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

Accepted Manuscript Title: Dipeptidyl peptidase-II from probiotic Pediococcus acidilactici: Purification and functional characterization Author: Dimpi Gandhi Preeti Chanalia Pooja Attri Suman Dhanda PII: DOI: Reference:

S0141-8130(16)31545-8 http://dx.doi.org/doi:10.1016/j.ijbiomac.2016.09.023 BIOMAC 6489

To appear in:

International Journal of Biological Macromolecules

Received date: Revised date: Accepted date:

18-4-2016 4-9-2016 7-9-2016

Please cite this article as: Dimpi Gandhi, Preeti Chanalia, Pooja Attri, Suman Dhanda, Dipeptidyl peptidase-II from probiotic Pediococcus acidilactici: Purification and functional characterization, International Journal of Biological Macromolecules http://dx.doi.org/10.1016/j.ijbiomac.2016.09.023 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Dipeptidyl peptidase-II from probiotic Pediococcus acidilactici: Purification and functional characterization Dimpi Gandhi, Preeti Chanalia, Pooja Attri, Suman Dhanda* Department of Biochemistry, Kurukshetra University, Kurukshetra

Dimpi Gandhi E mail: [email protected] Preeti Chanalia E mail: [email protected] Pooja Attri E mail: [email protected]

**To whom all correspondence should be made: Dr. Suman Dhanda Assistant Professor Department of Biochemistry Kurukshetra University, Kurukshetra Email: [email protected] Fax: 0091-01744-238277

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Abstract Dipeptidylpeptidase-II (DPP-II, E.C. 3.4.14.2), an exopeptidase was purified 15.4 fold with specific activity and yield of 15.4 U/mg/mL and 14.68% respectively by a simple two step procedure from a probiotic Pediococcus acidilactici. DPP-II is 38.7 KDa homodimeric serine peptidase with involvement of His and subunit mass of 18.9 KDa. The enzyme exhibited optimal activity at pH 7.0 and 37 oC with activation energy of 24.97 KJ/mol. The enzyme retained more than 90% activity upto 50 oC thus adding industrial importance. DPPII hydrolysed Lys-Ala-4mβNA with KM of 50 µM and Vmax of 30.8 nmoles/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 HPLCanalysis 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. Keywords: Pediococcus acidilactici; Dipeptidyl peptidase-II; Meat Tenderization

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1. Introduction Lactic acid bacteria (LAB) are traditionally and widely used for food, meat and milk fermentation and subject of continued research because of their potential health related benefits. LAB are nutritionally fastidious and require preformed amino acids for their growth. Proteolytic system of LAB is well developed and comprised of endopeptidases and exopeptidases. Endopeptidases generate oligopeptides which are then hydrolysed by exopeptidaes to generate free amino acids for growth and bioactive peptides. Pediococcus acidilactici is a gram positive LAB belonging to Lactobacillaceae that contain both endopeptidases and exopeptidases [1, 2] and possess probiotic attrbutes [3]. It is a facultative anaerobe being used worldwide in fermentation of vegetables and meat based products (dry sausages). Proteolytic activities of LAB are known to play key role in cheese ripening [4] and affecting flavor and texture of these products. Special attention was paid to extracellular enzymes of P. acidilactici as they are involved in keeping the cells alive by providing amino acids for nutrition and play major role in depolarizing activity. Only 16% of total proteins are secreted in external environment by bacteria. Dipeptidyl peptidases (DPPs) are exopeptidases that selectively release dipeptide moieties from amino terminal from oligopeptides/proteins. Their study is significant for basic, biotechnological and industrial domains. Amongst DPPs, DPP-II (E.C.3.4.14.2) mainly hydrolyse Lys-Ala-, Phe-Pro- and X-Pro-Pro- dipeptides from amino terminus. DPP-II is a serine protease that belongs to clan SC, family S28 along with PCP (prolyl carboxypeptidase). It exhibited N-terminal sequence similarity with PCP and also selectivity for prolyl bonds [5, 6]. Structure of DPP-II from Rattus kidney showed significant similarity to that of Mus and Homo sapiens QPP (93% and 79% respectively), to PCP (38%) and to the N-terminal a.a. sequence of Porcine DPP-II (76%) but low similarity to that of DPP-IV. The primary sequence of Homo sapiens DPP-II (492 a.a.) possessed 8 cysteine residues, 6 potential N-glycosylation sites, the consensus sequence for the active site Ser162 of serine proteases and its associated a.a. (Asp418 and His443), and a leucine zipper motif. DPP-II is synthesized with a cleaved signal peptide and/or propeptide [5, 7]. The native DPP-II is a glycoprotein containing mannose and glucosamine but having little or no sialic acid [8, 9, 10]. N glycosylation is necessary for DPP-II's enzymatic activity but not for its subcellular localization [7].

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Its proline hydrolyzing activity may play a key role in debitterning protein hydrolysates and other food products and generation of bioactive peptides. DPP-II is also known as quiescent cell proline dipeptidase and plays a major role in maintaining the cell in Go phase of cell cycle and thus in regulation of cell cycle [11]. To study the physiological and industrial role of DPP-II, its purification and characterization is of great significance. This is the first report of DPP-II from probiotic P. acidilactici. In this work, the purification, biochemical and in-silico characterization and its role in meat tenderization is reported. This is the part of larger project that focuses on characterization of metabolic traits of probiotic bacteria. 2. Materials and Methods 2.1. Materials Fast Garnet GBC, Sephadex G-100 and various substrates viz Lys-Ala-4mβNA, Gly-Phe-βNA, LeuAla-βNA, Asp-Arg-βNA, Ser-Met- βNA, Ser-Tyr- βNA, Gly-Arg-4mβNA, Phe-Arg-βNA, Ala-Ala-βNA, GlyPro-Leu- βNA, collagen from bovine Achilles tendon were procured from Sigma Chemical Co., St. Louis, MO, USA. Ammonium chloride, barium chloride, dimethyl sulphoxide, glacial acetic acid, hydrochloric acid, potassium chloride and zinc chloride were purchased from Rankem. Molecular weight markers (14.3-97.4 kDa) were obtained from Bangalore Genei India. Ammonium sulphate, gelatin and all other chemicals were purchased from himedia. 2.2. Culture P. acidilactici was purchased from National Dairy Research Institute (NDRI), Karnal. It was maintained on MRS agar plates. 2.3. Chicken Sample Chicken sample was purchased from local slaughter house, Kurukshetra, Haryana. 2.4. Methodology 2.4.1. Enzyme Assay DPP-II activity was calculated by using Lys-Ala-4mβNA substrate. Reaction mixture consisted of 875 µL of assay buffer (50mM Tris-HCl pH 7.4) and 30 µg of enzyme. Incubation was done at 37°C for 10 min followed by addition of 25µL substrate (8mg/mL) with 20 min incubation. The reaction was stopped with 1.0 mL of stopping reagent (1 M Sodium acetate buffer, pH 4.2) and 0.5 mL coupling reagent (0.1% Fast Garnet

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GBC in H2O). Absorbance was recorded at 520 nm against blank in which enzyme was added after addition of coupling reagent. One unit of enzyme activity was defined as that amount of enzyme which released one nmole of 4-methoxy-β-naphthylamine per min from substrate under assay conditions.

