Endophytic bacteria isolated from Phaseolus vulgaris produce phytases with potential for biotechnology application

Currently, endophytic microorganisms have become a good source of different enzymes and others metabolites of industrial interest. Among a huge spectral of molecules, enzymes as phytases have been emphasized by the ability to hydrolyze the phytic acid that represents the largest storage form of inorganic phosphorus in cereals, which are the staple diet of monogastric animals such as swine and poultry. Moreover, phytic acid acts as an antinutrient by chelating divalent metal ions, and it is interesting provide phytase as an animal feed supplement for those monogastric animals. In the current study, 158 endophytic bacteria isolated from the leaves of three cultivars of Phaseolus vulgaris were assessed for the ability to produce phytase. Among them, four isolates belonging to the Pseudomonas, Stenotrophomonas, Microbacterium and Rhodococcus genera were highlighted, due their phytase production. The phytase produced by Microbacterium foliorum BAC1157 exhibited activity at 70 °C and stability in the presence of divalent cations, indicating that this phytase has a promising use in the animal feed industry. To the authors’ knowledge, this is the first report on phytase production by bacteria of the Microbacterium genera.


Introduction
Endophytes are microorganisms that can be isolated from surfacedisinfested healthy plant tissue and do not harm the host plant (Hallmann et al., 1997).These microorganisms are considered potential sources of compounds such as antibiotics, antioxidants, anticancer drugs and enzymes (Strobel and Daisy, 2003).Phytase activity were detected in the endophytic bacteria belonging the genera Bacillus, Streptomyces, Acinetobacter, and Rhizobium isolated from the seeds and roots of Phaseolus vulgaris (common bean) (López-López et al., 2010).The phytases, or myo-inositol hexakisphosphate phosphohydrolases (E.C. 3.1.3.8; 3.1.3.26; 3.1.3.72), are derived from various sources, including animals, plants and microorganisms (Konietzny and Greiner, 2002;Vohra and Satyanarayana, 2003).These enzymes are notable for being able to hydrolyze phytate making available the inorganic phosphorus (Pi) and phosphorylated myo-inositols (Ariza et al., 2013).Phytic acid (myo-inositol hexakisphosphate) and the mixture of the cationic salts of phytic acid, which is commonly known as phytate, are organic phosphorus compounds that are widespread in nature.Approximately 60% to 90% of the Pi in vegetables, oilseeds and cereals is stored in the form of phytic acid (Coelho et al., 2002;Rao et al., 2009).
Monogastric animals such as swine and poultry are unable to degrade the phytic acid in food (Schroder et al., 1996).Therefore, the diet of these animals should be supplemented with Pi to meet their nutritional needs.However, approximately 70% of the Pi supplemented in diets is excreted into the environment, triggering the eutrophication of bodies of water near farm areas (Turner and Haygarth, 2000).Furthermore, phytic acid acts as an antinutritional factor by chelating divalent cations (such as Zn 2+ , Fe 2+/3+ , Ca 2+ , Mg 2+ , Mn 2+ and Cu 2+ ), proteins, starch and lipids, thus preventing their absorption throughout the gastrointestinal tract (Urbano et al., 2000).Thus, the importance to add feed enzymes on animal diet is that it can have an effect on the microbiome of the gastrointestinal tract promoting gut absorption of Pi and other cations for the animals (Kiarie et al. 2013;Tortola et al., 2013).
The global market for feed enzyme has growing in the next years; it has valued at $899.19 Million in 2014 and is estimated to reach $1,371.03Million by 2020 according to market research firm -MarketsandMarkets (http://www.marketsandmarkets.com).Among the major enzymes applied, phytases correspond for 60% to 80% of the market for animal nutrition (Corrêa et al., 2015).Thus, endophytic microorganisms that are promising sources of phytases have been evaluated.Accordingly, the aim of this study was to assess the phytases produced by the endophytic bacteria isolated from three varieties of the common bean, Phaseolus vulgaris (Costa et al., 2012), and to characterize the enzymes from these promising bacterial isolates.

