Fungal Phytase Research Articles

Isolation of a Phytase with Distinctive Characteristics from an Edible Mushroom, Pleurotus eryngii

From the fresh fruiting bodies of the mushroom Pleurotus eryngii a phytase with a molecular mass of 14 kDa was isolated. The isolation protocol entailed ion exchange chromatography on DEAE-cellulose and CM-cellulose, affinity chromatography on Affi-gel blue gel, and ion exchange chromatography on Q-Sepharose. The phytase was unadsorbed on DEAE-cellulose, CM-cellulose and Affi-gel blue gel, and adsorbed on Q-Sepharose. It appeared as a single band in SDSPAGE. It exhibited maximal activity at around 37°C. Its activity underwent little changes over the range of pH 3.0 to 9.0. The aforementioned characteristics are different from those of animal, plant and bacterial phytases. The low molecular mass and pH stability of P. eryngii phytase also distinguish it from mushroom phytases and other fungal phytases reported earlier. The purified enzyme exhibited a broad substrate specificity on a range of phosphorylated compounds, and the phytase demonstrated the N-terminal sequence ADNVYRHDNN which shows little homology to known phytases. It inhibited proliferation of human nasopharyngeal carcinoma CNE2 cells, hepatoma HepG2 cells and breast cancer MCF7 cells with an IC50 of 1.9 µM, 2.9 µM, and 1.0 µM, respectively. Keywords: Phytas isolation, Pleurotus eryngii, antiproliferative, fruiting bodies, mushroom, chromatography, fungal phytase, nasopharyngeal carcinoma, hepatoma

Application of Bifidobacterial Phytases in Infant Cereals: Effect on Phytate Contents and Mineral Dialyzability

Phytase activity was recently described in probiotic bifidobacterial strains, opening the possibilities for their use in foods, due to the generally regarded as safe/qualified presumption of safety status of these bacteria. Two raw materials for infant cereals (multicereal and gluten-free) were examined by measuring the myo-inositol phosphates content and the in vitro Ca, Fe, and Zn availability after a dephytinization process with purified phytases from Bifidobacterium longum spp. infantis and Bifidobacterium pseudocatenulatum. Treatment with both enzymes reduced the contents of phytate as compared to control samples (untreated or treated with fungal phytase) and led to increased levels of myo-inositol triphosphate. Dephytinization followed by an in vitro model of intestinal digestion increased the solubility of Zn. However, phytase treatment did not increase significantly the mineral dialyzability as compared to untreated samples. This is the first example of the application of purified bifidobacterial phytases in food processing and shows the potential of these enzymes to be used in products for human consumption.

Influence of Added Enzymes and Bran Particle Size on Bread Quality and Iron Availability

ABSTRACTThe aim of this investigation was to study the influence of different bran proportions and particle sizes, addition of fungal phytase, and α‐amylase addition on bread quality and phytate levels, and how these treatments affect availability of iron to intestinal epithelial (Caco‐2) cells. Potential mineral contributions to dietary reference intakes and phytate‐to‐mineral molar ratios were also evaluated. Wheat bran supplementation significantly affected bread quality. Smaller bran particle size affected crumb firmness negatively, whereas the use of α‐amylase, in some cases in combination with phytase, could improve technological bread quality. The use of phytase in the formulation significantly reduced the level of phytates, and phytate hydrolysis also led to smaller bran particle size. Increasing the bran proportion used in the bread formulation increased the iron concentration in bread samples by 18.9%. Phytase addition proved to be a useful strategy to improve iron dialyzability; however, incomplete dephytinization still had an inhibitory effect on iron uptake, with the exception of samples formulated with 10% bran. The inhibitory effect of phytate could be predicted from the values of the phytate‐to‐iron ratios. Reduction of particle size did not improve iron availability or uptake by Caco‐2 cells.

Ethanol Tolerance in <i>Aspergillus niger</i> and <i>Escherichia coli</i> Phytase

Despite yeast having its own native phytase, the high levels of phytate found in DDGS, a byproduct of ethanol (ETOH) fermentation, suggest that its activity is diminished in the presence of ETOH. Ethanol, a product of grain fermentation, is known to inactivate several hydrolytic enzymes but its effect on phytases is relatively unknown. In this study, two phytases, Aspergillus niger (PhyA) and Escherichia coli (AppA2), were tested for ETOH tolerance. The E. coli phytase displayed greater ethanol tolerance over fungal phytase in the 5% to 10% range. However, ETOH inactivation was found to be reversible for both the enzymes. These differences in ETOH tolerance do suggest that there is a potential to achieve higher ETOH tolerance in phytases by 'structure-function' studies to lower phytic acid levels in DDGS and for other applications.