OD520 x 109 x 2.0 x10-3 x10 Activity (nmoles/min/mL) =

---------------------------------εxt

ε= molar extinction coefficient of 4mβNA/ βNA under assay conditions t= reaction time in min 2.0 x 10-3 = Vol of n-butanol in litres 10= multiplication factor for calculating enzyme activity as 0.1 mL enzyme used for reaction 109 = used for converting moles into nanomoles

2.4.2. Protein Estimation Protein content in crude and purified enzyme samples was determined by Lowry’s method [12] using BSA as standard. Amount of protein was calculated from the standard curve.

2.5. Purification of enzyme 2.5.1. Collection of culture filtrate P. acidilactici was grown in MRS containing 1% gelatin for 36 h at 32oC upto O.D.600.nm of 1.696 which corresponded to approximately 1.36x 109 cells/ml and supernatant was collected by centrifugation at 4800 rpm for 45 min. 2.5.2. Ammonium Sulphate Fractionation The proteins of culture filtrate were precipitated by saturating the supernatant upto 80% by ammonium sulphate precipitation. Pellets were redissolved in 3 mL of Tris-HCl buffer (50 mM, pH 7.4) and dialyzed with same buffer (100 mL) for 24 h with 3-4 changes of buffer. 2.5.3. Gel Filtration Chromatography

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The dialyzed sample (340 mg) was loaded on Sephadex G-100 column (75 x 2.5 cm) which was preequilibrated with Tris-HCl buffer (50 mM, pH 7.4). The column was run below 10oC at a flow rate of 0.5 mL/min under hydrodynamic pressure maintained by Mariotte flask and fraction of 5 mL each were collected. The protein content of each fraction was analysed spectrophotometrically by recording A 280 and activity of DPPII was screened by its standard assay. 2.5.4. Concentation of protein Fractions exhibiting DPP-II activity were pooled and concentrated by ultrafiltration Amicon stirred cell with YM 10 membrane under nitrogen pressure and stored at 4oC for further studies. 2.5.5. Davis gel electrophoresis The apparent homogeneity and purity of DPP-II was analysed by 12% Davis gel electrophoresis by loading 5 µg of purified enzyme [13]. 2.5.6. In-situ gel assay It was also performed by 12% Davis gel electrophoresis. The gel was pre-electrophoresed for 2 h in refrigerator before loading sample (10 µg of purified enzyme). After gel was run, it was cut into two halves. One half was proceeded using standard staining procedure the other half was stained for enzyme activity by adding 2 mL of substrate (8 mg/mL, DMSO) in the gel. It was incubated for 1 h (substrate permeation in gel requires time) at 37oC. Then gel was dipped in GBC solution for colour development. 2.6. Molecular weight determination Molecular weight was determined using SDS-PAGE and MALDI-TOF analysis. 2.6.1. SDS-PAGE Molecular weight and subunit composition of purified DPP-II was determined using 12% SDS-PAGE by method of Laemmli, 1970 [14]. 2.6.2. MALDI-TOF Purified DPP-II was digested with trypsin (MALDI grade). Trypsin digested sample was treated with equal volume of matrix solution (α-cyano-4-hydroxy cinnamic acid (HCCA) (10 mg/mL) in 70% acetonitrile and 0.03% trifluoric acid) and dried at room temperature. Peptide mass spectra was obtained using MALDI-

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TOF/TOF mass spectrometer (Bruker Ultraflex III TOF/TOF) and generated peptide mass list was used to match protein identity. Sequence alignment of tryptic fragments of purified protein and homology search was done

with

BLAST

(http://blast.ncbi.nlm.nih.gov/Blast.cgi)

and

Clustal

W

(http://www.ebi.ac.uk/Tools/msn/clustalw2). 2.7. Physicochemical characterization 2.7.1. pH “optima” and stability pH “optima” and stability of purified DPP-II were determined in pH range 4.0-10.5 using 50 mM sodium acetate (4.0-5.5), sodium-phosphate (6.0-6.5), Tris-HCl (7.0-8.5), glycine-NaOH (9.5-10.5) as assay buffers. For pH “optima”, the purified DPP-II (30 µg) was added to buffers of different pH and assayed by adding substrate at 37oC for 20 min. Percent activity was calculated by considering maximum activity as 100%. pH stability was determined by incubating purified DPP-II (30 µg) with buffers of different pH for 10 min at 37oC. The enzyme was assayed at optimum temperature. 2.7.2. Temperature “optima” and stability Temperature “optima” of purified DPP-II (30 µg) was determined by assaying it at different temperature in the range of 0° to 75°C. Temperature stability was assessed by incubating enzyme at different temperatures for 10 min and then assayed at optimum temperature using Lys-Ala-4mβNA (8 mg/mL). Activation energy was calculated from Arrhenius plot. 2.7.3. Kinetic parameters Apparent Michaelis-Menten constatnt (KM) and maximum velocity (Vmax) were calculated by LineWeaver Burk plot and Hanes plot using Lys-Ala-4mβNA by standard assay procedure. Substrate was used in concentration range of 1-500 µM. Specificity constant (Kcat/KM) is used to measure how efficiently an enzyme converts substrate into products. 2.7.4. Substrate hydrolysis study The substrate specificity of purified DPP-II was determined using various chromogenic substrates. The enzyme was incubated with different substrate at 37 oC viz. Lys-Ala-4mβNA, Gly-Phe-βNA, Leu-Ala-βNA, Asp-Arg-βNA, Ser-Met-βNA, Ser-Tyr-βNA Gly-Arg-4m βNA, Phe-Arg-βNA Ala-Ala-βNA and Gly-Pro-Leu-

