Molecular Cloning of Phytase Gene from ASUIA279 and Its Expression in Pichia pastoris System
Mohd Hafidz Mahamad MaifiahParveen JamalTamrin NugeNor Soleha Mohd DaliAnis Shobirin Meor HussinAbd‐ElAziem FaroukAnd Hamzah Mohd. Salleh
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Phytases catalyze the hydrolysis of phytate (myo-inositol hexakisphosphate), one of the major storage form of phosphate in plants, with subsequent release of myo-inositol, phosphate and phytate-bound minerals. Non-ruminant animals such as chicken, swine and fish can't use the organic phosphorus and minerals from their diet because there is no phytase activity in their digestive tract. Phytate degrading enzyme is added to the animal feed diet to improve phosphorus availability from the dietary phytate and at the same time this lessen the phosphate pollution level in areas of intensive animal production as the phosphate would not be excreted out to the environment. ASUIA279, a bacterial strain isolated from Malaysian soil has potentially shown good phytase activity. In the present work, the gene encoding for phytase has been amplified from the plasmid DNA of recombinant ASUIA279(5) obtained from a previous study (unpublished data) by a polymerase chain reaction (PCR) methodology. The amplified phytase gene was extracted, purified then cloned into the pPICZï¡A plasmid and transformed into Pichia pastorisX-33 strain for enzyme production.ABSTRAK: Fitase pemangkinan hidrolisis fitat (myo-inositol hexakisphosphate), merupakan salah satu cara penyimpanan utama fosfat dalam tumbuhan, dengan pelepasan berturut myo-inositol , fosfat dan galian terikat fitat. Haiwan bukan ruminan seperti ayam, khinzir dan ikan tidak dapat memanfaatkan fosforus organik serta galian yang diperolehi daripada makanan kerana tidak mempunyai aktiviti fitase di dalam saluran pencernaan mereka. Enzim pengecilan fitase dicampurkan ke dalam pemakanan haiwan untuk mempertingkatkan keperolehan fosforus dari fitat diet. Pada masa yang sama ia dapat mengurangkan tahap pencemaran fosfat di kawasan yang terdapat penternakan haiwan secara intensif agar fosfat tidak dikumuhkan ke persekitaran. ASUIA279, satu strain bakteria yang diasingkan daripada tanih di Malaysiamenunjukkan aktiviti fitase yang memberansangkan. Buat masa kini, pengekodan gen fitase telah diperkuatkan dengan plasmid DNA dari ASUIA279(5), rekombinan yang diperolehi daripada kajian terdahulu (data tak diterbitkan) berdasarkan kaedah tindak balas berantai polimerase (polymerase chain reaction (PCR)). Gen fitase yang diperkuatkan, ditulenkan dan kemudian diklonkan menjadi plasmid pPICZαA dan berubah menjadi strain Pichia pastorisX-33 untuk penghasilan enzim.KEYWORDS: Phytase, animal feed, pollution, polymerase chain reaction (PCR), Pichia pastorisKeywords:
Phytase
Phytase are enzymes capable of hydrolyzing phytic acid to less-phosphorylated myo-inositol derivates and inorganic phosphate. The addition of phytase to feed can resolve the antinutrition of phytic and increase the bioavailablity of protein and minerals. It also can dispel the pollution of phosphorus in animal wastes. This paper summarized the research progress of phytase from aspects such as the source of phytase, zymetology nature, genetic engineerings, and discussed the problem and the prospect of phytase.
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본 연구는 산란계에 대한 phytase의 적정 첨가수준 및 생산성에 대한 효과를 구명하고자 TCP 4수준 0 %(무기 태인 0.11 %), 0.5 % (0.20 %), 1.0 % (0.29 %), 1.5 %(0.38 %)과 phytase 4수준 0, 300, 500, 1000 DPU에 대하여 4×4 요인 시험을 12주간 실시하였다. 산란율은 phytase 첨가가 무첨가에 비하여 증가한 것으로 나타났으며(P＜0.05), 사육기 간이 지속될수록 증가폭이 커지는 것으로 나타났다. Phytase 첨가수준에서는 차이가 없는 것으로 나타났다. TCP 수준에서는 0.5 % 수준 이상 처리에서 무첨가에 비하여 증가하였으며 (p＜0.05), 6주 이후 TCP 첨가에 의한 산란율의 차이가 심화되는 것으로 나타났다. 난중은 phytase, TCP 수준간 차이가 없는 것으로 나타났다. 산란량은 산란율과 비슷한 경향을 보였는데 phytase 첨가가 무첨가에 비하여 유의성은 없었으나 증가하는 경향을 보였으며, TCP는 0.5 %(0.20 % NPP) 이상 함유한 처리에서 0 % (0.11 % NPP) 비하여 산란량이 증가하는 것으로 나타났다(P＜0.05). 사료섭취량은 phytase 수준별 차이가 없었으며, TCP 수준이 증가할수록 섭취량이 증가하는 것으로 나타났다(P＜0.05). 사료요구율은 phytase 첨가구가 무첨가구에 비하여 개선되었으며(P＜0.05), TCP 수준간에는 차이가 없었다. 영양소소화율은 phytase를 첨가가 무첨가에 비하여 향상되는 경향을 보였으나, 통계적 유의성은 인정되지 않았다. 인의 소화율은 phytase 첨가구가 무첨가에 비하여 특히 개선되었으며(P＜0.05), phytase 수준에 있어서는 차이가 없었다. 무기태인의 수준이 낮을 때 phytase에 의한 영양소 소화율이 향상되었으며, 무기태인의 수준이 권장량 수준일 경우는 phytase에 의한 향상효과가 낮았다. 무기태인의 수준별 비교에서는 뚜렷한 차이가 없었다. 난각질은 시험 경과 6주부터 난각강도 빚 난각두께가 향상되었으며(P＜0.05), 12주도 비슷하게 나타났다. 난각질 역시 무기태인의 수준이 낮을 때 phytase 첨가에 의한 효과가 크게 나타났다. TCP 0%수준에서 난각질이 떨어지는 것으로 나타났으며, 0.5 %(0.20 % NPP)수준이상에서는 비슷하게 나타났다(P＜0.05), 경골 회분과 칼슘 및 인의 함량은 phytase 첨가구가 약간 높았으며, 칼슘함량은 유의적으로 무첨가구보다 높았다(P＜0.05). TCP 수준에 의한 차이는 없었다. 이상의 결과로 무기태인의 수분이 낮은 사료에 phytase 첨가시 생산성 및 영양소 이용성을 향상시킬 수 있었는데 이는 부족되는 무기태인의 수준을 보충해 주기 때문으로 생각된다. 또, 산란사료에 phytase 300 DPU 수준 첨가시 무기태인 수준을 낮은 농도로(0.11 %) 급여할 수 있다고 판단된다.