A Single Mutation in the Hepta-Peptide Active Site of <i>Aspergillus niger</i> PhyA Phytase Leads to Myriad Biochemical Changes

The active site motif of proteins belonging to "Histidine Acid Phosphatase" (HAP) contains a hepta-peptide region, RHGXRXP. A close comparison among fungal and yeast HAPs revealed the fourth residue of the hepta-peptide to be E instead of A, which is the case with A. niger PhyA phytase. However, another phytase, PhyB, from the same microorganism has a higher turnover number and it shows E in this position. We mutated A69 residue to E in the fungal PhyA phytase. The mutant phytase shows a myriad of new kinetic properties. The pH profile shifted 0.5 pH unit in both 5.0 and 2.5 bi-hump peaks. The optimum temperature shifted down from 58℃ to 55℃. However, the greatest difference was observed in the mutant protein's reaction to GuCl at a concentration of 0.1 to 0.2 M. The activity of the mutant phytase jumped 100% while the wild type protein showed no activity enhancement in the same concentration range of GuCl. The kinetics performed at higher concentration of GuCl also contrasted the difference between the wild type and mutant phytase. While Km was least affected, the Vmax increased for the mutant and decreased for the wild type. The sensitivity towards myo-inositol hexasulfate, a potent inhibitor, was decreased by the mutation. All in all, A69E mutation has affected a multitude of enzymatic properties of the protein even though the residue was thought to be non-critical for phytase's catalytic function notwithstanding its location in the conserved hepta-peptide region of the biocatalyst.

In vitro assessment of the effects of phytate and phytase on nitrogen and phosphorus bioaccessibility within fish digestive tract

The aim of the present work was to assess different possible interactions between phytate (IP6), dietary protein and digestive proteases, as well as the preventive effect of phytase in the formation of protein-IP6 complexes, through the use of a gastrointestinal model simulating conditions existing in the digestive tract of rainbow trout ( Oncorhynchus mykiss). Two commercial phytases, expressed in P. lycii and E. coli, were evaluated. Fungal phytase showed an optimum at pH 5.5 and bacterial phytase showed two peaks at pH 2.5 and 4.5. These phytases evidenced different resistance against gastric proteolytic degradation; E. coli phytase retained 90% of its initial activity after 4 h incubation in the presence of either porcine pepsin or fish stomach proteases, while the dephosphorylation activity of P. lycii phytase was reduced by 90% (P<0.001). The presence of IP6 determined a reduction of 80% in protein solubility (P<0.001) when casein was used as substrate of fish acid proteases. The formation of IP6-protein complexes resulted in lower amino acids liberation from casein (P<0.05). Addition of phytase to soybean meal prevented the formation of such IP6-protein complexes and improved amino acid release by 60% (P<0.01). In contrast, IP6 had not effect on protein solubilisation or amino acid liberation during the intestinal digestion. Under conditions simulating gastric digestion a great amount of IP6 was combined to either porcine pepsin or soluble protein in fish stomach crude extract, precipitating 68 and 100% of such proteins, respectively. A similar, but lower reduction in soluble protein from either pancreatin or fish intestinal extract was observed when simulating intestinal digestion (47 and 33%, respectively). The formation of IP6-protein complexes reduced the activity of all gastric and intestinal proteases assayed (P<0.01) with the exception of chymotrypsin. A positive effect (P<0.05) of low pH on IP6-dephosphorylation was measured when the effect of bacterial phytase (2500 FTU/kg) on the release of soluble P from native IP6 present in soybean meal was tested under different digestion temperatures (6 and 16 °C) and pH (2.0, 3.0 and 4.0). A positive effect of temperature was also observed when the reaction was carried out at pH 2.0 and 3.0 (P<0.001), but not at 4.0, suggesting the existence of a limit in the solubility of IP6 and therefore in its availability for phytase action at this last pH value.