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βNA each at final concentration of 50 µM. Assay was done using standard assay procedure. The relative enzyme activity was calculated with respect to Lys-Ala-4mβNA. Control was run separately for each studied substrate. 2.7.5. Investigation of Active site 2.7.5.1. Effect of inhibitors Amino acids involved in enzyme’s catalysis were predicted by pre-incubating enzyme (30µg) with different concentration of different chemical inhibitors viz. PMSF, AEBSF (serine protease inhibitor), DEPC (histidine modifier), 4-Nitrophenyl Iodoacetate, Iodoacetate, PCMB, NEM (cysteine peptidases) puromycin (serine and metalloprotease), pepstatin (aspartyl protease), DTNB (thiol modifier), and EDTA (metalloprotease) for 10 minutes. The inhibitory effect was measured by running the standard enzyme assay with Lys-Ala-4mβNA (8 mg/mL) in comparison to control. 2.7.5.2. Active site investigation by varying pH A graph of log Vmax vs pH was plotted to investigate the active site amino acid residues. The curve was extrapolated to determine the pKa of amino acids involved in enzyme catalysis. 2.7.6. Effect of metal ions Purified enzyme (30µg) was incubated with 0.05-0.2 mM concentration of chloride salt of different metal ions viz. FeCl3, KCl, NaCl, CuCl2, HgCl2, ZnCl2 and CoCl2 for 10 min and activity was calculated by standard assay using Lys-Ala-4mβNA (8 mg/mL) in comparison to control. 2.7.7. Effect of chloride ions Effect of chloride ions was studied in salt (NaCl) free buffer. Purified DPP-II (30µg) was incubated with different concentration of NaCl (0 to 1M) for 10 min. The activity was calculated by standard assay using Lys-Ala-4mβNA (8 mg/mL) in comparison to control. 2.7.8. Effect of thiol compounds Effect of thiol compounds viz. DTT and β-ME was studied by incubating with purified enzyme (30 µg) for 10 min and residual activity was calculated using Lys-Ala-4mβNA (8 mg/mL) by standard assay in comparison to control.

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2.7.9. Effect of organic solvents Organic solvents, ethanol and DMSO are generally used as solvents and also possess industrial significance. So effect of both was studied on activity of DPP-II. Purified enzyme (30 µg) was incubated with DMSO and ethanol in concentration range of 0-12% for 10 min and then assayed by using Lys-Ala-4mβNA (8 mg/mL) by standard assay procedure. 2.8. Phylogenetic analysis The evolutionary history was determined by using the Maximum Likelihood method based on the JTT matrix-based model [15]. 2.9. In-silico characterization 3-D structure of protein was studied by homology analysis by comparing query protein with existing protein structures. For this 3-D models of DPP-II and HPBP were rendered graphically by the PyMOL visualization tool along with the sequences. Secondary structural analysis and comparative modeling was executed by PDBsum server and LOMETS (LOcal MEta-Threading-Server) respectively. Energy minimization was carried out using the GROMOS 943a1 force field by swiss-PDB viewer. Validity of modeled protein was assured by PROCHECK [16], PSVS [17], ERRAT [18], PROVE [19] and SAVES (the Structure Analysis and Verification Server) (http://nihserver.mbi.ucla.edu). 3DLigandSite is a server that automates a successful manual method for the prediction of protein ligand binding residues in CASP8 [20]. 2.10. Application Studies of purified DPP-II 2.10.1. Collagen hydrolysis by DPP-II The reaction mixture of collagen degradation contained 50-mg collagen from bovine achilles tendon (Sigma), purified DPP-II (10 U mL-1), and neutral protease (50mg, 100U) in 50mM Tris-HCl (pH 7.5) and the final volume was made 50 mL. Reaction mixture was agitated at 37oC for 1 h and reaction was terminated by heating in a boiling water bath for 20 min. The supernatant was collected by centrifugation at 8300 rpm at 4oC for 10 min [21]. Amino acids in the supernatant were qualitatively analysed by Ninhydrin test. 2.10.1.1. Determination of free amino acid Free amino acids were determined by Ninhydrin method [22]. Absorbance was noted at 570 nm with reference to blank and standard curve of glycine was used for amino acid determination.

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2.10.1.2. Thin layer chromatography (TLC) of collagen hydrolysate Sample was further detected on TLC. Silica gel plates (5 X 20 cm) were prepared by dissolving silica in water followed by air drying. Plates were activated at 100oC for 30 min. One microliter standard proline solution (1mg/mL in 10% isopropanol) was spotted along with samples to be analysed. Plates were air dried and subjected to TLC using butanol: acetic acid: water (4:1:5) as mobile phase. Plates were then dried and sprayed with 0.25% ninhydrin (0.25g in acetone) and then heated for 10 min at 110oC in oven and color was recorded. 2.10.1.3. Amino acid analysis by HPLC HPLC was done by Agilent 1100 series. Sample preparation: To 1 mL of sample 4 mL of methanol was added and vortexed. Sample was kept overnight at -20oC and then centrifuged. Supernatant was collected and subjected to nitrogen flow for 1 h at 45 oC. To the dried sample coupling reagent [Phenyl Iso Thio Cyanate (10 µL), methanol (70 µL), Triethyl amine (10 µL), Filtered MQ (10 µL)] was added and kept in thermomixer for 1 h at 45oC. To this sample 200 µL of A-buffer (Sodium acetate) was added and injection volume was 20 µL. Agilent TC-C18 column was used with column dimensions 4.6 x 250mm 5-Micron at flow rate 1 mL/mL. Gradient system was used with buffer A : 10 mM sodium acetate (pH 6.4) with 6% acetic acid and buffer B: 10mM sodium acetate & 60% acetonitrile adjusted to pH 6.4 with 6% acetic acid and analysis was done at 254 nm wavelength. 2.10.2. Meat tenderization studies on chicken myofibrillar proteins Purified sample was checked for proteolytic activity on myofibrillar proteins. Meat tenderization was done by dehydration of meat followed by enzyme treatment. Meat was cut and dehydrated in sucrose (high osmotic pressure) after covering it with a semipermeable membrane thus allowing selective permeation of water. It was placed in cold conditions for 18h. After dehydration each sample was placed for 3 h at 4oC in two volumes of 50mM Tris-HCl (pH 7.4) containing purified DPP-II (10 U mL-1), neutral endoprotease (50mg, 100U) and both in combination. Control was dipped in distilled water instead of an enzyme solution. After that each sample was stored for 24 h at 4oC [23]. Myofibrils were made from each muscle according to the procedures described by Busch et al, 1972 [24]. The ground muscle was suspended in 6 volumes (w/v) of 50 mM Tris-HCl buffer (pH 7.4) containing 100 mM KCl and 5 mM EDTA by using a Blendor for 15 s and sedimented at 2600 rpm for 10 min. This process was repeated four more times. After the fifth wash, the myofibrils suspended in same buffer were passed through nylon net to remove connective tissue. The strained

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myofibrils were sedimented at 2600 rpm for 10 min washed three times in 1000 mM KCl and finally suspended in 100 mM KCl and 1 mM NaN3.

