Characterization of a putative metal-dependent PTP-like phosphatase from Lactobacillus helveticus 2126 / Caracterización de una supuesta fosfatasa similar a PTP dependiente de metales de Lactobacillus helveticus 2126

To date, there are very limited reports on sequence analysis and structure-based molecular modeling of phosphatases produced by probiotic bacteria. Therefore, a novel protein tyrosine-like phosphatase was characterized from L. helveticus 2126 in this study. The purified bacterial phosphatase was subjected to mass spectrometric analysis, and the identity of constructed sequence was analyzed using peptide mass fingerprint. The 3-D structure of protein was elucidated using homology modeling, while its stability was assessed using Ramachandran plot, VERIFY 3D, and PROCHECK. The bacterium produced an extracellular phosphatase of zone diameter 15 ± 0.8 mm on screening medium within 24 h of incubation. This bacterial phosphatase was highly specific towards sodium phytate as it yielded the lowest Km value of 299.50 ± 4.95 μM compared to other phosphorylated substrates. The activity was effectively stimulated in the presence of zinc, magnesium, and manganese ions thereby showing its PTP-like behavior. The phosphatase showed a molecular mass of 43 kDa, and the corresponding M/Z ratio data yielded 46% query coverage to Bacillus subtilis (3QY7). This showed a 61.1% sequence similarity to Ligilactobacillus ruminis (WP_046923835.1). The final sequence construct based on these bacteria showed a conserved motif “HCHILPGIDD” in their active site. In addition, homology modeling showed a distorted Tim barrel structure with a trinuclear metal center. The final model after energy minimization showed 90.9% of the residues in the favorable region of Ramachandran’s plot. This structural information can be used in genetic engineering for improving the overall stability and catalytic efficiency of probiotic bacterial phosphatases.(AU)

Characterization of a putative metal-dependent PTP-like phosphatase from Lactobacillus helveticus 2126.

To date, there are very limited reports on sequence analysis and structure-based molecular modeling of phosphatases produced by probiotic bacteria. Therefore, a novel protein tyrosine-like phosphatase was characterized from L. helveticus 2126 in this study. The purified bacterial phosphatase was subjected to mass spectrometric analysis, and the identity of constructed sequence was analyzed using peptide mass fingerprint. The 3-D structure of protein was elucidated using homology modeling, while its stability was assessed using Ramachandran plot, VERIFY 3D, and PROCHECK. The bacterium produced an extracellular phosphatase of zone diameter 15 ± 0.8 mm on screening medium within 24 h of incubation. This bacterial phosphatase was highly specific towards sodium phytate as it yielded the lowest Km value of 299.50 ± 4.95 µM compared to other phosphorylated substrates. The activity was effectively stimulated in the presence of zinc, magnesium, and manganese ions thereby showing its PTP-like behavior. The phosphatase showed a molecular mass of 43 kDa, and the corresponding M/Z ratio data yielded 46% query coverage to Bacillus subtilis (3QY7). This showed a 61.1% sequence similarity to Ligilactobacillus ruminis (WP_046923835.1). The final sequence construct based on these bacteria showed a conserved motif "HCHILPGIDD" in their active site. In addition, homology modeling showed a distorted Tim barrel structure with a trinuclear metal center. The final model after energy minimization showed 90.9% of the residues in the favorable region of Ramachandran's plot. This structural information can be used in genetic engineering for improving the overall stability and catalytic efficiency of probiotic bacterial phosphatases.

Probiotic Validation of a Non-native, Thermostable, Phytase-Producing Bacterium: Streptococcus thermophilus.

Phytate-linked nutritional deficiency disorders have plagued poultry for centuries. The application of exogenous phytases in poultry feed has served as a solution to this problem. However, they are linked to certain limitations which include thermal instability during prolonged feed processing. Therefore, in this study, Streptococcus thermophilus 2412 based phytase stability was assessed at higher temperatures up to 90 °C. This was followed by probiotic validation of the same bacterium in an in vitro intestinal model. Bacterial phytase showed thermostability up to 70 °C with a recorded activity of 9.90 U. The bacterium was viable in the intestinal lumen as indicated by the cell count of 6.10 log(CFU/mL) after 16 h. It also showed acid tolerance with a stable cell count of 5.01 log(CFU/mL) after 16 h of incubation at pH 2. The bacterium displayed bile tolerance yielding a cell count of 6.36 log(CFU/mL) in the presence of 0.3% bile. Bacterial susceptibility was observed toward all tested antibiotics with a maximum zone of 20 mm against clindamycin. The maximum antagonistic activity was observed against Staphylococcus aureus, Serratia marcescens, and Escherichia coli with inhibition zone diameters up to 10 mm. The above characteristics prove that S. thermophilus 2412 can be used as an effective phytase-producing poultry probiotic.

