Purification and characterization of a phospholipid-hydrolyzing phosphoesterase produced by Pediococcus acidilactici isolated from Gouda cheese.

Lipolysis occurs during ripening of dairy products as a result of esterase or lipase activity. Lactic acid bacteria (LAB) are considered to be weakly lipolytic bacteria compared with other species. In cheeses with extended ripening periods, lipolytic LAB may have several advantages. Pediococcus acidilactici is a LAB frequently found in fermented dairy products, but no previous reports exist on their production of esterases or lipases. Our interest in the relationship of LAB and enzymatic characterization is due to the multiple reports of the benefits of LAB in the gut microbiome, particularly at the intestinal membrane. Pediococci have been characterized as probiotic and especially active in membrane interactions. The aim of this project was to purify, characterize, and identify the phosphoesterase produced by P. acidilactici originally isolated from Gouda cheese and determine its phospholipid (PL) hydrolysis profile, with a focus on increased absorption of these compounds in the human gut. Native zymograms were performed to identify a protein with lipolytic activity in the intracellular fraction of P. acidilactici. The enzyme was purified via size-exclusion HPLC, concentrated via ultrafiltration, and identified using sequence analysis in liquid chromatography (LC)-MS/MS. The purified fraction was subjected to biochemical characterization as a function of pH, temperature, ion concentration, hydrolysis of different substrates, and PL. A single protein with a molecular weight of 86 kDa and esterase activity was detected by zymography. Analysis of the LC-MS/MS results identified a putative metallophosphoesterase with a calculated molecular weight of 45.5 kDa, suggesting that this protein is active as a homodimer. The pure protein showed an optimal activity between pH 8.0 to 9.0. The optimal temperature for activity was 37°C, and the enzyme lost 15% of activity after incubation at 90°C for 1 h. This enzyme showed activity on short-chain fatty acids and exhibited high hydrolysis of phosphatidylinositol. It also hydrolyzed phosphatidylserine, phosphatidylcholine, and sphingomyelin. Phosphatidylethanolamine was hydrolyzed but with less efficiency. The characteristics and lipolytic actions exerted by this protein obtained from LAB hold promise for a potential strain of esterase or lipase that may exert human health benefits through increased digestibility and absorption of nutrients found in dairy products.

FMN-dependent oligomerization of putative lactate oxidase from Pediococcus acidilactici.

Lactate oxidases belong to a group of FMN-dependent enzymes and they catalyze a conversion of lactate to pyruvate with a release of hydrogen peroxide. Hydrogen peroxide is also utilized as a read out in biosensors to quantitate lactate levels in biological samples. Aerococcus viridans lactate oxidase is the best characterized lactate oxidase and our knowledge of lactate oxidases relies largely to studies conducted with that particular enzyme. Pediococcus acidilactici lactate oxidase is also commercially available for e.g. lactate measurements, but this enzyme has not been characterized in detail before. Here we report structural characterization of the recombinant enzyme and its co-factor dependent oligomerization. The crystal structures revealed two distinct conformations in the loop closing the active site, consistent with previous biochemical studies implicating the role of loop in catalysis. Despite the structural conservation of active site residues, we were not able to detect either oxidase or monooxygenase activity when L-lactate was used as a substrate. Pediococcus acidilactici lactate oxidase is therefore an example of a misannotation of an FMN-dependent enzyme, which catalyzes likely a so far unknown oxidation reaction.

Probiotic properties of a phytase producing Pediococcus acidilactici strain SMVDUDB2 isolated from traditional fermented cheese product, Kalarei.

The nutritional challenge faced by the monogastric animals due to the chelation effects of phytic acid, fuel the research on bioprospecting of probiotics for phytase production. Pediococcus acidilactici SMVDUDB2 isolated from Kalarei, exhibited extracellular phytase activity of 5.583 U/mL after statistical optimization of fermentation conditions viz. peptone (1.27%); temperature (37 °C); pH (6.26) and maltose (1.43%). The phytase enzyme possessed optimum pH and temperature of 5.5 and 37 °C, respectively and was thermostable at 60 °C. The enzyme was purified 6.42 fold with a specific activity of 245.12 U/mg with hydrophobic interaction chromatography. The purified enzyme had Km and Vmax values of 0.385 mM and 4.965 µmol/min respectively, with sodium phytate as substrate. The strain depicted more than 80% survival rate at low pH (pH 2.0, 3.0), high bile salt concentration (0.3 and 0.5%), after gastrointestinal transit, highest hydrophobicity affinity with ethyl acetate (33.33 ± 0%), autoaggregation (77.68 ± 0.68%) as well as coaggregation (73.57 ± 0.47%) with Staphylococcus aureus (MTCC 3160). The strain exhibited antimicrobial activity against Bacillus subtilis (MTCC 121), Mycobacterium smegmatis (MTCC 994), Staphylococcus aureus (MTCC 3160), Proteus vulgaris (MTCC 426), Escherichia coli (MTCC 1652) and Lactobacillus rhamnosus (MTCC 1408). The amount of exopolysaccharide produced by the strain was 2 g/L. This strain having the capability of phytate degradation and possessing probiotic traits could find application in food and feed sectors.