2.10.2.1. SDS-PAGE Myofibrils were centrifuged at 2600 rpm for 10 min and were solubilized in 0.01 M Na-phosphate buffer (pH 7.0) containing 5% SDS and 1% β-mercaptoethanol in boiling water for 2 min with subsequent centrifugation at 8300 rpm for 15 min [23]. The clear supernatant was analyzed by 7.5% SDS-PAGE as described by Laemmli, 1970 [14]. 2.10.2.2. Micrscopic analysis Microscopy of above samples was carried out under plane polarized light with 40X resolution. 3. Result and discussion 3.1. Purification of dipeptidyl peptidase-II (DPP-II) Extracellular DPP-II of P. acidilactici was purified by simple two-step process. The enzyme was purified 15.4 fold with a yield of 14.68% with specific activity of 15.45 U/mg. The results are summarized in Table 1. The elution profile of DPP-II on Sephadex G-100 is shown in Fig. 1A. This is the first report of purification of DPP-II from Lactic Acid Bacteria (LAB). Purification fold for DPP-II is less than that of earlier reported for porcine seminal plasma (1700 fold) [25], rat kidney (130 fold), rat brain (2600 fold) [26, 27], bovine pituitary (100 fold) [28, 29], human placenta (341 fold), kidney (5000 fold) and seminal fluid (7300 fold) [30, 31, 32]. However percent yield was higher than for bovine pituatiary gland (0.02%), human kidney (5%), placenta (6%), porcine spleen (3%), seminal plasma (7.6%) and ovary (10%) [8, 9, 25, 28, 29, 30, 31] and lower than that of human seminal fluid (40%) and rat skin (28%) [32, 33]. Specific activity for DPP-II from human placenta was 307 units/mg which is much higher than microbial DPP-II. Apparent homogeneity of purified DPP-II was checked on 12% PAGE stained with Coomassie Briilliant Blue R-250 and in-situ gel assay i.e. activity staining (Fig. 1B). Single band on native PAGE corresponding to activity staining band confirmed the purity and apparent homogeneity of DPP-II. 3.2. Molecular weight determination 3.2.1. SDS-PAGE

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Molecular weight of purified enzyme was determined by 12% SDS-PAGE run under reducing and nonreducing conditions at pH 8.3 in comparison to molecular weight markers in range of (14.3-97.4 KDa). Single band of approximately 19 KDa on SDS-PAGE suggests the absence of disulfide bond between two subunits. The band of purified DPP-II in lane 1 corresponded to ~19 KDa. The molecular weight of purified enzyme calculated from graph of log molecular weight versus relative mobility was calculated to be ~ 18.9 KDa. 3.2.3. Peptide mass fingerprinting by MALDI-TOF Molecular weight determined from MALDI was 38.7 KDa (Fig. 2A). These results suggested protein to be a homodimer as it migrated as single band on SDS-PAGE. Besides its low molecular weight, DPP-II was eluted in starting fractions on Sephadex G-100 column because of its possibility of formation of high molecular aggregates which might results from interaction between exposed hydrophobic surfaces of neighboring molecules. This limits the quantity of water required for stabilizing these proteins. In Gram-positive bacteria, aggregation of extracellular enzymes/proteins was also reported with crude as well as purified lipase of Staphylococcus aureus [34] and in other bacteria [35, 36, 37, 38]. Earlier a monomeric dipeptidyl peptidase of 37 KDa was reported for Streptomyces species WM-23 [39]. However DPP-II from other sources possessed high molecular weight in the range 101-130 KDa and that of seminal plasma and rat brain DPP-II had even higher molecular weight in range of 185-220 KDa [5, 6, 8, 10, 25, 26, 27, 28, 29, 31, 40]. Purified DPP-II of P. acidilactici is a homodimer. DPP-II from most species was reported to be homodimeric with subunit’s molecular weight in the range of 50-64 kDa that varied with species [5, 6, 8, 10, 25, 26, 27, 28, 29, 31, 40] whereas DPP-II from porcine seminal plasma was trimeric [25]. About 52% of studied aminopeptidases display multimeric structure. Enzymes with two, four and six subunits are most prevalent. Peptide fragments peaks obtained on MALDI were further analysed on Matrix server and their identity with

other

peptidases

is

represented

in

Fig.

2B.

Nine

peptides

were

obta

ined after trypsin digestion. The enzyme showed maximum identity with full phosphate binding protein with 123 score as protein score of greater than 103 which is significant. DPP-II of P. acidilactici also had similarity with phosphate binding protein and it is discussed later in phylogenetic analysis. 3.3. pH optima and stability of purified DPP-II Purified DPP-II worked optimally at pH 7.0 with ~82% activity retained at pH 6.5 and 92% at 7.5 (Fig. 3A). Enzyme was active over a broad pH range with upto 70% activity at pH 5.5 and 9.0. It exhibited 50%

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activity even at pH 4.5. pH optima of purified DPP-II from Polyphyromonas gingivalis was in the range of 6.59.0 in different buffers [41]. Optimum pH for DPP-II from Streptomyces sp. was 7.0 [39]. pH optima of DPP-II in mammalian species ranged between 5.0-6.5 [5, 25, 28, 32, 42, 43]. DPP-II was stable over a broad pH range of 5.0-9.0 with more than 80-90% activity at 10 min of preincubation (Fig 3A). At pH 4.5 and pH 8.5 DPP-II retained 93% and 87% of activity respectively. The enzyme retained 40% activity at pH 4.0. pH stability over acidic to basic range broadens the applicability of the enzyme. DPP-II from Polyphyromonas gingivalis and Streptomyces sp. were also stable from neutral to alkaline pH range [41] whereas DPP-II from some other species was stable over acidic to alkaline range. DPP-II of porcine seminal plasma was stable in pH range of 3.5-10; ovary, 3-7.5 pH; rat brain, 4-8 pH; and rat kidney, 3.78.8 pH [8, 10, 25]. 3.4. Temperature Optima and stability of DPP-II Temperature optima of purified DPP-II was 37ºC and it exhibited more than 90% activity upto 45 oC and more than 80% activity at 30ºC and 50ºC (Fig 3B). Enzyme’s activity declined abruptly beyond 50oC and completely lost at 60ºC. Enzyme was active over a broad temperature range of 30o to 50oC. Same temperature optima was also reported for DPP-II of other species [5, 8, 25, 28, 32, 42, 43] while temperature optima of 50oC and 43ºC was reported for DPP-II from Streptomyces sp and P. gingivalis respectively [39, 41]. DPP-II was stable upto 55°C during 10 min incubation with more than 85% activity. Only 45% activity left upto 75°C (Fig 3B). Results are also in agreement to earlier studies as DPP-II was stable upto 50°C in Rattus species, Homo sapiens, Porcine and Bos taurus [6, 28, 29, 33]. DPP-II homologs were stable upto 5560°C depending on source [25, 42, 43]. Activity over a broad temperature range and stability upto 50 oC adds industrial significance to this bacterial enzyme. This bacterial enzyme can be used for the processes which need to be carried out even at high temperature (upto 55 oC). Activation energy (Ea) of DPP-II calculated using Arrhenius plot was 24.97 KJ/mol for Lys-Ala4mβNA substrate (Fig 3C). Ea calculated for plant DPP-II was 32.77 KJ/mol [44]. 3.5. Kinetic characterization of purified DPP-II KM and Vmax for Lys-Ala-4mβNA substrate were determined to be 50 µM and 30.8 nmoles/mL/min respectively (Fig 4 A, B and C). The enzyme exhibited affinity in micromolar range for the substrate. KM of DPP-II varied from 40 to 930 µM for DPP-II of different species [5, 8, 25, 26, 31, 32, 45, 46]. KM values were

