Characterization of Extracellular Phytase Produced from Bacillus subtilis

Background: Cereals, legumes, and oilseed crops are very important crops as nutrition for human and animals. Phytate (myo inositol hexa kis phosphate) is the main storage form of phosphorus in these crops. These crops are major source of nutrients for humans and animals including fish, poultry and pig. Phytic acid is the nutritional constituent of animal diet but it is not digested by monogastric animals because they do not contain phytase enzyme in their intestines to break the phytic acid and due to this, phytic acid acts as an antinutritional chelating agent for various metal ions like Ca, Mg, Fe, Zn, and etc., so that reduced the nutritive quality of food. Objectives: Phytase is an important enzyme in the food/feed industry; therefore, isolation of phytase producing bacteria and optimization of phytase production on different parameters were performed in this study. Materials and Methods: The present study was conducted in Biotechnology laboratory, Motilal Nehru National Institute of Technology, Allahabad, Uttar Pradesh, India. To isolate phytase producing bacteria from different soil samples like cattle shed, pulse crop field, poultry farms, and etc. 0.1 gr of the soil samples were streaked on phytase screening medium. The qualitative screening of the isolates was performed on phytase screening medium plate with 1.5% agar, and phytase activity was determined by using shaking flask method. The best phytase producer was optimized using different parameters of phytase production. Results: We isolated 32 phytase producing bacteria on phytase screening media. Upon screening of these strains, one of the best strain (DR6) which showed a 39 mm clear zone on phytase specific medium (PSM) was identified as Bacillus subtilis. So this strain was selected for further enzymatic assay and optimization. This strain showed 378U/mL enzymatic activity upon enzymatic assay, the result of optimization of this best strain was performed at different parameters, and this strain showed best results at pH 5.5, Temp 50 °Cwith Glucose + Sucrose as Carbon source and Yeast extract as Nitrogen source. Conclusions: The present study suggests that the enzyme obtained from strain B. subtilis can be used as feed supplement in animal diet also for reduction of phosphorus pollution problem in areas of livestock production.

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.

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.

Phytase, a phytate hydrolysing enzyme has substantial application in food, feed and agriculture sector for improvising the availability of phosphorus and reduces the environmental pollution. The present study emphasizes on cost effective production of bacterial phytase. Total 25 agroresidues were screened for economic production of phytase using bacteria. A total of 30 bacterial isolates were obtained from poultry and goat shed soil and litter sample showing highest phytase activity. The efficient isolates SP-46 and SP-52 were screened for selection of efficient combination bacterial isolate and agroresidues for cost-effective production of phytase. The results showed that utilization of agroresidues not only serve as efficient substrate but also contribute to sustainable agro-waste valorisation and reduces need of expensive synthetic media for phytase production.

BackgroundPhytase catalyses the breakdown of complex organic forms of phosphorous into simpler forms by sequential hydrolysis of phosphate ester bonds to liberate the inorganic phosphate. Supplementation of feeds with bacterial phytase therefore could enhance the bioavailability of phosphorus and micronutrients. Hence, the aim of this study was to isolate and characterize phytase producing bacteria from rhizosphere soil, fresh poultry excreta, and cattle shed to evaluate their potential in improving poultry feeds. Phytase producing bacteria were isolated using wheat bran extract medium.ResultsA total of 169 bacterial isolates were purified and screened for phytase activity. Out of these, 36 were confirmed as positive for phytase enzyme activity. The bacterial isolates were identified by cultural, morphological, and biochemical features. The isolates were also identified by using 16 S rRNA gene sequencing. The bacterial isolates (RS1, RS8, RS10 and RS15) were provided with gene bank database accession numbers of MZ407562, MZ407563, MZ407564 and MZ407565 respectively. All isolates increased phytase production when cultured in wheat bran extract medium (pH 6) supplemented with 1% (wt/v) galactose and 1% (wt/v) ammonium sulphate incubated at 50oC for 72 h. Proximate composition analysis after supplementation of phytase showed that phytase supplementation improved bioavailability of phosphorus, calcium, potassium and sodium in poultry feed.ConclusionsOverall, this study showed that the nutritional value of poultry feed can be improved using microbial phytase enzyme which reduces the cost of supplementation with inorganic phosphate.