Next generation sequencing, biochemical characterization, metabolic pathway analysis of novel probiotic Pediococcus acidilactici NCDC 252 and it's evolutionary relationship with other lactic acid bacteria.

Pediococcus acidilactici NCDC 252 is a facultative anaerobe of dairy origin that possessed all studied in vitro probiotic attributes and several useful enzyme activities. Its whole genome was sequenced and analysed for its evolutionary relationship with other lactic acid bacteria (LAB). This is a novel sequence and first report of genome sequence of P. acidilactici of dairy origin. Its genome is relatively larger than other studied genomes of P. acidilactici and is comprised of 40 scaffolds that totals to 3,243,337 bases and 44.5% GC content. A total of 3054 coding sequences (CDS) were identified by RAST and DIAMOND servers. The genome also encoded different enzyme activities required for utilization of various carbohydrates. This was also confirmed by carbohydrate utilization studies. The genome also encoded genes for probiotics properties. The phylogenetic analysis of P. acidilactici NCDC 252 genome was done using Maximum Parsimony and Maximum Likelihood methods to study its evolution and relatedness to other LABs based upon their 16S rDNA sequences. The strain exhibited highest resemblance to Lactobacillus plantarum WCFS1 and is also much close to P. acidilactici based on similarity of ribosomal protein. The strain seems to have acquired some genes for its adaptation in dairy/environmental niche. This genome sequence is novel with genome more similar to L. plantarum and biochemical and phenotypic characteristics of P. acidilactici.

Engineering D-Lactate Dehydrogenase from Pediococcus acidilactici for Improved Activity on 2-Hydroxy Acids with Bulky C3 Functional Group.

Engineering D-lactic acid dehydrogenases for higher activity on various 2-oxo acids is important for the synthesis of 2-hydroxy acids that can be utilized in a wide range of industrial fields including the production of biopolymers, pharmaceuticals, and cosmetic compounds. Although there are many D-lactate dehydrogenases (D-LDH) available from a diverse range of sources, there is a lack of biocatalysts with high activities for 2-oxo acids with large functional group at C3. In this study, the D-LDH from Pediococcus acidilactici was rationally designed and further engineered by controlling the intermolecular interactions between substrates and the surrounding residues via analysis of the active site structure of D-LDH. As a result, Y51L mutant with the catalytic efficiency on phenylpyruvate of 2200 s-1 mM-1 and Y51F mutant on 2-oxobutryate and 3-methyl-2-oxobutyrate of 37.2 and 23.2 s-1 mM-1 were found, which were 138-, 8.5-, and 26-fold increases than the wild type on the substrates, respectively. Structural analysis revealed that the distance and the nature of the interactions between the side chain of residue 51 and the substrate C3 substituent group significantly affected the kinetic parameters. Bioconversion of phenyllactate as a practical example of production of the 2-hydroxy acids was investigated, and the Y51F mutant presented the highest productivity in in vitro conversion of D-PLA.

A novel endo-ß-1,4-xylanase from Pediococcus acidilactici GC25; purification, characterization and application in clarification of fruit juices.