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much higher as reported for Homo sapiens kidney (250 µM), Porcine seminal plasma (360 µM) and Rattus sp. kidney (555 µM). For Rattus sp brain and Porcine ovary KM values were 87µM and 99 µM respectively. Variation in reported KM may be due to varied chromogen and enzyme source. Vmax for DPP-II from porcine seminal plasma was 1.43 µmole/mg/min [25]. The reported Vmax varied from 1.43 to 29 µmoles/mg/min for different species [5, 8, 10, 25, 26, 31, 32). Kcat/KM for Lys-Ala-4mβNA substrate determined was 1.1688 µM-1 min-1. It is used to measure preference of enzyme for different substrates. Higher the specificity constant, preferred is the substrate. 3.6. Substrate hydrolysis by DPP-II Substrate hydroysis was determined with Lys-Ala-4mβNA hydrolyzing activity as reference. DPP-II exhibited maximum hydrolysing activity with Gly-Phe-βNA followed by Leu-Ala-βNA over Lys-Ala-4mβNA alongwith Asp-Arg-βNA, Ser-Met-βNA and Ser-Tyr-βNA. Negligible activity was reported for Gly-Arg-4m βNA. No activity was detected with Phe-Arg-βNA and Ala-Ala-βNA (Table 2). It did not hydrolyse studied tripeptide substrate i.e. Gly-Pro-Leu- βNA. However hydrolysis of tripeptide substrate was reported by DPP-II from porcine, rat and human [8, 42, 43]. No systematic studies are available for DPP-II substrate selectivity. There are various contradictory reports in literature [40]. Enzyme exhibited broad substrate specificity. Maximum rates were observed for Lys-Ala- and Lys-Pro- derivatives with several differences in preferential cleavage. So hydrolysis of Lys-Ala-4mβNA can be used conveniently for detection of this enzyme. In previous studies maximum hydrolysis was seen for derivatives of Lys-Ala-, Lys- Pro-, Gly-Pro-, Arg-Pro, Ala-Ala-, ArgAla-, Leu-Ala-, Phe- Pro- [28, 29, 31, 33, 47, 48, 49]. DPP-II from the porcine ovary preferred Phe-Pro-βNA over Lys-Ala-βNA [8] and porcine skeletal muscle DPP-II hydrolyzed Gly-Pro-MCA better than Lys-Ala-MCA [42, 43]. Relative rates or specific activities could be influenced by variations in KM. 3.7. Active site investigation 3.7.1. Effect of inhibitors PMSF, AEBSF and DEPC had strong inhibitory effect on DPP-II. The enzyme is a serine protease and completely inhibited by 1mM PMSF and 5% DEPC and significant inhibition at higher concentration of AEBSF (Table 3). Inhibition by PMSF is comparable to that of DPP-II from Rattus sp.brain, Porcine ovary and Homo sapiens [8, 10, 27, 31] but it was not much effective on DPP-II of Porcine seminal plasma [25]. Inhibition by AEBSF was also reported for DPP-II from Rattus sp kidney [6] and Porcine seminal plasma [25]. Inhibition by

14

DEPC suggests involvement of Histidine in enzyme catalysis. DEPC selectively modifies Histidine by substitution of N- of imidazole ring in pH range of 5.5 to 7.5 [50]. EDTA decreased DPP-II activity by 40% at 10 mM EDTA whereas only 7% activity was lost at 1mM concentration. Similar observations were reported for DPP-II from other species [27, 44]. Enzyme was inhibited only upto 47% by 1mM DTNB. Though DTT and βME had no effect on enzyme activity but there are some contradictory reports of involvement of sulfhydrl groups in the catalytic mechanism of DPP-II. DPP-II activity of bovine brain was activated by DTT [5], while that of rat astroglial cells and brain was unaffected [27, 52]. Pepstatin modifies aspartate residues. DPP -II was inhibited slightly by pepstatin at 0.5 and 1.0 mM concentration. Pepstatin is transition state analog and highly specific inhibitor for aspartic proteases with K i in the range of 10-6 to 10-12 M. DPP-II of skeleton muscle, porcine ovary and plant DPP-II were also slightly inhibited by pepstatin. Puromycin did not affect the enzyme under study whereas DPP-II of plant and rat brain DPP-II were slightly inhibited and that of porcine ovary was strongly inhibited by puromycin. Like DPP-II of other species, iodoacetate, PCMB and 4-nitrophenyl iodoacetate also did not affect DPP-II of Pediocoocus acidilactici. This suggests that –SH are neither involved in catalysis nor in enzyme regulation. Thus serine, histidine and aspartate are present at active site. 3.7.2. Investigation of catalytic/active site amino acid residues pKa values of amino acids involved in catalysis were 6.3 and 9.2 that were in range of pKa of His and Ser [53] respectively (Fig. 5). His is present at enzyme’s active site in many enzymes and polarizes and deprotonates the nucleophile to initiate nucleophilic reaction inititated by serine/cysteine [54]. Results are also in agreement with the inhibition studies. Histidine acts as a proton donor/acceptor depending upon microenvironment and also coordinate metal ions and thus participate in enzyme catalysis. 3.8. Effect of metal ions on DPP-II Metal ions had slight modifying effect (not significant) on purified DPP-II of P. acidilactici at range of 0.05-0.2 mM concentration (Table 4). Enzyme activity was slightly enhanced in the presence of all studied metal ions thereby indicating lack of any absolute requirement of metal ion at active site. Though DPP-II activity from other species was inhibited by several divalent cations viz Cu 2+, Fe2+ and Hg2+ and moderately by Zn2+, Co2+, Pb2+ and Cd2+ [6, 8, 9, 10, 25, 26, 27, 33, 42, 43, 52, 55, 56]. 3.9. Effect of chloride ions

