Survey of Microorganisms for the Production of Extracellular Phytase

A culture enrichment technique was used to isolate phytase-producing microorganisms. Also, microorganisms from various culture collections were tested for their phytase-producing ability. A number of the Aspergillus niger group produced extracellular phytase which dephosphorylated calcium phytate in acidic solution. A soil isolate, A. ficuum NRRL 3135, produced the most active phytase in a cornstarch-based medium. Production of phytase was strongly repressed by inorganic phosphates and required a high carbon to phosphorus ratio in the medium.     

Characterization and identification of phytase-producing bacteria isolated from the gastrointestinal tract of four freshwater teleosts

Isolation and enumeration of phytase-producing bacteria in the proximal intestine (PI) and distal intestine (DI) of four freshwater teleosts, Nile tilapia (Oreochromis niloticus), murrel (Channa punctatus), climbing perch (Anabas testudineus), and stinging catfish (Heteropneustes fossilis) have been carried out following enrichment culture technique. The bacterial isolates were screened on the basis of their phytase-producing ability. In modified phytase screening medium (MPSM), phytase-producing strains were recorded at higher densities in the PI of Nile tilapia and climbing perch and at a minimum in the DI of catfish. Out of 32 isolates, 20 phytase-producing strains (9 from the DI and 11 from the PI) were primarily selected on the basis of qualitative assay on MPSM plates. Among these isolates, 3 strains (2 from the PI and 1 from the DI) were selected as potent phytase producers according to quantitative enzyme assay. Maximum phytase activity was detected in the bacterial strain ONF2 isolated from the PI of O. niloticus followed by CPF6 and CPH6, isolated from the PI and DI, respectively of C. punctatus. All the three selected phytase-producing strains were Gram-positive rods, capable of forming endospores, and could tolerate a wide range of temperature (25–42 °C) and pH (6–10). The strain CPF6 was able to grow at temperatures up to 55 °C. On the basis of 16S rDNA sequence analysis, isolates ONF2, CPF6 and CPH6 were identified as Bacillus licheniformis. The strain ONF2 showed 100 % similarity to B. licheniformis strain LCR32 (Accession no. FJ976541.1) whereas CPF6 and CPH6 showed 99 % similarity to B. licheniformis strain LCR32 (Accession no. FJ976541.1).     

Phytate degradation by immobilized<Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> phytase in soybean-curd whey

Saccharomyces cerevisiae CY phytase-producing cells were immobilized in calcium alginate beads and used for the degradation of phylate. The maximum activity and immobilization yield of the immobilized phytase reached 280 mU/g-bead and 43%, respectively. The optimal pH of the immobilized cell phytase was not different from that of the free cells. However, the optimum temperature for the immobilized phytase was 50°C, which was 10°C higher than that of the free cells; pH and thermal stability were enhanced as a consequence of immobilization. Using the immobilized phytase, phytate was degraded in a stirred tank bioreactor. Phytate degradation, both in a buffer solution and in soybean-curd whey mixture, showed very similar trends. At an enzyme dosage of 93.9 mU/g-phytate, half of the phytate was degraded after 1 h of hydrolysis. The operational stability of the immobilized beads was examined with repeated batchwise operations. Based on 50% conversion of the phytate and five times of reuse of the immobilized beads, the specific degradation (g phytate/g dry cell weight) for the immobilized phytase increased 170% compared to that of the free phytase.     

中性植酸酶菌株的筛选及性质

从天然土壤中筛选出产胞外中性植酸酶的细菌20余株,通过钼蓝法对菌种进行复筛,确定phy7为研究菌。通过16SrDNA测序分析方法鉴定该菌株属于绿脓假单胞菌属。结果表明：该菌株分泌中性植酸酶,其最适反应pH为7．0、最适反应温度为40℃;在37℃时以植酸钠为底物的Km为0．26mmol／L,max为0．0506nmol／min。金属离子zn2＋、Al3＋、Cu2＋和Mn2＋等对该酶有抑制作用,而Fe2＋等则有一定的激活作用。     

Phytases: microbial sources,production, purification,and potential biotechnological applications

The review deals with phytase-producing microorganisms along with optimum conditions for its production. Various methods used for purifying phytases and their characteristics are discussed. Heterologous gene expression, cost-effective large-scale phytase production, and various biotechnological applications of the enzyme in animal feed and food industries are also discussed.     