Transgenic microalgae expressing Escherichia coli AppA phytase as feed additive to reduce phytate excretion in the manure of young broiler chicks

Microbial phytases are widely used as feed additive to increase phytate phosphorus utilization and to reduce fecal phytates and inorganic phosphate (iP) outputs. To facilitate the process of application, we engineered an Escherichia coli appA phytase gene into the chloroplast genome of the model microalga, Chlamydomonas reinhardtii, and isolated homoplasmic plastid transformants. The catalytic activity of the recombinant E. coli AppA can be directly detected in the whole-cell lysate, termed Chlasate, prepared by freeze-drying the transgenic cell paste with liquid nitrogen. The E. coli AppA in the Chlasate has a pH and temperature optima of 4.5 and 60°C, respectively, which are similar to those described in the literature. The phytase-expressed Chlasate contains 10 phytase units per gram dry matter at pH 4.5 and 37°C. Using this transgenic Chlasate at 500 U/kg of diet for young broiler chicks, the fecal phytate excretion was reduced, and the iP was increased by 43% and 41%, respectively, as compared to those of the chicks fed with only the basal diet. The effectiveness of the Chlasate to break down the dietary phytates is compatible with the commercial Natuphos fungal phytase. Our data provide the first evidence of functional expression of microbial phytase in microalgae and demonstrate the proof of concept of using transgenic microalgae as a food additive to deliver dietary enzymes with no need of protein purification.

Vanadate Inhibition of Fungal PhyA and Bacterial AppA2 Histidine Acid Phosphatases

The fungal PhyA protein, which was first identified as an acid optimum phosphomonoesterase (EC 3.1.3.8), could also serve as a vanadate haloperoxidase (EC 1.11.1.10) provided the acid phosphatase activity is shut down by vanadate. To understand how vanadate inhibits both phytate and pNPP degrading activities of fungal PhyA phytase and bacterial AppA2 phytase, kinetic experiments were performed in the presence and absence of orthovanadate and metavanadate under various acidic pHs. Orthovanadate was found to be a potent inhibitor at pH 2.5 to 3.0. A 50% activity of fungal phytase was inhibited at 0.56 μM by orthovanadate. However, metavanadate preferentially inhibited the bacterial AppA2 phytase (50% inhibition at 8 μM) over the fungal phytase (50% inhibition at 40 μM). While in bacterial phytase the K(m) was not affected by ortho- or metavanadate, the V(max) was reduced. In fungal phytase, both the K(m) and V(max) was lowered. The vanadate exists as an anion at pH 3.0 and possibly binds to the active center of phytases that has a cluster of positively charged Arg, Lys, and His residues below the enzymes' isoelectric point (pI). The active site fold of haloperoxidase was shown to be very similar to fungal phytase. The vanadate anions binding to cationic residues in the active site at acidic pH thus serve as a molecular switch to turn off phytase activity while turning on the haloperoxidase activity. The fungal PhyA phytase's active site housing two distinct reactive centers, one for phosphomonoesterase and the other for haloperoxidase, is a unique example of how one protein could catalyze two dissimilar reactions controlled by vanadate.

Agrobacterium-mediated transformation of cotton (Gossypium hirsutum L.) with a fungal phytase gene improves phosphorus acquisition

A phytase gene (phyA), isolated from Aspergillus ficuum (AF537344), was introduced into cotton (Gossypium hirsutum L.) by Agrobacterium-mediated transformation to increase the phosphorus (P) acquisition efficiency of cotton. Southern and Northern blot analyses showed that the phyA was successfully incorporated into the cotton genome and expressed in transgenic lines. After growing for 45 days with phytate (Po) as the only P source, the shoot and root dry weights of the transgenic plants all increased by nearly 2.0-fold relative to those of wild-type plants, but were similar to those of transgenic plants supplied with inorganic phosphorus. The phytase activities of root extracts prepared from transgenic plants were 2.4- to 3.6-fold higher than those from wild-type plants, and the extracellular phytase activities of transgenic plants were also 4.2- to 6.3-fold higher. Furthermore, the expressed phytase was secreted into the rhizospheres as demonstrated by enzyme activity staining. The transgenic plants accumulated much higher contents of total P (up to 2.1-fold after 30 days of growth) than the wild-type plants when supplied with Po. These findings clearly showed that cotton plant transformed with a fungal phytase gene was able to secret the enzyme from the root, which markedly improved the plant’s ability to utilize P from phytate. This may serve as a promising step toward the development of new cotton cultivars with improved phosphorus acquisition.