15

DPP-II activity increased with increase in NaCl concentration with maximum activity at 500 mM. Further increase in NaCl had no effect on enzyme activity (Fig. 6A). Contrarily activity of plant, human, bovine and rat DPP-II depicted varied inhibitory effect upto 100 mM NaCl [8, 25, 27, 28, 29, 32, 42, 43, 44, 57]. 3.10. Effect of organic solvents on DPP-II As substrate of DPP-II is dissolved in DMSO so effect of its different concentrations on enzyme activity was studied. DPP-II activity initially increased with increase in DMSO concentration upto 2% but further increase in DMSO concentration was accompanied with decreased enzyme activity (Fig 6B). It retained upto 50% activity at 8% DMSO concentration. Increased activity at low DMSO concentration might be due to increased substrate solubility. High DMSO concentration may severely disturb hydrophobic residues conformation and thus decrease enzyme activity [58]. DMSO concentration may also affect the protein structure in many complex ways, leading to variations in activity during biochemical screenings [59]. Interaction between substrate and protein binding sites are dependent on energy of protein bound conformations. Diprotic solvents like DMSO can also facilitate the nucleophilic reactions. Ethanol is used as solvent for inhibitors and in industries so its effect on enzyme activity was studied. The stability of protein structure is dependent upon nature of its solvent environment. Activity was maximum at 1% concentration and decreased thereafter with further increase in ethanol concentration. DPP-II retained 50% activity at 10% concentration (Fig 7B). The favourable effects of organic solvents result in better affinity of substrate for formation of enzyme-substrate complex. DPP-II of plant also showed similar response to organic solvents [44]. 3.11. Phylogenetic analysis of DPP-II The tree with the highest log likelihood (850.7947) is shown in Fig. 7. A discrete Gamma distribution was used to model evolutionary rate differences among sites (5 categories (+G, parameter = 0.5186). Pblast of DPP-II protein from P. acidilactici showed similarity with 23 protein sequences. There were a total of 117 positions in the final dataset. Proteins were divided in two domains. Domain one having alkaline phosphatase from Pseudomonas species, human phosphate binding of protein (HPBP) and Domain two mainly contains DPP-II protein under study, Phosphate binding proteins and alkaline phosphatases from Pseudomonas and periplasmic binding proteins. In domain two, DPP-II of P. acidilactici form a clade with Human Phosphate Binding Protein (HPBP). These proteins also had the highest alignment score of 203 with DPP-II. Results of phylogeny thus revealed evolutionary relationship between DPP-II and Human Phosphate Binding Protein (HPBP).

16

3.12. In-silico characterization of DPP-II protein 3-D structure was determined by comparative modeling of purified DPP-II with known sequences of 135 amino acids and Human phosphate binding protein (HPBP) from Protein Data Bank (PDB code: 4m1va ) at 1.30 Å resolution with 81% sequence identity and Z score 18 with DPP-II was the best template for query (Fig. 8A). Energy minimization for 3D structure was 3818.434 KJ/Mol. In Ramachandran plot phi–psi angles of 79.1% of residues were found in most favored regions, 16.5% in additional allowed region, 3.5% in generously allowed regions and 0.9% residues in disallowed regions (Fig. 8B). Model was accepted because overall G-factor scores for model was equal to recommended value (−0.5) with overall average positive scores (cut-off score was >0.2) which also indicated reliability of the proposed model. The Z-score RMS values analyzed by PROVE program [19] for DPP-II was 1.881 (RMS value of ~1.0 indicates good resolution of structures). The 3D models of DPP-II and HPBP were depicted graphically by the PyMOL visualization tool along with the sequences (Fig 8C). Secondary structural analysis was performed by PDBsum

server

(Fig

8D).

Ligand binding analysis by COFACTOR server revealed ligand binding homology with hypothetical ABC-type phosphate transport (PDB: 1twyc) (Fig 8E) with confidence score and TM-score 0.05 and 0.470 respectively. Arginine, serine and theronine were predicted to be binding residues. Structural confidence score for MG (ligand) was found to be 11.2 on comparison with 1twy_A from structural library with Mammoth. No ligand binding site was revealed. 3.13. Application Studies 3.13.1. Collagen hydrolysis DPP-II exhibited a broad range of substrate specificity and also cleaved near –Pro residues [40]. Pro hydrolyzing enzymes are of particular interest in food industry especially in removing bitterness. Collagen is rich in proline and hydroxyproline residues. Protein hydrolyzates of collagen with antioxidant properties are of great interest for pharmaceutical and food processing industries. These peptides can also induce synthesis of new collagen fibres by stimulating fibroblast cells. Meat is rich in collagen and meat’s tenderization is desired in meat industry. Collagen hydrolysis was studied in terms of release of free amino acids. DPP-II from P. acidilactici is an enzyme from LAB which is expected to be non-immunogenic and safe. LAB have been

17

conferred GRAS (generally regarded as safe) status and thus LAB and their products are expected to be nonimmunogenic. So collagen hydrolysis was studied with purified DPP-II and also in combination of neutral endoprotease with DPP-II. 3.13.1.1. Qualitative and quantitative test Released amino acids were evaluated qualitatively by ninhydrin test. Appearance of pale yellow colour indicated presence of proline. Ninhydrin method was used to estimate released free amino acid (s) with reference to glycine. Free amino acid content increased when collagen was treated with combination of neutral endoprotease and DPP-II. Content was 41.47 mg/L with control, 73.73 mg/L with DPP-II, 81.22 mg/L with neutral endoprotease (NP) and was most prominent with combination of DPP-II and NP (89.28 mg/L). 3.13.1.2. Thin layer chromatographic analysis TLC of collagen hydrolysis with DPP-II and neutral endoprotease revealed that collagen was hydrolysed in presence of all enzymes. Collagen hydrolysis was relatively less when treated only with DPP-II and spot intensity increased when enzymes were used in combination. This is because neutral endoprotease converts protein to oligopeptides which are better hydrolysed by exopeptidases and thus by DPP-II. 3.13.1.3. HPLC Analysis HPLC is an important analytical method for analysis of amino acids and other metabolites. Amino acids released by collagen hydrolysis are the part of structural and functional amino acid pool. Gelatin (irreversibly hydrolysed collagen) is used for treatment of bones and skin complications. The enzyme treated collagen sample was analysed by HPLC and results are summarized in Table 5. The combined action of neutral endoprotease and DPP-II has resulted in release of several amino acids/ or metabolites of biological /nutritional significance. Several essential amino acids are released viz Phe, Ile, Lys. Essential amino acids are important component of diet. Ile regulates blood sugar and Phe is precursor of catecholamines that regulate central and peripheral nervous system. Released phosphoethanolamine and phosphoserine help in good muscle growth. Glycine and serine are also released by action of DPP-II. 3-methyl Histidine is reduced and 1-methyl Histidine increases. Both are markers of muscle damage. 1-methyl Histidine is derived from anserine. β-aminobutyric acid, hydroxyl lysine and 3-methyl Histidine were released only by action of neutral endoprotease. DPP-II hydrolysed products didn’t have Trp, Asp, Val, OH-Lys, Leu and Tyr. This might be due to substrate selectivity