Screening, cloning and overexpression of Aspergillus niger phytase (phyA) in Pichia pastoris with favourable characteristics

AIMS: Using gene cloning and overexpression to obtain a potential industrial phytase as a feed additive to upgrade the nutritional quality of phytate-rich seed-based animal feed. METHODS AND RESULTS: A phyA gene from a high extracellular phytase-producing Aspergillus niger sp. was cloned and overexpressed in Pichia pastoris GS115 using the secretive expression vector pPICZalphaA. After cultivation for 4 days in buffered methanol complex medium (BMMY) containing methanol for induction, catalytically active phytase was secreted as a predominantly extracellular protein. The activity of the expressed phytase in fermented broth was 30 000-fold higher than that of native phytase with a specific activity of 503 U mg(-1). The Lineweaver-Burk plot indicated K(m) values of 0.196 mmol l(-1) for sodium phytate and 18.16 mmol l(-1) for p-nitrophenylphosphate (pNPP). Thermostability studies showed that recombinant phytase retained 70% activity after exposure to 90 degrees C for 5 min and 65% activity after 30 min, much higher than for commercial phytase. CONCLUSIONS: The higher activity and high thermostability of recombinant phytase enable it to withstand the temperatures of the feed pelleting process. SIGNIFICANCE AND IMPACT OF THE STUDY: The characteristics of this recombinant phytase, especially the good thermostability, are likely to render it of potential industrial importance.     

植酸酶产生菌的选育及固态产酶条件研究

植酸酶催化植酸,并将其盐类水解成肌醇和磷酸,因此植酸酶的使用可以提高植酸磷的吸收利用率,降低饲料成本,同时还可保护生态环境.经分离和亚硝基胍诱变选育,得到一株植酸酶高产菌株绿色木霉LH374,并对该菌株固态发酵产植酸酶的条件和扩大生产进行了研究.结果表明,固态发酵的最佳条件:稻草和米糠的比例为8:2,培养基起始pH为6.5,最适温度为30℃,最适培养时间为96 h,含水量为60%,硫酸铵的流加量为2%.绿色木霉LH37在上述最适条件下生产植酸酶平均可达1 580 U·g^-1.     

Impact of oxygen supply on rtPA expression in <Emphasis Type="Italic">Escherichia coli</Emphasis> BL21 (DE3): ammonia effects

A phytase with high activity at neutral pH and typical water temperatures (∼25°C) could effectively hydrolyze phytate in aquaculture. In this study, a phytase-producing strain, Pedobacter nyackensis MJ11 CGMCC 2503, was isolated from glacier soil, and the relevant gene, PhyP, was cloned using degenerate PCR and thermal asymmetric interlaced PCR. To our knowledge, this is the first report of detection of phytase activity and cloning of phytase gene from Pedobacter. PhyP belongs to beta-propeller phytase family and shares very low identity (∼28.5%) with Bacillus subtilis phytase. The purified recombinant enzyme (r-PhyP) from Escherichia coli displayed high specific activity for sodium phytate of 24.4 U mg⁻¹. The optimum pH was 7.0, and the optimum temperature was 45°C. The K _m, V _max, and k _cat values were 1.28 mM, 71.9 μmol min⁻¹ mg⁻¹, and 45.1 s⁻¹, respectively. Compared with Bacillus phytases, r-PhyP had higher relative activity at 25°C (r-PhyP (>50%), B. subtilis phytase (<8%)) and hydrolyzed phytate from soybean with greater efficacy at neutral pH. These characteristics suggest that r-PhyP might be a good candidate for an aquatic feed additive in the aquaculture industry.     

Identification of β-propeller phytase-encoding genes in culturable Paenibacillus and Bacillus spp. from the rhizosphere of pasture plants on volcanic soils

Phytate is one of the most abundant sources of organic phosphorus (P) in soils, but must be mineralized by phytase-producing bacteria to release P for plant uptake. Microbial inoculants based on Bacillus spp. have been developed commercially, but few studies have evaluated the ecology of these bacteria in the rhizosphere or the types of enzymes that they produce. Here, we studied the diversity of aerobic endospore-forming bacteria (EFB) with the ability to mineralize phytate in the rhizosphere of pasture plants grown in volcanic soils of southern Chile. PCR methods were used to detect candidate phytase-encoding genes and to identify EFB bacteria that carry these genes. This study revealed that the phytate-degrading EFB populations of pasture plants included species of Paenibacillus and Bacillus, which carried genes encoding β-propeller phytase (BPP). Assays of enzymatic activity confirmed the ability of these rhizosphere isolates to degrade phytate. The phytase-encoding genes described here may prove valuable as molecular markers to evaluate the role of EFB in organic P mobilization in the rhizosphere.     