An in vitro chicken gut model for the assessment of phytase producing bacteria.

An in vitro simulated chicken gut model was proposed for studying the phytase activity of selected bacteria such as Streptococcus thermophilus, Sporosarcina pasteurii, Sporosarcina globispora, and Sporosarcina psychrophila using known probiotic bacterium, Lactobacillus helveticus as a control. The selected bacteria were viable in the intestinal lumen and produced extracellular phytase at optimal phytate concentration of 6.25 mM when compared to 3.125 mM and 12.5 mM. These bacteria demonstrated significantly higher (p < 0.05) phosphate liberation (up to 387 µM) due to better phytase activity in the production medium, when compared to the growth medium (339 µM). The phytase activity showed a steady increase in phosphate liberation up to 150 min after which it became constant. This trend is observed for the selected bacteria at pH 5, 6 and 7. However, the liberation of phosphates showed no significant difference (p > 0.05) at the tested pH. Among the analyzed bacteria, the members of the genus Sporosarcina showed better phytate degradation when compared to S. thermophilus. The proposed model can be extended to analyze any extracellular enzymes produced by gut microbes.

A Preliminary Study on Probiotic Characteristics of Sporosarcina spp. for Poultry Applications.

Probiotics are well known for their wide range of beneficial activities. However, recent use of probiotic Bifidobacterium, Enterococcus, and Lactobacillus spp. has been plagued by certain disadvantages such as complex growth requirements, high maintenance cost, susceptibility to the gastrointestinal environment, pathogenic gene transfer, non-standardized dosage, cell lysis at extreme acidic pH, widespread antibiotic resistance, and lower bacterial viability due to the lack of spore formation. Therefore, spore-forming bacteria belonging to Sporosarcina genus such as pasteurii, globispora, and psychrophila were assessed for probiotic characteristics such as biofilm formation, intestinal adhesion, acid and bile tolerance, antibiotic sensitivity, and anti-pathogenic activity. This ensures bacterial viability under gastrointestinal conditions and enabled the same to colonize effectively in the intestinal lumen (in vitro). The bacterial cell counts ranging from 6.59 to 6.91 log(CFU/mL) was observed for Sporosarcina spp. after 16 h. This indicated that there is no significant difference in the cell counts (P-value = 0.90). The cell counts of Sporosarcina spp. ranging from 5.57 to 5.93 log(CFU/mL) displayed strong acid tolerance at pH 2. They were also viable at higher bile (0.5%) concentration. Among the Sporosarcina spp., pasteurii showed better tolerance (6.90 log(CFU/mL)) even after 16 h. Among the selected bacteria, Sporosarcina psychrophila was more susceptible to teicoplanin and meropenem with an inhibition zone of 30 mm. Maximum antagonistic activity was observed against Serratia marcescens (with inhibition zone up to 15 mm). Our results suggest that bacteria belonging to Sporosarcina genus possess all the required characteristics to be used as potential poultry probiotics.

Microbial degradation of myo-inositol hexakisphosphate (IP6): specificity, kinetics, and simulation.

Microbial degradation of myo-inositol hexakisphosphate (IP6) is crucial to deal with nutritional problems in monogastric animals as well as to prevent environmental phosphate pollution. The present study deals with the degradation of IP6 by microorganisms such as Sporosarcina spp. pasteurii, globiospora, psychrophila, Streptococcus thermophilus and Saccharomyces boulardii. These microbes were screened for phytase production under laboratory conditions. The specificity of the enzyme was tested for various phosphorylated substrates such as sodium phytate (IP6), sodium hexametaphosphate, phenyl phosphate, α-d-glucose-6 phosphate, inosine 5' monophosphate and pyridoxal 5' phosphate. These enzymes were highly specific to IP6. The influence of modulators such as phytochemicals and metal ions on the enzymatic activity was assessed. These modulators in different concentrations had varying effect on microbial phytases. Calcium (in optimal concentration of 0.5 M) played an important role in enzyme activation. The enzymes were then characterized based on their molecular weight 41~43 kDa. The phytase-producing microbes were assessed for IP6 degradation in a simulated intestinal setup. Among the selected microbes, Sporosarcina globiospora hydrolyzed IP6 effectively, as confirmed by colorimetric time-based analysis.