The production of phytase and biomass (estimated by the ergosterol content) by submerged fermentation with Aspergillus niger var. phoenicis URM 4924 was studied. Experimental assays were carried under different conditions of pH (4.0 to 8.0) and temperature (25 to 35 ºC), and the influence of these variables on the responses was studied through a 22 central composite design and response surface methodology. Phytase and biomass production were strongly affected by the pH and temperature used during fermentation. Phytase activity was increased in up to 7.8-fold (from 1.04 to 8.09 U/mL) and the ergosterol content was increased in up to 38-fold (from 9.3 to 354.09 µg/mL). The maximum values of both responses were achieved when using pH 4.0 and 30 ºC. Good correlation (second-order fit, R2 = 0.9875) was found between the data obtained for phytase activity and ergosterol content, suggesting that the phytase production depends on the biomass formation. These results are of interest since they contribute for the development of an industrial process for phytase production with elevated yields by submerged fermentation.

Microbial enzymes meet industrial demands. Over 60% of the total Phosphorus in cereal grains and oil seeds as well as their by-products is found as phytate, myo-inositol hexakisphosphate. Phytases are a group of enzymes that initiate the phosphate hydrolysis from phytate and produce myo-inositol and inorganic phosphate by catalyzing the stepwise removal of the phosphate groups. Microbial phytases effectively improve dietary phytate-P utilization. Extracellular phytase produced by Bacillus subtilis ATCC 6051 was purified by acetone precipitation, DEAE-sepharose and phenylsepharose column chromatographies. The molecular weight of the enzyme was estimated to be 48 kDa on gel filtration and 43kDa on SDS-polyacrylamide gel electrophoresis. Its optimum pH and temperature for phytase activity were pH 6.2 to 8.2 and 40°C without 10mM CaCl2 and pH 6.5 to 9.5 and 60° with 10mM CaCl2. The enzyme activity was stable from pH 6.5 to 10. The enzyme had an isoelectric point of 6.8. It was very specific for sodium phytate. The Km value for sodium phytate was 50μM. Its activity was inhibited by EDTA and metal ions such as Mn2+, Hg2+, Cu2+, Cr3+, Co2+, Ba2+ and Cd2+ ions. The enzyme has great potential in food industry, probiotics, animal feed supplement and transgenic crops.

Phytases are specialized phosphatases capable of releasing inorganic phosphate from myo-inositol hexakisphosphate (phytate), which is highly abundant in many soils. As inorganic phosphorus reserves decrease over time in many agricultural soils, genetic manipulation of plants to enable secretion of potent phytases into the rhizosphere has been proposed as a promising approach to improve plant phosphorus nutrition. Several families of biotechnologically important phytases have been discovered and characterized, but little data are available on which phytase families can offer the most benefits toward improving plant phosphorus intake. We have developed transgenic Arabidopsis thaliana plants expressing bacterial phytases PaPhyC (HAP family of phytases) and 168phyA (BPP family) under the control of root-specific inducible promoter Pht1;2. The effects of each phytase expression on growth, morphology and inorganic phosphorus accumulation in plants grown on phytate hydroponically or in perlite as the only source of phosphorus were investigated. The most enzymatic activity for both phytases was detected in cell wall-bound fractions of roots, indicating that these enzymes were efficiently secreted. Expression of both bacterial phytases in roots improved plant growth on phytate and resulted in larger rosette leaf area and diameter, higher phosphorus content and increased shoot dry weight, implying that these plants were indeed capable of utilizing phytate as the source of phosphorus for growth and development. When grown on phytate the HAP-type phytase outperformed its BPP-type counterpart for plant biomass production, though this effect was only observed in hydroponic conditions and not in perlite. Furthermore, we found no evidence of adverse side effects of microbial phytase expression in A. thaliana on plant physiology and seed germination. Our data highlight important functional differences between these members of bacterial phytase families and indicate that future crop biotechnologies involving such enzymes will require a very careful evaluation of phytase source and activity. Overall, our data suggest feasibility of using bacterial phytases to improve plant growth in conditions of phosphorus deficiency and demonstrate that inducible expression of recombinant enzymes should be investigated further as a viable approach to plant biotechnology.