Matherials and methods
One hundred fifty-eight strains of endophytic bacteria were isolated from the leaves of the Talismã, Ouro Negro and Vermelhinho cultivars of the common bean (Costa et al., 2012) and used for screening phytase production.The bacterial isolates were activated in 10 mL of 10% TSA culture media (Araujo et al., 2002) at 28 °C and 150 rpm for 48 to 72 h.Each bacterial isolate was inoculated into the phytase screening media plates described by Kim et al. (2003) Phytase production was assessed by the formation of a hydrolysis halo around the colonies 48 and 96 h after incubation.Bacterial isolates showing larger hydrolysis halos were selected for further testing.The strains selected were activated in TSA media as previously described to quantify phytase activity.An aliquot of the cell culture was used to inoculate 50 mL of phytase screening media (PSM), which consisted of 1.5 g L -1 glucose, 0.5 g L -1 NH 4 NO 3 , 0.05 g L -1 KCl, 0.05 g L -1 MgSO 4 .7H 2 O, 0.001 g L -1 FeSO 4 .7H 2 O, 0.001 g L -1 MnSO 4 .7H 2 O and 5.00 g L -1 phytic acid calcium salt (C 6 H 16 CaO 24 P 6 -P9539-Sigma ® ) at a pH of 5.5.The cultures were maintained at 28 °C and 150 rpm for 48 h.After this period, the broth was centrifuged at 3,000 g for 5 min, and the supernatant was used to determine the enzymatic activity.
In order to characterize the enzyme produced by the endophytic isolates, the phytase activity was determined by incubating 50 µL of supernatant in 350 µL of 0.1 M sodium acetate buffer, pH 5.0, with 2.5 mM phytic acid sodium salt (C 6 H 18 O 24 P 6 xNa + yH 2 O-P0109-Sigma ® ) at 50 °C for 15 min.The Pi released was quantified using the method described by Heinonen and Lahti (1981).A standard curve with several concentrations of KH 2 PO 4 was used to calculate the enzymatic activity.One unit of phytase activity was defined as the amount of enzyme required to release 1 µmol of phosphate per minute under the assay conditions.The effect of temperature on phytase activity was measured by incubating the culture filtrate with the substrate at temperatures ranging from 20 °C to 80 °C for 15 min.The effect of pH on enzyme activity was assessed by incubating the culture supernatant and the substrate in the following (0.1 M) buffer solutions: glycine-HCl (pH 1.0-3.5),sodium acetate (pH 4.0-6.0),Tris-HCl (pH 6.5-8.5) and glycine-NaOH (pH 9.0-12.0).The enzyme-substrate specificity was determined by replacing the phytic acid sodium salt with 2.5 mM ATP, ADP, pyrophosphate or β-glycerophosphate.The effect of divalent cations on phytase activity was determined by incubating the supernatant of each bacterial isolate with 1 mM, 5 mM, 10 mM or 20 mM CaCl2, CdSO 4 , CuSO 4 , FeSO 4 , KCl, MgCl 2 , MnCl 2 or ZnSO 4 for 1 h.The 500 mM stock solution of each cation was prepared as described in Tang et al. (2006).The untreated enzyme activity was set to 100% in all of the assays.The data were analyzed using analysis of variance and Tukey's test at 5% probability using SAEG software, version 9.1, Universidade Federal de Viçosa.
Table 1.The in vitro phytase production assay from the 158 endophytic isolates of the P. vulgaris leaf and hydrolysis halo size of the 45 isolates positive for phytase production.The phytase produced by the endophytic isolates was characterized for optimal activity temperature and pH, substrate specificity and stability in the presence of divalent cations.M. foliorum BAC1157 contained the phytase with the highest optimal temperature (70 °C), followed by R. erythropolis BAC2162 and S. maltophilia BAC2135 (50 °C) and P. aeruginosa BAC3115 (40 °C) (Figure 2).The pH studies revealed that M. foliorum BAC1157, P. aeruginosa BAC3115 and R. erythropolis BAC2162 exhibit two peaks of maximum phytase activity (pH 3.0 and 5.0, 3.0 and 8.0 and 4.5 and 8.0, respectively), while S. maltophilia BAC2135 showed a single peak of activity at pH 4.5 (Figure 3).To determine the specific phytase activity, different phosphate sources were used to measured phytase activity from the four endophytic bacteria isolates.