18

bearing these amino acid in scissile bond. Trp is not present in collagen so it is not present in hydrolysed products as well. Collagen hydrolysis is associated with softening/ tenderization of muscles (meat). 3.13.2. Meat tenderization Chicken chest muscles (meat) sample was treated with neutral endoprotease, DPP-II and combination of neutral endoprotease and DPP-II. 3.13.2.1. SDS-PAGE The samples were analysed on 7.5% SDS-PAGE. Relatively intense bands were observed in control as compared to bands in enzyme treated sample (Fig. 9A). So the enzymes in combination can be used for meat softening. Reports witness that ingestion of low molecular weight hydrolysed sternal cartilage extract helped in releiving the pain of osteoarthritis without any serious adverse effects [60]. 3.13.2.2. Micrscopic Analysis of Meat Sample Samples treated with different enzymes were also observed under microscope at 40X resolution under plane polarized light. The results are shown in Fig. 9 B. It is clear that enzyme treatment resulted in softening of muscles. Microscopic analysis of chicken meat sample revealed hydrolysis of muscle fibres and thus enhanced palatability. Though both neutral endoprotease and DPP-II separately resulted in hydrolysis in meat sample (Fig. 9B, 2 and 3) in comparison to control (Fig. 9B, 1) but results were better when both were used in combination (Fig 9B, 4). Treatment of meat sample with whole bacteria and only with extracellular sample also resulted in encouraging results (Fig. 9B, 5 and 6) because extracellular fluid contained both endoproteases and exopeptidases which work in sequential order So this bacteria can be used for meat fermentation and tenderization. Earlier Lactobacillus sakei is most widely used species of meat fermentation. Though studies are limited but Lactobacillus sp. exhibited the proteolytic activity on porcine muscle myofibrils and sarcoplasmic proteins [61]. Meat tenderization due to proteolysis is also associated with generation of small peptides and free amino acids including essential amino acids which may contribute to flavor and nutritional quality. This is also supported by studies of collagen hydrolysis. This will add to palatability and digestibility of meat. P. acidilactici is rich in different proteolytic activities [1]. Presence of aminopeptidase activity is very positive aspect of strains to be used as starter culture. Two membrane bound aminopeptidases (DPP-III and aminopeptidase B) have been purified and characterized from P. acidilactici [62] and studies will also be

19

focused on intracellular peptidases of this bacteria. DPP-II also reportedly plays important role in maintaining cells in Go phase of cell cycle in mammals [63]. Any similar role of DPP-II in prokaryotes needs further investigation. Conclusion The enzyme is purified to apparent homogeneity with a simple procedure. The enzyme is a homodimer active over a broad range of pH (5.0-9.0), temperature (0-55oC) and even salt concentration (100-1000 mM) for Lys-Ala-4mβNA substrate. The enzyme preferably hydrolysed Lys-Ala-4mβNA and also cleaved broad range of substrates with cleavage specificity towards hydrophobic residues. This adds industrial significance to the enzyme. Broad specificity of DPP-II may regulate various processes, generation of bioactive oligopeptides endorphins, neuropeptides, peptide hormones, chemokines etc. which may further lead to immunomodulation and modified biological activities. Studies of collagen hydrolysis and meat tenderization revealed good results and studies need to be applied and focused on use of these bacterial enzymes to improve meat quality. Acknowledgement Authors are thankful to UGC for funding the research in the form of Major research grant. Authors also extend their thanks to Dr Naresh Kumar, Assistant Professor, Department of Geology, Kurukshetra University, Kurukshetra for extending the facility of microscope. Conflict of interest The authors declare no conflict of interest. References 1.

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25

Tables

Table 1: Purification table of extracellular DPP-II from Pediococcus acidilactici.

Purification Steps

Crude extract (NH4) 2SO4

Volume

Total

Specific

Activity

Activity

(U)

(U/mg)

2139.00± 0.08

2147.54±0.02

340.00±0.10

20.40±0.03

Total Protein (mg)

(ml)

1000

Purification

Yield

(fold)

(%)

1.00±0.90

1.00±0.02

100.00±0.50

538.93±0.20

1.59±0.07

1.58±0.03

25.09±0.20

315.30±0.05

15.45±0.40

15.40±0.04

14.68±0.90

30

fractionation Sephadex G-100

60

Values are mean ± S.D. of three different experiments.

26

Table 2: Substrate hydrolysis study of DPP-II with different chromogenic substrates Substrates

Activity (nmoes/ml/min)

Lys-Ala-4mβNA

34.00±0.08

Gly-Phe-βNA

46.40± 0.07

Leu-Ala-βNA

38.69±0.03

Asp-Arg-βNA

30.29±0.80

Ser-Met-βNA

28.76±0.05

Ser-Tyr-βNA

9.12±0.34

Gly-Arg-4mβNA

1.00±0.80

Phe-Arg-βNA

Nil

Ala-Ala-βNA

Nil

Gly-Pro-Leu-βNA

Nil

Values are mean ± S.D. of three different experiments

27

Table 3: Effect of chemical inhibitors on DPP-II Inhibitor

Concentration

Control

-

PMSF

% Inhibition 0

0.1 mM

47.10±0.03

1 mM

100.00±0.09

0.1 mM

31.25±0.40

0.5 mM

35.00±0.70

1 mM

50.00±0.80

5 mM

75.00±0.40

1.00%

64.30±0.02

5.00%

100.00±0.09

1 mM

0

1 mM

0

0.1

0

1 mM

0

Puromycin

1 mM

0

DTNB

1 mM

47.20±0.40

Pepstatin

0.5mM

7.00±0.30

1.0 mM

12.00±0.70

NEM

1 mM

0

EDTA

1mM

7.00±0.08

10mM

40.00±0.20

DTT

0.5 mM

0

β-ME

0.5 mM

0

AEBSF

DEPC

4-Nitrophenyl Iodoacetate Iodoacetate PCMB

Values are mean ± S.D. of three different experiments

28

Table 4 Effect of metal ions on DPP-II Conc.

Activity

(mM)

(%)

Metal Ion

Control

FeCl3

KCl

NaCl

CuCl2

HgCl2

ZnCl2

CoCl2

100.00±0.30 0.05

100.00±0.05

0.1

113.00±0.09

0.2

121.00±0.10

0.05

114.50±0.40

0.1

122.50±0.08

0.2

124.10±0.30

0.05

125.80±0.60

0.1

125.80±0.09

0.2

125.80±0.02

0.05

119.30±0.10

0.2

119.00±0.06

0.5

119.00±0.03

0.05

104.83±0.40

0.1

112.90±0.10

0.2

117.70±0.09

0.05

119.30±0.02

0.1

124.10±0.30

0.2

116.10±0.30

0.05

125.80±0.50

0.1

122.50±0.40

0.2

103.00±0.07

Values are mean ± S.D. of three different experiments

29

Table 5: HPLC analysis of collagen degraded products

Content (µg/L) AMINO ACIDS

Neutral Control (1)

Aspartic acid

protease (2)

DPP-II (3)

NP+ DPP-II (4)