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Optimal condition, stability and Km of phytase which was from Aspergillus oryzae093 were studied. The optimal temperature and pH for sodium-phytate hydrolysis by Aspergillus oryzae093 phytase is 50°C and 5.5 respectively. Phytase has better heat stability as temperature below 45°C. The phytase activity remained 90% when pH is 5.0 to 6.0. Low concentration Fe2+ and Mg2+ can promote phytase activity, while Cu2+, Mn2+, Fe3+, Al3+ or high concentration Fe2+ and Mg2+ can inhibite the activity of phytase. The Km is about 4.8×10-5 mol•L-1 when sodium phytate as substrate. The enzyme properties of Aspergillus oryzae093 phytase make it could apply to food and feed to reduce the phosphorus exhausted to environment.
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Pure cotton fabric was pretreated with cellulase enzyme (Neutrox BDN 100 Powder) and the enzymatic conditions were optimized. These included pH of media 7, enzyme concentration 5% (o.w.f) and the treatment time of 75 minutes, optimized on the basis of effect of pre-treatment on physical properties. The cellulase treatment produces fabric which is more absorbent.
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To study the relevance of native phytase for phosphorus digestibility and for the relation between P-digestibility and the effectiveness of supplements of microbial phytase balance experiments with 5 pigs (40 - 70 kg LW) was designed. Diets based on cereals and soyabean meal were fed, including four treatments with untreated and phytase inactivated cereals, accomplished by extrusion. Both diets were supplemented with the microbial phytase with a dose of 800 U/kg. Reduction of phytase activity decreased P-digestibility of the diets from 45 % to 18 %. Microbial phytase supplements increased P-digestibility in the untreated diet by 7 %-U to 52 % and in the diet with inactivated phytase from 18 % to 47 %. The results document the effects of native phytase activity on P-digestibility and show that the efficacy of supplements of microbial phytase depends basicly on the amount of native phytase activity and correspondingly on P-digestibility.
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Phytase
Aspergillus niger
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Two experiments were conducted to compare a novel phytase with its commercial predecessor. In Exp. 1, Cobb 500 (n = 720) male broilers were weighed and fed one of 10 treatments from d 8 to 18 post-hatch. Treatments consisted of a nutrient adequate positive control (PC), a Ca and non-phytate P (nPP) deficient negative control (NC), the NC plus the novel phytase (phytase 1) at 187.5, 375, 750, 1,500 or 2,000 FYT/kg or the NC diet plus the commercially available phytase (phytase 2) at 500, 1,000 or 2,000 FYT/kg. In Exp. 2, Ross 308 (n = 1,008) male broilers were assigned to one of 7 treatments from d 1 to 36 post-hatch. The treatments consisted of a PC, a NC, the NC diet plus phytase 1 at 500, 1,000 or 2,000 FYT/kg or the NC diet plus phytase 2 at 1,000 or 2,000 FYT/kg. In both Exp. 1 and 2, birds fed the NC diet had reduced (P < 0.05) weight gain (WG), feed intake (FI), or tibia ash compared with birds fed the PC. Phytase supplementation at >187.5 FYT/kg feed improved WG and FI in Exp. 1 and 2, whereas phytase 1 at 750 to 2,000 FYT/kg or phytase 2 at 2,000 FYT/kg was needed to improve tibia ash comparable to the PC in Exp. 1. Supplementing broiler diets with a novel phytase at a lower dose compared with the commercial phytase significantly increased ileal phytate degradation and P digestibility resulting in an increase in tibia ash. Tibia ash, ileal phytate P, and P digestibility are more sensitive, compared with growth performance, to detect differences between phytases at different dietary concentrations.