Isolation of Phytate-Hydrolysing Microbial Strains From Traditional Waste Water of Rice Fermentation and Liquid Cattle Feeds

Traditional fermentation systems employed by rural people of India were initially screened for phytate-hydrolysing microbes on solid medium supplemented with calcium phytate. It was followed by enzyme assay of culture filtrates to differentiate the phytase-producing microbes. Three new microbial genera are added to the list of phytase-producing organisms.     

Phytate utilization by genetically engineered lysine-producing Corynebacterium glutamicum

Heterologous expression of a phytase gene (phyC) from Bacillus amyloliquefaciens DSM 7 enabled the growth of Corynebacterium glutamicum with phytate (myo-inositol-1,2,3,4,5,6-hexakisphosphate) as a new, sole source of phosphorus. Phytate was not used as a carbon source. During growth of the phyC-expressing amino acid (l-lysine)-producing strain C. glutamicum ATCC 21253 (pWLQ2::phyC) with phytate as the source of phosphorus, merely a small, transient accumulation of inorganic phosphate was observed in the fermentation broth. At the later stages of fermentation, free inorganic phosphate from phytate degradation was no longer detectable. Growth and l-lysine production by the phytase-producing strain C. glutamicum ATCC 21253 (pWLQ2::phyC) in phytate medium did not differ significantly from control experiments with strain C. glutamicum ATCC 21253 (pWLQ2) conducted with an excess of inorganic phosphate, demonstrating that there was no phosphate limitation when phytate was used as the phosphorus source. Under the expression conditions employed, only part of PhyC was secreted to the culture broth by C. glutamicum, but this did not significantly affect growth or lysine production.     

Multiple forms of phytase in germinating cotyledons of Cucurbita maxima

Multiple forms of phytase (myoinositol hexaphosphate phosphohydrolase, EC 3.1.3.8) have been isolated in highly purified forms from germinating Cucurbita maxima cotyledons using acetone and ammonium sulphate fractionation, Sephadex gel filtration and ion exchange chromatography on DEAE- and CM-cellulose. Gel filtration produced two peaks of phytase activity; phytase I (high MW) and phytase II (low MW). Phytase I was further resolved into 4 distinct species on CM-cellulose and these were designated phytase IA, IB, IC and ID, according to their elution order. On the other hand, phytase II remained as a single species with a purification of 35-fold. The MWs of each phytase I species were identical (MW 66 500 ± 4000) and they were twice the MW of phytase II (MW 32 400 ± 4000) indicating that I and II may be structurally related. The properties of various molecular forms were compared. The difference in properties between phytase II and phytase I isoenzymes (IA, IB, IC and ID) was more pronounced than that observed among the isoenzymes of phytase I alone.     

Enhanced submerged Aspergillus ficuum phytase production by implementation of fed-batch fermentation

Phytase is an important feed and food additive, which is both used in animal and human diets. Phytase has been used to increase the absorption of several divalent ions, amino acids, and proteins in the bodies and to decrease the excessive phosphorus release in the manure to prevent negative effects on the environment. To date, microbial phytase has been mostly produced in solid-state fermentations with insignificant production volumes. There are only a few studies in the literature that phytase productions were performed in submerged bench-top reactor scale. In our previous studies, growth parameters (temperature, pH, and aeration) and important fermentation medium ingredients (glucose, Na-phytate, and CaSO₄) were optimized. This study was undertaken for further enhancement of phytase production with Aspergillus ficuum in bench-top bioreactors by conducting fed-batch fermentations. The results showed that addition of 60 g of glucose and 10 g of Na-phytate at 96 h of fermentation increased phytase activity to 3.84 and 4.82 U/ml, respectively. Therefore, the maximum phytase activity was further enhanced with addition of glucose and Na-phytate by 11 and 40 %, respectively, as compared to batch phytase fermentations. It was also reported that phytase activity increased higher in early log stage additions than late log stage additions because of higher microbial activity. In addition, the phytase activity in fed-batch fermentation did not drop significantly as compared to the batch fermentation. Overall, this study shows that fungal phytase can be successfully produced in submerged fed-batch fermentations.     