Abstract. Suryani AE, Anggraeni AS, Istiqomah L, Damayanti E, Karimy MF. 2021. Isolation and identification of phytate-degrading yeast from traditional fermented food. Biodiversitas 22: 866-873. Application of phytase (myo-inositol hexakisphosphate phosphohydrolase) to catalyze the release of phosphate from phytates contained on grain-based feed has been used widely in poultry feed industry. In this study, yeast as phytase producer from traditional fermented food was isolated, screened and identified their morphological, biochemical, and molecular characteristics. Production of extracellular phytase from yeast was quantified using spectrophotometer. The results showed that among 8 yeast isolates that had phytase activity, there were two isolates with the highest phytase activity and specific activity which were TKd3 isolate (6.57 U/mL and 54.230 U/mg) and GF1 (6.07 U/mL and 53.68 U/mg). Morphological identification using Scanning Electron Microscope revealed that TKd3 cells isolated from soybean tempeh had an elongated oval cell structure, whereas the GF1 isolated from fresh gatot had a rounder cell structure. TKd3 isolate with accession number MW131530 had homology with Candida tropicalis ATCC 750 28S rRNA with 99.83% similarity and GF1 isolate with accession number MW131531 had homology with Candida tropicalis ATCC 750 28S rRNA with 100% similarity. It could be concluded that C. tropicalis yeast from traditional fermented food produced the extracellular phytase for further use of phytase in poultry feed additive.

1. Non-genetically modified (non-GM) phytase product derived from Aspergillus niger possesses various side active enzymes including α -amylase, protease, cellulase and hemicellulase. In contrast, the product of genetically modified (GM) phytase product has much less side active enzyme since the capacity of phytase production is reinforced by gene modification. In the present study we have tried to determine whether the difference of side enzyme activity of phytase product affects growth performances and nutritive value in chicks; in addition we tried to characterise the physiological change induced by the difference of side active enzymes. 2. Single Comb White Leghorn male chicks at 7 d of age were fed on experimental barley-based diets for 10 d. The feeding trial was of a factorial design (3 × 2 × 2), having three types of dietary phytase products (control, non-GM or GM phytase products derived from A. niger at 1000 U/kg diet), two levels of dietary available P supplement (0 or 6 g/kg diet) and two levels of dietary protein (CP 180 or 120 g/kg). 3. The non-GM phytase product caused a 6% increase in final body weight and feed efficiency com6 pared with the control and the GM phytase product without interacting with dietary protein and available P level. However, in birds given available P-free diet, both non-GM and GM phytase products induced a 20% increase in plasma P concentration, suggesting no difference in phytase activity between the non-GM and GM phytase products. 4. The balance study showed that the metabolisable energy of the non-GM phytase product (15.6 ± 0.05 kJ/g diet) was significantly higher among the treatments (control, 15.1 ± 0.05; GM phytase product 15.3 ± 0.07). The non-GM phytase product also increased the rate of food passage through the crop, and caused a drastic reduction in intestinal weight, perhaps as a consequence of digestion of non-starch polysaccharides. 5. We conclude that the side active enzymes in non-GM phytase product improve growth performance and nutritive value of the diet in chicks. However, the efficacy of phytase activity should not be different between non-GM and GM phytase products.

Barley genotypes grown in nutrient solution under P nutrient stress and sterile conditions were compared in activity of root-associated and root-released extracellular phytase. The activity of root-associated phytase of all genotypes was about 10 times higher than that of root-released phytase and the genotypes performed differently with regard to the activity of the enzymes. The winter barley genotype, Marinka had the highest activity of root-associated extracellular phytase which differed significantly from Alexis and Sonate, but not from Regatta. Alexis showed the lowest activity of root-released extracellular phytase which differed significantly from those of Marinka and Regatta, but not from Sonate. Generally, there was a significant correlation between the activity of root-associated and released extracellular phytase.

Five most efficient phytase producing fungi belonging to genus Aspergillus, Emericella,Gliocladium, Penicillium and Trichoderma were isolated from arid and semi-arid soils and testedfor their efficiency in hydrolyzing phytin-P compounds. Penicilliul11 purpurogellul11 accumulatedmore biomass closely followed by Trichoderma harzianum during the growth period. A strongnegative correlation (r = - 0.91, n = 20, P <0.01) was observed between development offungal mat and pH of the media. The extracellular phytase released by the organisms was12.7 times more than their intracellular phytase. The extracellular phytase activity was morein Emericella rugulosa, whereas intracellular phytase activity was more in Trichoderma harziallu11l.Emericella rugulosa was found to be the most efficient in hydrolyzing phytin-P (98.82J..lgg-l).The results indicated that Emericella rugulosa can be used as a biofertilizer under arid andsemi-arid environments for native P mobilization.Key words: Fungi, efficiency, phytase, phytin-P, hydrolysis.