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Ruminants have the ability to utilize phytate phosphate by rumen microorganisms.Until now,domestic research on ruminal microbe phytase is insufficient.In this paper,phytase origin and activity in the rumen was briefly reviewed,and five kinds of rumen bacteria were capable of producing phytase.Among these bacteria,phytase activity was particularly prevalent in Selenomonas ruminantium(199.1-703.6 U·mL-1).Based on their optimal pH and catalytic mechanisms,ruminal phytases are classified into acid phytase and cysteine phosphatases phytase,respectively.According to the stereospecificity of phytase,rumen microbes may exist 3-phytase and 5-phytase.Furthermore,kinetics of enzymatic reaction characteristics of phytases were summarized and discussed comprehensively.In a brief,phytase of S.ruminantium has a optimal temperature of 50-55 ℃,an optimal pH range of 4.0-5.5,and exhibits the highest specificity constant for myo-inositol hexakisphosphate and ATP.The optimal phytase activity of Megasphaera elsdenii is displayed at pH5.0 and 60 ℃,and it also displays strict specificity for myo-inositol hexakisphosphate.
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Phytases increase utilization of phytate phosphorus in feed. Since wheat is rich in endogenous phytase activity it was examined whether wheat phytases could improve phytate degradation compared to microbial phytases. Moreover, it was investigated whether enzymatic degradation of phytate is influenced by the matrix surrounding it. Phytate degradation was defined as the decrease in the sum of InsP₆ + InsP₅.Endogenous wheat phytase effectively degraded wheat Ins₆ + InsP₅ at pH 4 and pH 5, while this was not true for a recombinant wheat phytase or phytase extracted from wheat bran. Only microbial phytases were able to degrade InsP₆ + InsP₅ in the entire pH range from 3 to 5, which is relevant for feed applications. A microbial phytase was efficient towards InsP₆ + InsP₅ in different phytate samples, whereas the ability to degrade InsP₆ + InsP₅ in the different phytate samples ranged from 12% to 70% for the recombinant wheat phytase.Wheat phytase appeared to have an interesting potential. However, the wheat phytases studied could not improve phytate degradation compared to microbial phytases. The ability to degrade phytate in different phytate samples varied greatly for some phytases, indicating that phytase efficacy may be affected by the phytate matrix.
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Degradation
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본 시험은 Aspergillus ficuum으로부터 생산된 crude phytase가 육계의 생산성, 영양소이용율 및 경골과 혈장의 광물질 함량에 미치는 영향을 측정하고자 실시하였다. 갓 부화한 육계 수평아리 (Avian^®) 240수를 공시하여 2 수준의 phytase (0과 600 U/㎏)와 2 수준의 무기태 P(non-phytate P. NPP)의 요인실험으로서, P의 수준은 전기(1~21일)에 0.45 및 0.35% 이었고, 육성기(21~35일)에는 0.35 및 0.25% 이었다. 사료섭취량과 증체량은 phytase 첨가에 의해 유의하게 향상되었다 (P＜0.05). 사료효율과 사망율에 있어서는 처리간에 유의한 차이가 없었다. 영양소이용율은 P 수준과 phytase에 의해 유의한 영향을 받지 않았다. 저수준의 P과 phytase 첨가구의 P 배설량이 유의하게 감소되었다. 그러나 N의 배설량에는 유의한 차이가 없었다. 저 수준의 N 급여에 의해 Ca이용율이 향상되었으며 phytase 첨가에 의해 P, Mg 이용율은 향상되었으나 (P＜0.05) Fe과 Zn의 이용율은 감소되었다. 광물질 이용율에는 사료내 P 수준과 phytase 첨가간에 유의한 상호작용이 있었다. 경골의 무게는 저수준의 P 급여시 가볍고 짧았으며 phytase 첨가에 의해 무거워졌다. 경골내 Ca. P, Mg 함량은 저수준의 P 급여시 감소되었으며 phytase 첨가시 증가되었다. 경골의 회분함량은 phytase 첨가에 의해 증가되었다. 결론적으로 P 수준이 낮은 육계사료에 crude phytase 첨가시 생산성과 광물질이용율 (Ca, P 그리고 Mg)을 향상시키며 사료내 P 수준을 낮출 수 있으므로 P의 배설량을 효과적으로 감소시킬 수 있었다.
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