular phytase was isolated from livestock waste water, identified as a strain of Pseudomonas fragi and designated as Pseudomonas fragi Y9451. Under the phytase production medium, the activity of phytase reached the highest level after 120 hours of incubation. On the effect of carbon sources on the phytase production, the most favorable carbon source for phytase production was fructose. As for the effect of nitrogen sources, high levels of phytase activity were detected in the medium containing nutrient broth as the nitrogen source. Free PO₄^(3-) inhibited phytase production with increasing concentration of KH₂PO₄ and phytate in the media. The addition of CaCl₂ and MgSO₄ also resulted in the inhibition of phytase production. To investigate the effect of aeration on the phytase production, different volumes of culture broth Erlenmeyer flasks were incubated in rotary shaker at the speed of 200 rpm. As a result, a high level of phytase activity was detected at small volume of culture broth at compared to larger volume because of its more aerobic condition.

Wheat phytase potentially protects HT-29 cells from inflammatory nucleotides-induced cytotoxicity

Jeongmin An,Jaiesoon Cho Asian Australasian Association of Animal Productio 2023 Animal Bioscience Vol.36 No.10

Objective: The aim of this study was to investigate the protective effect of wheat phytase as a structural decomposer of inflammatory nucleotides, extracellular adenosine triphosphate (ATP), and uridine diphosphate (UDP) on HT-29 cells. Methods: Phosphatase activities of wheat phytase against ATP and UDP was investigated in the presence or absence of inhibitors such as L-phenylalanine and L-homoarginine using a Pi Color Lock gold phosphate detection kit. Viability of HT-29 cells exposed to intact- or dephosphorylated-nucleotides was analyzed with an EZ-CYTOX kit. Secretion levels of pro-inflammatory cytokines (IL-6 and IL-8) in HT-29 cells exposed to substrate treated with or without wheat phytase were measured with enzyme-linked immunosorbent assay kits. Activation of caspase-3 in HT-29 cells treated with intact ATP or dephosphorylated-ATP was investigated using a colorimetric assay kit. Results: Wheat phytase dephosphorylated both nucleotides, ATP and UDP, in a dose-dependent manner. Regardless of the presence or absence of enzyme inhibitors (L-phenylalanine and L-homoarginine), wheat phytase dephosphorylated UDP. Only L-phenylalanine inhibited the dephosphorylation of ATP by wheat phytase. However, the level of inhibition was less than 10%. Wheat phytase significantly enhanced the viability of HT-29 cells against ATP- and UDP-induced cytotoxicity. Interleukin (IL)-8 released from HT-29 cells with nucleotides dephosphorylated by wheat phytase was higher than that released from HT-29 cells with intact nucleotides. Moreover, the release of IL-6 was strongly induced from HT-29 cells with UDP dephosphorylated by wheat phytase. HT-29 cells with ATP degraded by wheat phytase showed significantly (13%) lower activity of caspase-3 than HT-29 cells with intact ATP. Conclusion: Wheat phytase can be a candidate for veterinary medicine to prevent cell death in animals. In this context, wheat phytase beyond its nutritional aspects might be a novel and promising tool for promoting growth and function of intestinal epithelial cells under luminal ATP and UDP surge in the gut.

Microbial Phytase와 무기태 인 수준별 급여가 산란 생산성, 소화율 및 계란 품질에 미치는 영향

김상호,유동조,박수영,이상진,박용윤,이원준 한국가금학회 2000 韓國家禽學會誌 Vol.27 No.3

The effect of supplemental microbial phytase and non - phytate phosphorus(NPP) levels on layer productivity and nutrient digestibility were conducted in 640 21 weeks - old HyLine brown layer for 12 weeks. Supplemented phytase levels were 0, 300, 500 and 1,000 DPU/kg diet. NPP levels were adjusted with tricalcium phosphate(TCP), which were 0(0.11% NPP), 0.5(0.20), 1.0(0.29) and 1.5%(0.38). ME, CP and Ca levels were maintained at 2,800㎉/kg diet, 16% and 3.5%, respectively. Egg production was increased with phytase compared to without phytase(P〈0.05). Increasement of egg production was higher latter of experimental period. Egg production was not different to phytase levels. Egg production in TCP levels were increased in above 0.5% compared to 0% TCP. Difference of egg production by TCP was higher after 6 week. Especially, egg production to supplemental phytase was higher in 0% TCP. Egg weight was not different to phytase and TCP levels. Egg mass was increased with phytase compared to without phytase, but not difference significantly. There was similar to phytase levels. Egg mass in TCP group was increased in TCP supplementation(P〈0.05). Feed intake was not different in phytase levels, and greater with increasing TCP levels(P〈0.05). Feed conversion was improved with phytase(P〈0.05), and not difference in TCP levels. All of nutrients digestibility tended to improve with phytase, P(P〈0.05), especially. There were not different among phytase levels. The effect of adding phytase was higher in low phosphorus diets compared normal levels. Eggshell breaking strength and eggshell thickness also improved in added phytase(P〈0.05). Tibial ash and P content were slightly increased with phytase, and Ca content also was higher(P〈0.05) compared without phytase. We concluded that supplemental phytase in low phosphorus diet was showed to increase laying performance, feed efficiency, nutrients digestibility, egg quality, and bone development. Phytase supplementation was able to compensate for low NPP diet. We also thought optimum phytase level is 300 DPU, and can decrease NPP supplementation adding phytase in later diet.

Response of broiler chickens to diets containing different levels of sodium with or without microbial phytase supplementation

( Marjina Akter ),( Hadden Graham ),( Paul Ade Iji ) 한국축산학회(구 한국동물자원과학회) 2019 한국축산학회지 Vol.61 No.2

Phytate induced excessive mineral excretion through poultry litter leads to poor performance and environmental pollution. Exogenous microbial phytase supplementation to poultry diets reduce the environmental excretion of nutrient and improve bird’s performance. However, excessive dietary sodium (Na) level may hinder the phytase-mediated phytate hydrolysis and negate the beneficial effects of phytase. Therefore, this experiment was conducted to investigate the effects of different concentration dietary Na on phytase activity and subsequent impact on broiler performance, bone mineralisation and nutrient utilisation. In this study, six experimental diets, consisting of three different levels of Na (1.5, 2.5, or 3.5 g/kg) and two levels of microbial phytase (0 or 500 U/kg) were formulated by using 3 × 2 factorial design. The six experimental diets were offered to 360 day-old Ross 306 male chicks for 35 days, where, each experimental diet consisted of 6 replicates groups with 10 birds. Along with growth performance, nutrient utilization, intestinal enzyme activity, dry matter (DM) content of litter and mineral status in bone were analysed. Dietary Na and phytase had no effect on bode weight gain and feed intake. Birds on the low Na diet showed higher (p < 0.05) feed conversion ratio (FCR) than the mid-Na diets. High dietary Na adversely affected (p < 0.001) excreta DM content. Phytase supplementation to the high-Na diet increased (p < 0.01) the litter ammonia content. High dietary Na with phytase supplementation improved (Na × phytase, p < 0.05) the AME value and ileal digestibility of Ca and Mg. The total tract retention of Ca, P, and Mg was reduced with high Na diet, which was counteracted by phytase supplementation (Na × phytase, p < 0.001). The diets containing mid-level of Na improved (p < 0.001) the function of Na-KATPase and Mg-ATPase in the jejunum. The overall results indicate that high dietary Na did not affect phytase activity but influenced the nutrient utilization of birds, which was not reflected in bird overall performance.