A novel extracellular xylanase was purified and characterized from Pediococcus acidilactici GC25 (GenBank number: MF289522). The purification was 4.6-fold with a yield of 43.61% through acetone precipitation, Q-Sepharose, and CM-Sepharose ion change chromatography. The molecular weight of the enzyme was 48.15 kDa, and the optimum pH and temperature were 7.0 and 40 °C, respectively. The maximum activity was observed between 20 and 50 °C. Although it was active within a wide pH range (pH 2.0-9.0), it retained over 85% of its activity after 24 h incubation; and over 70% of its activity after 168 h incubation in neutral and alkaline pH. It was observed that the enzyme showed high stability with K+, Ba2+, Cd2+, Co2+, Sr2+, Mg2+, Ca2+, Al3+, Zn2+, and Ni2+ ions. The Km and Vmax for the xylanase were 3.10 mg mL-1 and 4.66 U/mg protein, respectively. It was determined that treatment of different fruit juices with P. acidilactici GC25 xylanase improved the clarification. The highest increase in the reducing sugar amount and decrease in the turbidity was 24.47 ±â€¯1.08 and 21.22 ±â€¯0.58 for peach juice at 0.15 U/mL enzyme concentration. These results showed that the xylanase purified from P. acidilactici GC25 may have a wide potential in biotechnological processes of the food and baking industry.

Purification, kinetic and functional characterization of membrane bound dipeptidyl peptidase-III from NCDC 252: a probiotic lactic acid bacteria.

Pediococcus acidilactici is a probiotic lactic acid bacteria possessing studied in-vitro probiotic properties. Study of membrane proteins is crucial in developing technological and health applications of probiotic bacteria. Genome analysis of Pediococcus acidilactici revealed about more than 60 proteases/peptidases which need characterization. Dipeptidyl peptidase-III (DPP-III) is studied for first time in prokaryotes and it is a membrane protein in P. acidilactici that has been purified to apparent homogeneity. The enzyme was purified 81.66 fold with 36.75% yield. The specific activity of purified DPP-III was 202.67 U/mg. The protein moved as single band on native PAGE. The purity was also confirmed by in-situ gel assay. However SDS-PAGE analysis revealed it as high molecular weight heterotetramer with molecular weight of 108 kDa. The enzyme was maximally active at pH 8.5 and at 37 C. Purified DPP-III specifically hydrolyzed Arg-Arg-4-ßNA with micromolar affinity (Km = 9.0 µM) and none of studied endopeptidase and monopeptidase substrate was hydrolyzed. Inhibition study revealed purified DPP-III to be a serine protease with involvement of metal ion at active site. The significance of this enzyme as membrane protein is yet to be studied.

Purification and characterization of ß-galactosidase from probiotic Pediococcus acidilactici and its use in milk lactose hydrolysis and galactooligosaccharide synthesis.

ß-galactosidase is a commercially important enzyme that was purified from probiotic Pediococcus acidilactici. The enzyme was extracted from cells using sonication and subsequently purified using ammonium sulphate fractionation and successive chromatographies on Sephadex G-100 and Q-Sepharose. The enzyme was purified 3.06-fold up to electrophoretic homogeneity with specific activity of 0.883 U/mg and yield of 28.26%. Molecular mass of ß-galactosidase as estimated by SDS-PAGE and MALDI-TOF was 39.07 kDa. The enzyme is a heterodimer with subunit mass of 15.55 and 19.58 kDa. The purified enzyme was optimally active at pH 6.0 and stable in a pH range of 5.8-7.0 with more than 97% activity. Purified ß-galactosidase was optimally active at 50 °C. Kinetic parameters Km and Vmax for purified enzyme were 400 µM and 1.22 × 10-1 U respectively. Its inactivation by PMSF confirmed the presence of serine at the active site. The metal ions had different effects on enzyme. Ca2+, Mg2+ and Mn2+ slightly activated the enzyme whereas NH4+, Co2+ and Fe3+ slightly decreased the enzyme activity. Thermodynamic parameters were calculated that suggested that ß-galactosidase is less stable at higher temperature (60 °C). Purified enzyme effectively hydrolysed milk lactose with lactose hydrolysing rate of 0.047 min-1 and t1/2 of 14.74 min. This is better than other studied ß-galactosidases. Both sonicated Pediococcus acidilactici cells and purified ß-galactosidase synthesized galactooligosaccharides (GOSs) as studied by TLC at 30% and 50% of lactose concentration at 47.5 °C. These findings indicate the use of ß-galactosidase from probiotic bacteria for producing delactosed milk for lactose intolerant population and prebiotic synthesis. pH and temperature optima and its activation by Ca2+ shows that it is suitable for milk processing.

Recombinant laccase from Pediococcus acidilactici CECT 5930 with ability to degrade tyramine.