Isolate
Activity toward β-glycerophosphate and sodium pyrophosphate was detected in the culture supernatants of the four bacterial isolates when phosphate compounds and phytic acid were both used as substrates in the reaction.S. maltophilia BAC2135 was the only bacterial isolate that showed activity with ATP, while M. foliorum BAC1157 and R. erythropolis BAC2162 showed activity with ADP (Table 2).Bacterial phytases usually exhibit specificity for phytic acid and low activities toward other phosphorylated compounds, such as ADP, β-glycerophosphate and pyrophosphate (Shimizu, 1982;Casey and Walsh, 2003;Kim et al., 2003).As shown in Table 2, the enzymes produced by the endophytic isolates can be characterized as phytases and phosphatases with a narrow spectrum of action on other phosphorylated compounds.

Results and discussion
The effects of 1, 5, 10 and 20 mM Ca 2+ , Cd 2+ , Cu 2+ , Fe 2+ , K + , Mg 2+ , Mn 2+ and Zn 2+ on the phytase activity of the four bacterial isolates can be found in Table 3.The phytase activity of the P. aeruginosa BAC3115 supernatant increased with the Ca 2+ concentration, and 1 and 5 mM Cu 2+ .In contrast, 10 and 20 mM Mn 2+ and Zn 2+ decreased the enzymatic activity of this isolate.Ten millimolar Ca 2+ increased the phytase activity of the S. maltophilia BAC2135 isolate, although high Mn 2+ and Zn 2+ concentrations decreased the activity of this supernatant.The enzyme produced by M. foliorum BAC1157 showed the highest activity in high Ca 2+ concentrations, 1 to 20 mM Fe 2+ , high Mg 2+ and low Mn 2+ and Zn 2+ concentrations.The phytase produced by R. erythropolis BAC2162 was the most active in high Fe 2+ concentrations and low Mn 2+ concentrations.
Table 3.Effect of divalent cations on the activity of endophytic isolate-derived phytases.
The large number of phytaseproducing bacterial isolates found in this work suggests that endophytic bacteria are promising candidates for the production and study of this enzyme.While assessing the potential production of bacterial phytase in Malaysia, Hussin et al. (2009) reported that endophytic bacteria exhibited the highest phytase activity.Endophytic microorganisms can be vertically transmitted to seeds.Considering that phytic acid is the main storage form of Pi in grains and legumes, it is plausible that a selection of endophytic microorganisms capable of degrading phytic acid and assisting in the process of seed germination might exist (López-López et al., 2010).
Extracellular enzymes are excellent candidates for large-scale production and marketing because they eliminate the expenses associated with the extraction process (Anis Shobirin et al., 2009).The most production of bacterial phytases are intracellular, with the exception of those phytases that are derived from Bacillus subtilis, Lactobacillus amylovorus and Enterobacter sp. 4 species (Vohra and Satyanarayana, 2003).However, in the current study, those bacterial isolates presenting the largest hydrolysis halos around the colonies in solid media containing calcium phytate suggests an extracellular phytase activity.Anis Shobirin et al. (2009)  To assess the potential application of the phytases produced by P. aeruginosa BAC3115, S. maltophilia BAC2135, M. foliorum BAC1157 and R. erythropolis BAC2162 in the animal feed industry, the enzymes produced by these bacteria were characterized for optimal activity pH and temperature, substrate specificity and divalent cations effect.The phytase produced by the M. foliorum BAC1157 isolate was the most active at 70 °C.Furthermore, the phytase in question showed 90% activity at 40 °C, which is another desirable feature, as the body temperature of pigs is 39 °C (Casey and Walsh, 2003).The optimal temperature for most microbial phytases is approximately 50 °C (Simon and Igbasan, 2002); however, some microbial phytases have exhibited optimal temperatures above 60 °C (Berka et al., 1998;Matsui et al., 2000;Zamudio et al., 2001).M. foliorum BAC1157 isolate has a remarkable application and can be used as a supplement in swine feed because the pelleting process involves temperatures above 70 °C.