Coexpression of fungal phytase and xylanase utilizing the cis-acting hydrolase element in Pichia pastoris

Plant-based animal feed contains antinutritive agents, necessitating the addition of digestive enzymes in commercial feeds. Enzyme additives are costly because they are currently produced separately from recombinant sources. The coexpression of digestive enzymes in a single recombinant cell system would thus be advantageous. A coexpression system for the extracellular production of phytase and xylanase was established in Pichia pastoris yeast. The genes for each enzyme were fused in-frame with the α-factor secretion signal and linked by the 2A-peptide-encoding sequence. Each enzyme was expressed extracellularly as individual functional proteins. The specific activities of 2A-expressed phytase (PhyA-2A) and 2A-expressed xylanase (XylB-2A) were 9.3 and 97.3 U mg(-1) , respectively. Optimal PhyA-2A activity was observed at 55 degreesC and pH 5.0. PhyA-2A also exhibited broad pH stability from 2.5 to 7.0 and retained approximately 70% activity after heating at 90 degreesC for 5 min. Meanwhile, XylB-2A exhibited optimal activity at 50 degreesC and pH 5.5 and showed pH stability from 5.0 to 8.0. It retained >50% activity after incubation at 50 degreesC for 10 min. These enzyme properties are similar to those of individually expressed recombinant enzymes. In vitro digestibility test showed that PhyA-2A and XylB-2A are as efficient as individually expressed enzymes for hydrolyzing phytate and crude fiber in feedstuff, respectively.

Wholemeal wheat bread: A comparison of different breadmaking processes and fungal phytase addition

The effect of different breadmaking processes (conventional, frozen dough, frozen partially baked bread) and the effect of the storage period on the technological quality of fresh wholemeal wheat breads are investigated. In addition, the impact of the exogenous fungal phytase on the phytate content was also determined. Results showed that breadmaking technology significantly affected the quality parameters of wholemeal breads (specific volume, moisture content, crumb and crust colour, crumb texture profile analysis and crust flaking) and frozen storage affected to a different extent the quality of the loaves obtained from partially baked breads and those obtained from frozen dough, particularly crust flaking. Freezing and frozen storage of wholemeal bread in the presence of fungal phytase decreased significantly the phytate content in whole wheat breads. The combination of fungal phytase addition, breadmaking process and frozen storage could be advisable for overcoming the detrimental effect of bran on the mineral bioavailability.

Influence of Dietary Phytic Acid and Source of Microbial Phytase on Ileal Endogenous Amino Acid Flows in Broiler Chickens

The effects of phytic acid and 2 sources of exogenous phytase (bacterial vs. fungal) on the flow of endogenous amino acids at the terminal ileum of broilers were assessed using the enzyme-hydrolyzed casein method. Phytic acid (as the sodium salt) was included in a purified diet at 8.5 and 14.5 g/kg, and each diet was fed without or with a fungal (Aspergillus niger-derived) or a bacterial (Escherichia coli-derived) microbial phytase at 500 phytase units/kg of diet. Increasing the concentration of phytic acid in the diet from 8.5 to 14.5 g/kg increased (P < 0.001) the flow of all measured amino acids by an average of 68%, with a range from 17% for proline to 145% for phenylalanine. The flow of endogenous aspartic acid, serine, glutamic acid, glycine, leucine, tyrosine, phenylalanine, and histidine were increased by more than the mean, indicating changes in the composition of endogenous protein in response to the presence of higher concentrations of phytic acid. Supplementation of both phytases reduced (P < 0.001) the flow of endogenous amino acids, but the reduction (P = 0.06) was greater for the bacterial phytase compared with the fungal phytase. These data suggest that a substantial part of the amino acid and energy responses observed following phytase supplementation in broiler chickens stems from reduced endogenous amino acid flows and that the capacity of different phytases to counteract the antinutritive properties of phytic acid vary.