Recombinant,Rice-Produced Yeast Phytase Shows the Ability to Hydrolyze Phytate Derived from Seed-Based Feed,and Extreme Stability during Ensilage Treatment

When fresh rice leaves producing yeast Schwanniomyces occidentalis phytase were grounded and mixed with the whole extract of seed-based feed for pigs, the release of orthophosphate increased significantly. More specifically, phytate, a major source of phosphorus in the seeds, was hydrolyzed by heterologous phytase. Moreover, when transgenic rice plants were ensiled for up to 12 weeks, no decrease in the phytase activity of the heterologous enzyme was observed. This result strongly suggests that transgenic rice plants producing yeast phytase can be stored as silage without any loss of enzyme activity until usage as a feed additive.     

Biochemical characteristics of phytases from fungi and the transformed microorganism

Five sources of phytases were used to study their biochemical characteristics. Phytase E was from an original Escherichia coli (E. coli), phytase PI and PG from the transformed Pichia pastoris (P. pastoris) with phytase gene of E. coli, phytase B and R from Aspergillus niger (A. niger). The results showed that the relative phytase activities had no significant changes when temperature was below 60 °C (P>0.05), and then decreased significantly with temperature increasing (P<0.01). The fungal phytase with the phytase gene from A. niger had the higher thermostability than the bacterial phytase with the phytase gene from E. coli; i.e. at 70 °C, 27–58% of phytase activity (compared with 30 °C) was retained for the bacterial phytase, and 73–96% for the fungal phytase; at 90 °C, 20–47% was retained for the bacterial phytase, and 41–52% for the fungal phytase, especially for the most thermostable phytase R (P<0.01). The optimum pH ranges were 3.0–4.5 for the bacterial phytases and 5.0–5.5 for the fungal phytases (P<0.01). When pH levels were 1, 7 and 8, only 3–7% of phytase activity (compared with the maximum phytase activity at a pH point) was retained for both bacterial and fungal phytases. The amount of inorganic P released from soybean meal was significantly increased when the levels of phytase activity in the soybean meal increased from 0 to 1.0 U/g soybean meal (P<0.01), except for phytase PI. The maximum P released was obtained at 1 U/g soybean meal for all five kinds of phytases (P<0.01). The most economical phytase concentration for P released was 0.25 U/g for phytase PI and B, and 0.50–1.0 U/g for phytase PG, E and R. In addition, the linear and non-linear regression models were established to estimate phytase activity and its characteristics very easily and economically.     

Production,characteristics and applications of the cell-bound phytase of <Emphasis Type="Italic">Pichia anomala</Emphasis>

Among several yeasts isolated from dried flowers of Woodfordia fruticosa, Pichia anomala produced a high titre of cell-bound phytase. The optimization of fermentation variables led to formulation of media and selection of cultural variables that supported enhanced phytase production. The enzyme productivity was very high in fed batch fermentation in air-lift fermentor as compared to that in stirred tank fermentor. Amelioration in the cell-bound phytase activity was observed when yeast cells were permeabilized with Triton-X-100. The enzyme is thermostable and acid stable with broad substrate specificity, the characteristics that are desirable for enzymes to be used in the animal feed industry. The phytase-encoding gene was cloned and sequenced. The 3D structure of the enzyme was proposed by comparative modeling using phytase of Debaryomyces occidentalis (50% sequence identity) as template. When broiler chicks, and fresh water and marine fishes were fed with the feed supplemented with yeast biomass containing phytase, improvement in growth and phosphorus retention, and decrease in the excretion of phosphorus in the faeces were recorded. The cell-bound phytase of P. anomala could effectively dephytinize wheat flour and soymilk.     

Cell-surface phytase on Pichia pastoris cell wall offers great potential as a feed supplement

Cell-surface expression of phytase allows the enzyme to be expressed and anchored on the cell surface of Pichia pastoris . This avoids tedious downstream processes such as purification and separation involved with extracellular expression. In addition, yeast cells with anchored proteins can be used as a whole-cell biocatalyst with high value added. In this work, the phytase was expressed on the cell surface of P. pastoris with a glycosylphosphatidylinositol anchoring system. The recombinant phytase was shown to be located at the cell surface. The cell-surface phytase exhibited high activity with an optimal temperature at 50–55 °C and two optimal pH peaks of 3 and 5.5. The surface-displayed phytase also exhibited similar pH stability and pepsin resistance to the native and secreted phytase. In vitro digestibility test showed that P. pastoris containing cell-surface phytase released phosphorus from feedstuff at a level similar to secreted phytase. Yeast cells expressing phytase also provide additional nutrients, especially biotin and niacin. Thus, P. pastoris with phytase displayed on its surface has a great potential as a whole-cell supplement to animal feed.     