The endophytic isolates used in the here exhibited more than one optimal pH peak, suggesting the production of phytases with different characteristics.B. subtilis (natto) produces a phytase with an optimal activity between 6.0 and 6.5 (Shimizu, 1992), and the C. braakii phytase shows optimal activity at a pH of approximately 4.0 (Kim et al., 1998).The phytase produced by M. foliorum BAC1157 showed the highest enzymatic activity at a pH of 3.0 and merely 20% activity at pH 2.0.The pH in the swine stomach varies between 2.0 and 4.0 (Lindberg and Ogel, 2001) depending on the feed and other factors.This substantial loss of activity at pH 2.0 is a negative factor, but controlling animal feed can ensure that the stomach pH does not reach this value.However, the low pH in gastric phase promotes the environment where phytate is really soluble and most susceptible to hydrolyses.Although for some fungal phytases it seems to be a huge problem because of the range of optimal activity at high pH.On the other hand, isolating and characterizing the gene encoding the M. foliorum BAC1157 phytase may aid in the development of site-directed mutagenesis strategies designed to increase enzymatic activity at pH values below 4.0, similar to the phytase applied in the animal feed industry, as the produced by Aspergillus niger NRRL3135 (Mullaney et al., 2002).
Because the several divalent cations found in the diets administered to monogastric animals may interfere with the activity of supplemental enzymes added to these foods, it is necessary to predict the effect of such cations on phytase activity.The addition of cations to the enzymatic reaction changed the phytase activity of the four bacterial isolates.The M. foliorum BAC1157 bacterial isolate was only inhibited at high concentrations of Cd 2+ and Mn 2+ , in addition to showing phytase activity at 70 °C.Furthermore, increased phytase activity in the presence of Zn 2+ is an uncommon feature, especially considering that this metal inhibits several of the phytases reported in the literature (Yoon et al., 1996;Greiner et al., 1997;Greiner et al., 2009).Studies have reported that the presence of Ca 2+ did not affect the activity of A. niger 11T53A9 (Greiner et al., 2009) and C. braaki phytases (Kim et al., 1998).However, Ca 2+ inhibited the activity of a phytase produced by A. niger ATCC9142 (Casey and Walsh, 2003), and decreased the activity of a phytase produced by B. subtilis DS11 (Kim et al., 1998).Similarly, Fe 2+ and Fe +3 did not affect the activity of the phytase produced by A. niger ATC9142 (Casey and Walsh, 2003), despite inhibiting some other phytases (Kim et al., 1998;Greiner et al., 2009).Increased activity in the presence of cations such as Cu 2+ , Mg 2+ , Mn 2+ and Zn 2+ was another unusual feature noted in the phytase isolated by Casey and Walsh (Casey and Walsh, 2003).

Conclusion
The results of the current study suggest that P. aeruginosa BAC3115, R. erythropolis BAC2162, S. maltophilia BAC2135 and M. foliorum BAC1157 are promising candidates for the study and application of endophytic isolate-derived phytases in the feed industry.To our knowledge, this is the first report on phytase production by the bacteria Microbacterium foliorum BAC1157, in which this phytase could has a potential use in monogastric diets.

Figure 3 .
Figure 3. Optimal pH for the phytases derived from the four endophytic isolates.The optimal pH values were determined in phytase-inducing liquid media after 48 hours of culture.A -P. aeruginosa BAC3115, B -S. maltophilia BAC2135, C -M. foliorum BAC1157, D -R. erythropolis BAC2162.