Effect of supplemented fungal phytase on performance and phosphorus availability by phosphorus-depleted juvenile rainbow trout ( Oncorhynchus mykiss), and on the magnitude and composition of phosphorus waste output

The effect of a supplemental fungal phytase on performance and phosphorus availability by juvenile rainbow trout fed diets with a high inclusion of plant based protein and on the magnitude and composition of the waste phosphorus production was tested in a 2 × 3 factorial design at a temperature of 11 °C. Two factors comprised of two dietary fungal phytase levels (0 or 1400 U kg − 1 feed − 1 ), and three dietary total phosphorus levels (0.89, 0.97 or 1.12%). All fish were acclimated to the lowest total phosphorus diet for 16 days, which included 0.29% phytate-phosphorus and no supplemental fungal phytase, to ensure that they were depleted of phosphorus prior to the feeding trial. Growth and feed conversion ratios were not significantly affected by the increasing dietary phosphorus level or supplemental fungal phytase. Phosphorus availability increased significantly as a result of phytase supplementation, reaching an upper level of 74% at an available dietary phosphorus concentration of 0.71%. Adding fungal phytase to the diets improved the availability of phytate-phosphorus from an average of 6 to 64%. The fish retained 53–79% of the ingested phosphorus, while 24–44% was recovered in the faeces. The particulate phosphorus waste output was significantly higher in faeces from fish fed diets without fungal phytase compared to fish fed phytase supplemented diets. The dissolved/suspended phosphorus waste output represented 2–13% of the ingested phosphorus, and there was a significant increase in the dissolved/suspended phosphorus waste output from fish fed the phytase supplemented diet containing 0.71% available phosphorus, suggesting that the phosphorus requirement was reached at this phosphorus level. Consistent with this, there was a substantial increase in the dissolved/suspended phosphorus waste output from fish fed the phytase supplemented diet containing 0.81% available phosphorus, suggesting that the phosphorus requirement was exceeded in this group. This study demonstrated that phytase supplementation will be advantageous to the fish and the environment if supplemented to low-phosphorus diets containing a large share of plant-derived protein. Conversely, the results demonstrated that fungal phytase should not be supplemented to diets in which the available phosphorus level already meets the requirement of the fish, as this will lead to a significant increase in the dissolved/suspended phosphorus waste output.

Unfolding and Refolding of Aspergillus niger PhyB Phytase: Role of Disulfide Bridges

The role of disulfide bridges in the folding of Aspergillus niger phytase pH 2.5-optimum (PhyB) was investigated using dynamic light scattering (DLS). Guanidinium chloride (GuCl) at 1.0 M unfolded phytase; however, its removal by dialysis refolded the protein. The thiol reagent tris(2-carboxyethyl)phosphine (TCEP) reduces the refolding activity by 68%. The hydrodynamic radius (R(H)) of PhyB phytase decreased from 5.5 to 4.14 nm when the protein was subjected to 1.0 M GuCl concentration. The active homodimer, 183 kDa, was reduced to a 92 kDa monomer. The DLS data taken together with activity measurements could indicate whether refolding took place or not in PhyB phytase. The correlation between molecular mass and the state of unfolding and refolding is a very strong one in fungal phytase belonging to histidine acid phosphatase (HAP). Unlike PhyA phytase, for which sodium chloride treatment boosted the activity at 0.5 M salt concentration, PhyB phytase activity was severely inhibited under identical condition. Thus, PhyA and PhyB phytases are structurally very different, and their chemical environment in the active site and substrate-binding domain may be different to elicit such an opposite reaction to monovalent cations.