The appA gene that was previously shown to code for an acid phosphatase instead codes for a bifunctional enzyme exhibiting both acid phosphatase and phytase activities. The purified enzyme with a molecular mass of 44,708 Da was further separated by chromatofocusing into two isoforms of identical size with isoelectric points of 6.5 and 6.3. The isoforms had identical pH optima of 4.5 and were stable at pH values from 2 to 10. The temperature optimum for both phytase isoforms was 60 degrees C. When heated at different pH values the enzyme showed the greatest thermal resistance at pH 3. The pH 6.5 isoform exhibited K(m) and Vmax values of 0.79 mM and 3165 U.mg-1 of protein for phytase activity and 5.5 mM and 712 U.mg-1 of protein for acid phosphatase, respectively. The pH 6.3 isoform exhibited slightly lower K(m) and Vmax values. The enzyme exhibited similar properties to the phytase purified by Greiner et al. (1993), except the specific activity of the enzyme was at least 3.5-fold less than that previously reported, and the N-terminal amino acid sequence was different. The Bradford assay, which was used by Greiner et al. (1993) for determination of enzyme concentration was, in our hands, underestimating protein concentration by a factor of 14. Phytase production using the T7 polymerase expression system was enhanced by selection of a mutant able to grow in a chemically defined medium with lactose as the carbon source and inducer. Using this strain in fed-batch fermentation, phytase production was increased to over 600 U.mL-1. The properties of the phytase including the low pH optimum, protease resistance, and high activity, demonstrates that the enzyme is a good candidate for industrial production as a feed enzyme.

The appA gene that was previously shown to code for an acid phosphatase instead codes for a bifunctional enzyme exhibiting both acid phosphatase and phytase activities. The purified enzyme with a molecular mass of 44 708 Da was further separated by chromatofocusing into two isoforms of identical size with isoelectric points of 6.5 and 6.3. The isoforms had identical pH optima of 4.5 and were stable at pH values from 2 to 10. The temperature optimum for both phytase isoforms was 60°C. When heated at different pH values the enzyme showed the greatest thermal resistance at pH 3. The pH 6.5 isoform exhibited Kmand Vmaxvalues of 0.79 mM and 3165 U·mg-1of protein for phytase activity and 5.5 mM and 712 U·mg-1of protein for acid phosphatase, respectively. The pH 6.3 isoform exhibited slightly lower Kmand Vmaxvalues. The enzyme exhibited similar properties to the phytase purified by Greiner et al. (1993), except the specific activity of the enzyme was at least 3.5-fold less than that previously reported, and the N-terminal amino acid sequence was different. The Bradford assay, which was used by Greiner et al. (1993) for determination of enzyme concentration was, in our hands, underestimating protein concentration by a factor of 14. Phytase production using the T7 polymerase expression system was enhanced by selection of a mutant able to grow in a chemically defined medium with lactose as the carbon source and inducer. Using this strain in fed-batch fermentation, phytase production was increased to over 600 U·mL-1. The properties of the phytase including the low pH optimum, protease resistance, and high activity, demonstrates that the enzyme is a good candidate for industrial production as a feed enzyme.

In this study, three strains of bacteria were isolated from soil. Among the three isolated strains, one was identified morphologically and confirmed by the molecular techniques as Bacillus subtilis MJA with high phytase activity. The phytase-producing bacteria were isolated using phytate screening agar media (PSM) with only 1.5% glucose and 0.5% sodium phytate as only source for carbon. In order to optimize the phytase production by B. subtilis MJA, different factors were studied. A combination of 0.5% glucose and 0.5% sucrose showed to be the best carbon source. Also, malt extract used as a source of nitrogen gave the highest phytase production. Also, the maximum phytase production was detected after incubation for four days (720 U/ml) at an optimum pH value of 7. The produced phytase was purified through various chromatographic techniques. The estimated enzyme molecular mass was about 38 kDa and the phytase had an optimal temperature and pH of 37°C and 5 to 6, respectively. On the other hand, studying the enzyme stability showed that enzyme was stable at low temperature, and had good pH stability by retaining 80% of its initial activity over a wide range of pH from 2 to 8. Kinetic values of V max and K m for the purified enzyme were 510 U/mg and 0.485 mM, respectively. The phytase activity was affected by different divalent metal ions. Cations such as Cu 2+ or Fe 2+ showed an inhibition effect on the phytase activity and the effect was in a dose dependent manner while, cations such as Mg 2+ or Ca 2+ showed an increase in the phytase activity. On the other hand, among different matrices used to immobilize the cells for phytase production, agar-agar matrix indicated a promising immobilization matrix used for phytase production by B. subtilis MJA. Keywords : Phytase, microbial sources, optimization, purification, characterization, immobilization African Journal of Biotechnology Vol. 12(20), pp. 2957-2967