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.

Extraction, purification and characterization of low molecular weight Proline iminopeptidase from probiotic L. plantarum for meat tenderization.

Membrane bound proline iminopeptidase (PIP) from lactic acid bacteria (LAB) L. plantarum was extracted and purified using CM-sephadex, Sephadex G-100 and Q-sepharose column chromatography. PIP was purified with purification fold 7.13 and 33.5% yield. SDS-PAGE and MALDI-TOF revealed it as homodimer with molecular weight of 37.9 kDa and subunit of mass 18.9 kDa. Purified enzyme exhibited maximum activity at 45 °C and pH 7.0. Km and Vmax of purified PIP were 65 µM and 25.9 nm/min/ml respectively. Inhibition by PMSF confirmed it a serine protease. Metal ions and EDTA showed no effect on enzyme activity. The enzyme mainly hydrolysed Pro-4mßNA. The effectiveness of enzyme in purified form, membrane bound form and in combination with other enzymes to degrade collagen resulting in pharmaceutically significant collagen hydrolysates and in meat tenderization marks its industrial importance. There are very few PIPs are characterized from LAB, and therefore this study is industrially significant and brings some new knowledge into this area.

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.

Biochemical, kinetic, and in silico characterization of DING protein purified from probiotic lactic acid bacteria Pediococcus acidilactici NCDC 252.

DING proteins are intriguing proteins characterized by conserved N-terminal sequence. In spite of unusually high sequence conservation even between distantly related species, DING proteins exhibit outstanding functional diversity. An extracellular caseinolytic alkaline enzyme was purified to homogeneity from a probiotic lactic acid bacteria Pediococcus acidilactici NCDC 252 using a simple procedure involving ammonium sulphate precipitation and gel filtration chromatography. This was purified 45.72-fold with a yield and specific activity of 43.5 % and 250 U/mg, respectively. The calculated molecular weight was 38.7 and 38.9 kDa by MALDI and SDS-PAGE, respectively, and pI was 7.77. The enzyme exhibited optimal activity at pH 8.0 and 40 °C. It was considerably stable up to pH 12. For casein, the enzyme had K m of 20 µM with V max of 26 U/ml. The enzyme was resistant to organic solvents but sensitive to DTNB and EDTA that confirmed it as thiol protein with involvement of metal ions in catalysis. Its tryptic peptide fragments showed 95 % similarity with eukaryotic DING, i.e., human phosphate binding protein (HPBP). Homology-based structure evaluation using HBPB as template revealed both to be structurally conserved and also possessing conserved phosphate binding motifs.

Enkephalin degrading enzymes: metalloproteases with high potential for drug development.

Enkephalins play a great role in management of pain, blood pressure, hypertension and cardiovascular diseases. Enkephalins are short-lived molecules being rapidly hydrolyzed following their synaptic release by enkephalin degrading enzymes. The inhibitors of enkephalin degrading enzymes are able to prolong the duration of action of enkephalins. This review will focus on the inhibitors of enkephalin degrading enzymes as a novel therapeutic approach for cancer itself and also in cancer and neuropathic pain management with discussion on the present status and future directions for a new class of drugs.

Activity Staining and Inhibition Characterization of Dipeptidylpeptidase-III Enzyme from Goat Brain.

Dipeptidylpeptidase-III (DPP-III) from goat brain was purified and characterized using Arginyl-Arginyl-4-methoxy-ß-naphthylamide (Arg-Arg-4mßNA) substrate. This enzyme retained its activity in native 10% polyacrylamide gel when stained using Arg-Arg-4mßNA. The activity was significantly increased by 100 mM chloride. Studies for its inhibition with some peptides and chemical inhibitors revealed that Leu-Trp-Met-Arg-Phe-Ala was most potent inhibitor followed by Arg-Phe-Ala and Gly-Phe-Leu. All the studied chemical inhibitors caused 40-50% inhibition at 1 mM. Metal ions helped to regain activity of EDTA pretreated enzyme. ZnCl(2) at 50 µM almost completely restored the enzyme activity. Further ZnCl(2) and CoCl(2) exerted protective effects on EDTA pretreated enzyme for its susceptibility to DTNB inhibition. Therefore, DPP-III is a metalloprotease with the involvement of cysteine residues either located at the catalytic site or involved in regulation.