Bacterial 및 Fungal Phytase의 단일 및 혼합 급여가 산란계의 생산성과 영양소 이용에 미치는 영향

본 시험에서는 bacteria 또는 fungi로부터 유래된 phytase를 단일 및 혼합으로 45주령 Hy-Line Brown 산란계 450수에 12주 동안 급여하였다. 공시계는 시험개시 전 일주일 동안 산란조사를 하여 처리간에 비슷한 산란율이 되도록 하였다. 대조구(Control)사료는 nonphytate phosphorus 함량이 0.3% 되게 하였으며, 처리구는 bacterial phytase(BP)와 fungal phytase (FP) 2종류를 각각 T1(300 DPU, 0 DPU), T2(300 DPU, 300 DPU), T3(300 DPU, 3000 DPU), T4(0 DPU, 3000 DPU)로 하여 급여하였다. 처리구들의 nonphytate phosphorus 함량은 Control의 60%로 하였다. 사료와 물은 전 기간 자유채식토록 하였고, 점등은 17L7D로 하였다. 산란율은 T4가 Control에 비해 유의하게 낮았으며(P<0.05), 두 종류 phytase의 동시 급여 효과는 없었다. 평균 난중과 1일 산란량은 처리구간에 통계적인 차이는 없었으나, 후자의 경우 시험 후반기에 T4에서 Control에 비해 유의하게 낮았다(P<0.05). 사료 섭취량, 사료요구율, 계란의 품질은 처리구간에 차이가 없었다. 건물, 조단백질 및 지방 소화율은 처리구간 유사하였으며, 무기물, 칼슘 및 인 이용율은 뚜렷한 경향이 없었다. 본 실험 결과에서 산란계 사료에 BP 300 DPU 수준일 때 NPP 첨가량을 NRC (1994) 권장량 대비 약 40%가 저감되었으며, 인 이용율 역시 개선되었으나 FP와의 혼합 급여로 인한 개선 효과는 나타나지 않았다. This study was conducted to investigate the effect of single or mixed supplementation of bacterial and fungal phytase using 45-wk-old 450 Hy-Line Brown laying hens housed in individual cages for 12-wk period. The birds were reallocated to have similar egg productivity by examining the egg production for one wk before starting the experiment. Two sources of phytase, bacterial (BP) and fungal (FP), were used either in single or mixture to determine the effects of these phytase. Five dietary treatments consisted of control (BP 0, FP 0), T1 (BP 300, FP 0), T2 (BP 300, FP 300), T3 (BP 300, FP 3000), and T4 (BP 0, FP 3000). The DPU was used for phytase activity in this experiment. The nonphytate phosphorus (NPP) content of control was 0.30%, and those of phytase treatments were set to 60% of the Control. Experimental diets were fed ad libitum throughout the experimental period. The lighting schedule of 17L7D was employed. The egg production was not different between control and bacterial phytase treatments, but the T4 showed significantly low productivity compared to control (P<0.05). No difference was found in average egg weight among all treatments. The daily egg mass did not show any statistical differences among all treatments: however, it was significantly low in T4 compared to ther control during the latter half of the experiment (P<0.05). No significant difference was found among treatments in terms of feed intake, feed conversion and egg quality. The digestibilities of dry matter, crude protein, and fat digestibility were similar regardless of the treatments. No significant trends were detected in Ca and P availability. In conclusion, the BP level of 300 DPU contributed to achieve 40% reduction of recommended nonphytate phosphorus addition. The synergistic effect of bacterial and fungal phytase was not confirmed.