Effects of phytase and 25-hydroxyvitamin D3 inclusions on the performance, mineral balance and bone parameters of grower-finisher pigs fed low-phosphorus diets

Two experiments, a performance experiment and a mineral balance study, were conducted on grower-finisher pigs (42 to 101 kg live weight) to investigate the effects of Peniophora lycii phytase enzyme and 25-hydroxyvitamin D3 (25-OHD3) on growth performance, carcass characteristics, nutrient retention and excretion, and bone and blood parameters. The two experiments were designed as a 2 × 2 factorial (two levels of phytase and two levels of 25-OHD3). The four diets were T1, low-phosphorous diet; T2, T1 + phytase; T3, T1 + 25-OHD3 and T4, T1 + phytase + 25-OHD3 diet. In all, 25 μg of 25-OHD3 was used to replace 1000 IU of vitamin D3 in diets T3 and T4. Diets were pelleted (70°C) and formulated to contain similar concentrations of energy (13.8 MJ DE/kg), lysine (9.5 g/kg) and digestible phosphorus (P; 1.8 g/kg). Neither the inclusion of phytase nor 25-OHD3 in the diet had any effect on pig performance. There was an interaction between phytase and 25-OHD3 on calcium (Ca) and P retention (P < 0.01) and on the apparent digestibility of ash (P < 0.01), P (P < 0.001) and Ca (P < 0.001). Pigs offered phytase diets only, had a higher retention of Ca and P and digestibility of ash (P < 0.01), P (P < 0.001) and Ca (P < 0.01) compared with pigs offered unsupplemented diets. However, when the combination of phytase and 25-OHD3 were offered, no effects were detected compared with 25-OHD3 diets only. Pigs fed phytase diets had higher bone ash (P < 0.01), bone P (P < 0.01) and bone Ca (P < 0.05) concentrations compared with pigs offered non-phytase diets. In conclusion, pigs offered phytase diets had a significantly increased bone ash, Ca and P than pigs offered unsupplemented phytase diets. However, there was no advantage to offering a combination of phytase and 25-OHD3 on either bone strength or mineral status compared to offering these feed additives separately.     

Barley HvPAPhy_a as transgene provides high and stable phytase activities in mature barley straw and in grains

The phytase purple acid phosphatase (HvPAPhy_a) expressed during barley seed development was evaluated as transgene for overexpression in barley. The phytase was expressed constitutively driven by the cauliflower mosaic virus 35S‐promoter, and the phytase activity was measured in the mature grains, the green leaves and in the dry mature vegetative plant parts left after harvest of the grains. The T₂‐generation of HvPAPhy_a transformed barley showed phytase activity increases up to 19‐fold (29 000 phytase units (FTU) per kg in mature grains). Moreover, also in green leaves and mature dry straw, phytase activities were increased significantly by 110‐fold (52 000 FTU/kg) and 57‐fold (51 000 FTU/kg), respectively. The HvPAPhy_a‐transformed barley plants with high phytase activities possess triple potential utilities for the improvement of phosphate bioavailability. First of all, the utilization of the mature grains as feed to increase the release of bio‐available phosphate and minerals bound to the phytate of the grains; secondly, the utilization of the powdered straw either directly or phytase extracted hereof as a supplement to high phytate feed or food; and finally, the use of the stubble to be ploughed into the soil for mobilizing phytate‐bound phosphate for plant growth.     

A comparative study on permeabilization treatments for in situ determination of phytase of Rhodotorula gracilis

Rhodotorula gracilis , a red yeast, was shown to produce phytase and the cultural conditions were standardized for phytase production. Permeabilization treatments using detergents, organic solvents, enzymes and physical methods were performed and compared by measuring the phytase activity of whole cells. None of the permeabilization methods except the freeze-thaw method enhanced phytase activity. The enzyme activity of a yeast suspension subjected to 15 cycles of freezing and thawing was 77·26 U g⁻¹ cells. Phytate in feeds is an anti-nutritional factor which complexes with many minerals, rendering it unavailable to monogastric animals. Rhodotorula gracilis subjected to freeze-thawing can be used as a source of phytase in feeds to enhance phosphorus availability.