Biogenic amines degradation by bacterial laccases is little known, so we have cloned and heterologously expressed, in E. coli, a new laccase from Pediococcus acidilactici CECT 5930 (Lpa5930), a lactic acid bacterium commonly found in foods able to degrade tyramine. The recombinant enzyme has been characterized by physical and biochemical assays. Here we report the optimization of expression and purification procedures of this laccase. DNA encoding sequence of laccase from P. acidilactici was amplified by PCR and cloned into the expression plasmid pET28a for induction by isopropyl-ß-D-thiogalactoipyranoside. Protein expression was performed in E. coli BL21(DE3) harboring pGro7 plasmid expressing a chaperone folding assistant induced by arabinose. Purification was performed by column metal-chelating chromatography on Ni-NTA-agarose. The laccase enzyme obtained has an apparent molecular mass of ∼60 kDa, an optimum temperature activity toward 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) of 28°C, and was quickly inactivated at temperatures higher than 70°C. The apparent Km value for ABTS was 1.7 mM and the Vmax obtained was 24 U/mg. In addition to ABTS, recombinant Lpa5930 laccase degraded the biogenic amine tyramine at pH 9.5 and pH 4.0 with or without ABTS as a mediator. Tyramine degradation by laccases could solve the problems generated in food due to the presence of this toxic compound.

In vitro assessment of Pediococcus acidilactici Kp10 for its potential use in the food industry.

BACKGROUND: Selection of a microbial strain for the incorporation into food products requires in vitro and in vivo evaluations. A bacteriocin-producing lactic acid bacterium (LAB), Pediococcus acidilactici Kp10, isolated from a traditional dried curd was assessed in vitro for its beneficial properties as a potential probiotic and starter culture. The inhibitory spectra of the bacterial strain against different gram-positive and gram-negative bacteria, its cell surface hydrophobicity and resistance to phenol, its haemolytic, amylolytic and proteolytic activities, ability to produce acid and coagulate milk together with its enzymatic characteristics and adhesion property were all evaluated in vitro. RESULTS: P. acidilactici Kp10 was moderately tolerant to phenol and adhere to mammalian epithelial cells (Vero cells and ileal mucosal epithelium). The bacterium also exhibited antimicrobial activity against several gram-positive and gram-negative food-spoilage and food-borne pathogens such as Listeria monocytgenes ATCC 15313, Salmonella enterica ATCC 13311, Shigella sonnei ATCC 9290, Klebsiella oxytoca ATCC 13182, Enterobacter cloaca ATCC 35030 and Streptococcus pyogenes ATCC 12378. The absence of haemolytic activity and proteinase (trypsin) and the presence of a strong peptidase (leucine-arylamidase) and esterase-lipase (C4 and C8) were observed in this LAB strain. P. acidilactici Kp10 also produced acid, coagulated milk and has demonstrated proteolytic and amylolactic activities. CONCLUSION: The properties exhibited by P. acidilactici Kp10 suggested its potential application as probiotic and starter culture in the food industry.

Dipeptidyl peptidase-II from probiotic Pediococcus acidilactici: Purification and functional characterization.

Dipeptidylpeptidase-II (DPP-II, E.C. 3.4.14.2), an exopeptidase was purified 15.4 fold with specific activity and yield of 15.4U/mg/mL and 14.68% respectively by a simple two step procedure from a probiotic Pediococcus acidilactici. DPP-II is 38.7KDa homodimeric serine peptidase with involvement of His and subunit mass of 18.9KDa. The enzyme exhibited optimal activity at pH 7.0 and 37°C with activation energy of 24.97kJ/mol. The enzyme retained more than 90% activity upto 50°C thus adding industrial importance. DPP-II hydrolysed Lys-Ala-4mßNA with KM of 50µM and Vmax of 30.8nmol/mL/min. In-silico characterization studies of DPP-II on the basis of peptide fragments obtained by MALDI-TOF revealed an evolutionary relationship between DPP-II of prokaryotes and phosphate binding proteins. Secondary and three-dimensional structure of enzyme was also deduced by in-silico approach. Functional studies of DPP-II by TLC and HPLC-analysis of collagen degraded products revealed that enzyme action released free amino acids and other metabolites. Microscopic and SDS-PAGE analysis of enzyme treated analysis of chicken's chest muscle (meat) hydrolysis revealed change and hydrolysis of myofibrils. This may affect the flavor and texture of meat thereby suggesting its role in meat tenderization. Being a protein of LAB (Lactic acid bacteria), it is also expected to be safe.