산란계 사료 내 칼슘 및 무기태 인 수준에 따른 Bacterial Phytase 급여 효과

본 실험은 사료 내 칼슘 수준과 fungal phytase의 상호 관계를 구명하고자 21주령 HyLine Brown 산란계 720수를 공시하여 시험을 실시하였다. 시험구는 phytase 첨가 수준 0, 300, 1000 DPU/kg, 칼슘 2수준(3.5, 4.0%), 무기태 인 수준 무첨가(0.095 NPP), 0.5(0.185 NPP), 1.0%(0.275%)를 조합한 <TEX>$3{\times}2{\times}3$</TEX> 요인 시험으로 하였다. 실험 사료는 CP 16%에 ME 2,800 kcal/ kg 수준으로 급여하였다. 사료는 실험 전 기간 자유 채식토록 하였으며, 점등은 17시간으로 하였다. 조사 항목은 산란계의 생산 능력, 계란의 품질이다. 산란율은 fungal phytase 300 DPU 첨가구에서 증가하는 경향을 보였으며, 연.파란율은 감소하는 경향을 보였다. 처리구간별 산란율은 사료의 칼슘 수준에 따른 차이가 없었지만, NPP 무첨가구인 0.095% 첨가구에서 다른 처리구에 비하여 낮았다(P<0.05). 평균 난중 및 1일 산란량은 fungal phytase 300 DPU 첨가구에서 가장 높게 나타났다. 평균 난중은 사료 칼슘 수준이 3.5% 처리구에서 높게 나타났지만(P<0.05) 1일 산란량은 처리구간에 차이가 없었다. 평균 난중 및 1일 산란량은 NPP 무첨가구인 0.095% 첨가구에서 다른 처리구에 비하여 매우 낮았다(P<0.05). 사료 섭취량은 사료의 fungal phytase 및 칼슘 수준에 관계없이 비슷한 경향을 보였지만, 사료내 NPP 0.275, 0.165% 함유된 처리구에서 0.095% 처리구보다 통계적으로 높게 나타났다(P<0.05). 사료 요구율은 사료내 칼슘 및 NPP 수준간에 차이가 없었지만, fungal phytase 300DPU 첨가구에서 가장 크게 개선되었다(P<0.05). 난각 강도와 두께는 사료의 칼슘 수준이 높을수록 향상되는 결과를 보였고, NPP 0.275, 0.165%가 함유된 처리구에서 0.095% 처리구에 비하여 향상되었으며, 사료내 fungal phytase 수준에 따른 차이는 없었다. Haugh unit은 처리구간에 큰 차이 없이 비슷하게 나타났다. 본 실험의 결과, 산란계 사료에서 fungal phytase의 적정 수준은 300 DPU이며, 이때 사료내 TCP 첨가량은 권장량 대비 40% 이상 저감 효과가 있었으며, 사료의 칼슘 수준을 높게 하였을지라도 fungal phytase 첨가에 대한 개선 효과는 나타나지 않았다. This study was conducted to identify the correlation of bacterial phytase (<TEX>$Transphos^{(R)}$</TEX>) to the calcium level in feed. Of all 21-week-old 720 HyLine brown laying hens, 2 birds of similar weight were placed on each individual cage. The experiment was conducted by <TEX>$3{\times}2{\times}3$</TEX> factorial design with including 3 different levels of phytase (0, 300, and 1,000 DPU/kg), 2 different levels of calcium (3.5% and 4.0%), and 3 different levels of no NPP addition 0% (0.095 NPP), 0.5% (0.185% NPP), and 1.0% (0.275% NPP). The feeding trial maintained the ME level of 2,800 kcal/kg and 16% for crude protein. The diet was fed ad libitum and 17 hours of lighting was provided throughout the experimental period. Egg production seemed to increase, in the 300 DPU of bacterial phytase added group and the cracked egg tended to reduce in Transphos added group. The egg productivity between treatment groups did not show significant difference by dietary calcium level, whereas non NPP added group (0.095% NPP) was found to be low compared to NPP added groups (P<0.05). The highest mean egg weight and the highest daily egg mass were detected in 300 DPU phytase added group. Although the mean egg weight was significantly higher in treatment groups fed with 3.5% calcium containing feeds (P<0.05), daily egg mass was no among treatment groups. The mean egg weight and daily egg mass were the lowest in non NPP added group (0.095% NPP) compared to other treatment groups (P<0.05). The feed intake showed similar pattern regardless of the bacterial phytase and calcium levels in the diet. However, the treatment groups fed diets containing NPP level of 0.275% and 0.165% showed significantly higher feed intake than the group fed with 0.095% NPP (P<0.05). Although the feed conversion was not affected by calcium and NPP levels in the diet, the most improved result was obtained from 300 DPU phytase added group (P<0.05). The eggshell breaking strength and thickness increased as dietary calcium level increase the level of calcium increases in diet. The treatment groups fed diet containing 0.275% and 0.165% NPP revealed to show improvement in eggshell breaking strength and yolk color index compared to the NPP non added (0.095% NPP) treatment group. The result of the present study suggests that the appropriate level of microbial phytase is 300 DPU and at this level, tricalciumphosphate supplementation in feed can be reduced to 40% of NRC recommendation. Higher calcium level in feed fail to show synergistic effect by adding microbial phytase.

