Identification of Pantoea Phytate-Hydrolyzing Rhizobacteria Based on Their Phenotypic Features and Multilocus Sequence Analysis (MLSA)

Abstract—: Accurate strain identification within the Pantoea genus is difficult due to homologous recombination, which may affect the species boundaries. An integrated approach is presently the most effective one in determining the species of bacteria. Biochemical identification using the API20E system, phylogenetic analysis of the 16S rRNA gene sequences, and MLSA analysis based on partial sequences of the fusA, pyrG, leuS, gyrB, and rpoB genes showed that the soil phytate-hydrolyzing isolates belonged to the genus Pantoea, specifically to the species Pantoea brenneri. It was also established that phytate-hydrolyzing activity of the strains was accompanied by their ability to fix atmospheric nitrogen

Expression of Pantoea sp. 3.5.1 AgpP Phytase in Three Expression Systems

Nowadays, microbial phytases have been widely used as feed additives that increase the bioavailability of phosphorus for monogastric animals. Still, there is an active search for new phytases, development of effective production systems, and study of their properties. In this study, we compared the biochemical characteristics of bacterial histidine acid phytase of Pantoea sp. 3.5.1 produced in three different expression systems: Escherichia coli, methylotrophic yeast Pichia pastoris, and yeast Yarrowia lipolytica. The maximum activity of the recombinant phytase AgpP-P expressed by P. pastoris occurred at pH 4.0, while the pH optimum of the recombinant AgpP-Y phytase expressed by Y. lipolytica is shifted towards more acidic pH 3.0. The recombinant AgpP-P enzyme is stable at pH values ​​from 2.0 to 5.0, while the AgpP-Y remains active at pH values ​​from 3.0 to 7.0; however, at pH above 8.0, the enzyme becomes inactive. The temperature optimum of recombinant AgpP-P phytase corresponded to 50 °С, while the temperature optimum for AgpP-Y was at 45 °С. The recombinant enzymes AgpP-P and AgpP-Y retained activity at temperatures from 4 to 70 °C and from 4 to 60 °C, respectively. Bivalent metal ions at a concentration of 1 mM had the same effect on the activity of recombinant phytases from E. coli, P. pastoris, and Y. lipolytica: Ca2 +, Mg2 +, and Mn2 + ions more than doubled the activity of enzymes, while Co2 + did not affect the activity of phytases, while ions Zn2 +, Cu2 +, and Fe2 + inhibited the activity of enzymes

Bacterial enzymes as potential feed additives in poultry farming

The problem of obtaining economic benefits in poultry breeding from complete absorption of feeds by domestic fowls through increasing the digestibility of nutrients remains urgent. The addition of exogenous enzymes to broiler rations enhances the energy and protein intake by improving the substrate bioavailability. The presence of antinutritional dietary factors in poultry feeds (non-starch polysaccharides, protease inhibitors, lectins, and phytates) indicates the possibility of using exogenous enzymes. We assessed the potential of histidine acid phytase Pantoea sp. 3.5.1 and subtilisin-like proteinase B. pumilus as feed additives. When the enzymes pass through the digestive tract, they are exposed to elevated body temperatures of the fowl (40 ?C), pH changes in different parts of the digestive tract, and the influence of bile. Thus, it was important to study the stability of the enzymes under these conditions. It was shown that proteinase maintains its activity throughout the gastrointestinal tract of the fowl: in a weakly acidic environment (pH 5.5, goiter), the enzyme activity remained full (100%); at pH 2.9 (stomach), its decreased by 40%; and under the alkaline conditions of pH 6.5–8.0 (small and large intestine), its values were restored and increased by 13% relative to the control group. When exposed to bile at the concentrations from 0.01% to 0.25% for 1 h, phytase retained more than 50% of its activity (p < 0.05). At the concentrations of bile from 0.01% to 0.05% (1-h exposure), the activity of proteinase remained at the control level. The data obtained show that microbial enzymes in the digestive tract of domestic fowls retain catalytic activity. In the course of the balance experiment, the group of chickens treated with proteinase at the concentration of 10 U/kg demonstrated the highest nitrogen and calcium digestibility coefficients (56.4% and 59.5%, respectively). The use of phytase (1000 U/kg) had a significant impact on the absorption of phosphorus (the digestibility coefficient was 40.5% (0.30 g)). Thus, B. pumilus proteinase and Pantoea sp. 3.5.1 phytase have the potential to be used as feed additives for chickens