Salt Effect on the pH Profile and Kinetic Parameters of Microbial Phytases

The pH profiles of two microbial phytases were determined using four different general purpose buffers at different pH values. The roles of calcium chloride, sodium chloride, and sodium fluoride on activity were compared in these buffers. For Aspergillus niger phytase, calcium extended the pH range to 8.0. A high concentration of sodium chloride affected the activity of fungal phytase in the pH 3-4 range and shifted the pH optimum to 2.0 from 5.5 in Escherichia coli phytase. As expected, both of the microbial phytases were inhibited by sodium fluoride at acidic pH values. Because the Km for phytate increased nearly 2-fold for fungal phytase while Vmax increased about 75% in a high concentration of sodium chloride, it is possible that salt enhanced the product to dissociate from the active site due to an altered electrostatic environment. Modeling studies indicate that while the active site octapeptide's orientation is very similar, there are some differences in the arrangements of alpha-helices, beta-sheets, and coils that could account for the observed catalytic and salt effect differences.

Effect of wheat bran and enzyme addition on dough functional performance and phytic acid levels in bread

Effects of bran concentration, bran particle size distribution, and enzyme addition – fungal phytase, fungal alpha-amylase – on the mixing and fermentative behaviour of wheat dough and on the amount of phytic acid remaining in bread have been investigated using a factorial design of samples 2 4. Bran concentration and bran particle size significantly affected all Farinograph parameters, whereas enzyme effects were particularly observed on both the water absorption of the flour and the parameters characterizing the overmixing. Water absorption was maximized in doughs with higher fine bran addition and/or in doughs with no enzymes, and was minimized in blends containing coarse added bran and alpha-amylase and/or alpha-amylase and phytase. alpha-Amylase addition had a significant positive effect on dough development and gassing power parameters during proofing. At low bran addition, phytate hydrolysis takes place to a greater extent than at high bran addition levels. Combination of bran with amylolytic and phytate-degrading enzymes could be advisable for overcoming the detrimental effect of bran on the mineral availability (phytase) or on the technological performance of doughs (alpha-amylase).

Transgenic maize plants expressing a fungal phytase gene

Maize seeds are the major ingredient of commercial pig and poultry feed. Phosphorus in maize seeds exists predominantly in the form of phytate. Phytate phosphorus is not available to monogastric animals and phosphate supplementation is required for optimal animal growth. Undigested phytate in animal manure is considered a major source of phosphorus pollution to the environment from agricultural production. Microbial phytase produced by fermentation as a feed additive is widely used to manage the nutritional and environmental problems caused by phytate, but the approach is associated with production costs for the enzyme and requirement of special cares in feed processing and diet formulation. An alternative approach would be to produce plant seeds that contain high phytase activities. We have over-expressed Aspergillus niger phyA2 gene in maize seeds using a construct driven by the maize embryo-specific globulin-1 promoter. Low-copy-number transgenic lines with simple integration patterns were identified. Western-blot analysis showed that the maize-expressed phytase protein was smaller than that expressed in yeast, apparently due to different glycosylation. Phytase activity in transgenic maize seeds reached approximately 2,200 units per kg seed, about a 50-fold increase compared to non-transgenic maize seeds. The phytase expression was stable across four generations. The transgenic seeds germinated normally. Our results show that the phytase expression lines can be used for development of new maize hybrids to improve phosphorus availability and reduce the impact of animal production on the environment.

Increase of calnexin gene dosage boosts the secretion of heterologous proteins byHansenula polymorpha

The type I membrane protein calnexin is a conserved key component of the quality control mechanism in the endoplasmic reticulum. It functions as a molecular chaperone that monitors the folding state of nascent polypeptides entering the endoplasmic reticulum. Calnexin also behaves as a lectin, as its chaperoning activity involves binding of oligosaccharide moieties present on newly imported glycoproteins. We isolated the calnexin gene (HpCNE1) from the methylotrophic yeast Hansenula polymorpha, and used HpCNE1 expression plasmids for supertransformation of H. polymorpha strains secreting target proteins of biotechnological interest. The elevated dosage of HpCNE1 enhanced secretion of the four proteins tested: three glycoproteins and one unglycosylated product. Secretion of bacterial alginate epimerase AlgE1 was increased threefold on average, and secretion of both human interferon-γ and fungal consensus phytase twofold. With phytase and AlgE1 this improvement was all the more remarkable, as the secretion level was already high in the original strains (g L−1 range). The same approach improved secretion of human serum albumin, which lacks N-linked glycans, about twofold. Glycosylation of the pro-MFα1 leader may account for the effect of calnexin in this case. Our results argue that cooverexpression of calnexin can serve as a generally applicable tool for enhancing the secretion of all types of heterologous protein by H. polymorpha.