0

0

0

0

Carnosine

0.6400

0

0

0

3-Methyl histidine

0.6458

0.0032

0

0

Anserine

9.0619

3.7592

1.5130

0

Tyrosine

54.7987

0

0

0

1-methyl histidine

0

0

1.9314

2.2882

Phenyl alanine

0

39.6887

44.8582

56.9342

Leucine

0

49.7264

0

0

Isoleucine

0

0

6.0534

43.2689

Phosphoenolamine

0

0

0.8852

2.0561

Glycine

0

0

0.9125

1.6920

Tryptophan

0

0

0

0

Phoshoserine

10.0275

0.8975

6.9488

12.3900

OH Lysine

95.4362

71.4263

0

0

Serine

0

2.7608

0.3325

3.9542

beta amino butyric acid

0

5.9358

0

0

Lysine

61.9331

0

13.8581

37.0039

Valine

0

0

0

0

2.3952

1.0470

0.8824

0

OH Proline

30

Fig. 1. Protein (A280nm) and activity profile of Lys-Ala-4mβNA hydrolysis on gel filtration chromatographic column (A), Davis Gel Electrophoresis, lane 1- after staining with Coomassie Brilliant Blue, lane 2- In-situ gel assay using Lys-Ala-4mβNA substrate (B). Fig. 2. Intact Mass determination (A), Matrix server results by MALDI-TOF. Trypsin digested fragments (highlighted in red) with their spectra. (B). Fig. 3. pH optima and stability of DPP-II (A), Temperature optima and stability of DPP-II (B), Arrhenius plot for DPP-II (C) (T: absolute temperature (Kelvin), Ea: activation energy). Fig. 4. Determination of kinetic parameters (KM and Vmax) for hydrolysis of Lys-Ala-4mβNA by DPP-II. Michaelis–Menton plot (A) Lineweaver-Burk plot (B) Hanes plot (C). Fig. 5. log Vmax vs. pH for DPP-II Fig. 6. Effect of chloride ions on DPP-II (A), Effect of organic solvents on DPP-II (B). Fig. 7. Evolutionary tree of DPP-II from Pediococcus acidilactici by PDBsum with domain one having alkaline phosphatase from Pseudomonas species, human phosphate binding protein and domain two mainly contains DPP-II protein under study, Phosphate binding proteins and alkaline phosphatases from Pseudomonas and periplasmic binding proteins. Fig. 8. 3D structure of DPP-II from P. acidilactici using HPBP (PDB ID: 4m1va) as a template (A), Ramachandaran plot by PDBsum (B), Superimposition of predicted DPP-II with HPBP (C), Secondary structure analysis (D), Ligand binding analysis by COFACTOR (E).

Fig 9 7.5% SDS-PAGE: Chicken myfibril proteins, lane 1-Control, lane 2- myofibrils treated with Neutral protease (NP), lane 3- treated with DPP-II, lane 4- treated with NP+DPP-II (A). Microscopic images of enzyme treated meat under plane polarized light. 1- Control, 2- sample treated with Neutral endoprotease (NP) , 3- sample treated with DPP-II, 4- sample treated with DPP-II and NP, 5- sample with extracellular, 6- sample with whole cells (B).

31

1 O.D. (280 nm)

2

Activity (nmoles/min/mL)

0.16

2 1.8

0.14

1.6

O.D. (280 nm)

1.4 0.1

1.2

0.08

1 0.8

0.06

0.6 0.04 0.4 0.02

Activity (nmoles/min/mL)

0.12

0.2

0

Vo

0 0

10

20 30 40 Fraction Number

50

60

1(A)

1(B)

Fig. 1. Protein (A280nm) and activity profile of Lys-Ala-4mβNA hydrolysis on gel filtration chromatographic column (A), Davis Gel Electrophoresis, lane 1- after staining with Coomassie Brilliant Blue, lane 2- In-situ gel assay using Lys-Ala-4mβNA substrate (B).

32

3(A)

(A)

33

Mascot Score Histogram Protein score is -10*Log(P), where P is the probability that the observed match is a random event. Protein scores greater than 90 are significant (p<0.05). Top Score:123 for gi|152032648, RecName: Full=Phosphate-binding protein; Short=HPBP [unidentified prokaryotic organism].

Fig. 2. Intact Mass determination (A), Matrix server results by MALDI-TOF. Trypsin digested fragments (highlighted in red) with their spectra. (B)

1 DINGGGATLP QKLYLTPDVL TAGFAPYIGV GSGKGKIAFL ENKYNQFGTD 51 TTKNVHWAGS DSKLTATELA TYAADKEPGW GKLIQVPSVA TSVAIPFRKA 101 GANAVDLSVK ELCGVFSGRI ADWSGITGAG RSGPIQVVYR AESSGTTELF 151 TRFLNAKCTT EPGTFAVTTT FANSYSLGLT PLAGAVAATG SDGVMAALND 201 TTVAEGRITY ISPDFAAPTL AGLDDATKVA RVGKGVVNGV AVEGKSPAAA 251 NVSAAISVVP LPAAADRGNP DVWVPVFGAT TGGGVVAYPD SGYPILGFTN 301 LIFSQCYANA TQTGQVRDFF TKHYGTSANN DAAIEANAFV PLPSNWKAAV 351 RASFLTASNA LSIGNTNVCN GKGRPQ

(B)

34

120

pH Optima

pH Stability Stability

90 % Activity

80 % Activity

Optima

110

100

60 40

70 50 4(C) 30

20 10 0

0 4

5

6

pH

7

8

9

10

11

10

20

30 40 Temp°C

50

60

70

80 Ea= 24.97 KJ/mol

4(A)

4(B)

Fig. 3. pH optima and stability of DPP-II (A), Temperature optima and stability of DPP-II (B), Arrhenius plot for DPP-II (C) (T: absolute temperature (Kelvin), Ea: activation energy).

35

40

A

Activity of DPP-II (nmoles /mL/min)

35

30

25

20

15

10

5

0 0

KM

100

200 Substrate (µM)

36

300

400

500

0.5 0.45 0.4 0.35

B

1/V

0.3 0.25 0.2 0.15 0.1 0.05 0 -0.05 0 -0.02

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0.4

0.45

1/[S]

16

14

12

C |S|/V

10

8

6

4

2

KM= 50µM -100

-50

0 0

50

100

150

200

250

300

350

400

450

500

550

Substrate (µM)

Fig. 4. Determination of kinetic parameters (KM and Vmax) for hydrolysis of Lys-Ala-4mβNA by DPP-II. Michaelis–Menton plot (A) Lineweaver-Burk plot (B) Hanes plot (C).

37

1.2 1

log Vmax

0.8 0.6 0.4 0.2 0 0

2

4

6 pH

8

10

12

Fig. 5. log Vmax vs. pH for DPP-II

120 120 100

DMSO

100

Ethanol % Activity

% Activity

80 60 40 20

80 60 40 20

0

0 0

200

400

600

800

1000

0

2

4

6

8

% Conc Conc. of Cl- (mM)

6(A)

6(B)

Fig. 6. Effect of chloride ions on DPP-II (A), Effect of organic solvents on DPP-II (B)

38

10

12

Fig. 7. Evolutionary tree of DPP-II from Pediococcus acidilactici by PDBsum with domain one having alkaline phosphatase from Pseudomonas species, human phosphate binding protein and domain two mainly contains DPP-II protein under study, Phosphate binding proteins and alkaline phosphatases from Pseudomonas and periplasmic binding proteins.

8(A)

8(B)

39

8(C)

8(D)

8(E) Fig. 8. 3D structure of DPP-II from P. acidilactici using HPBP (PDB ID: 4m1va) as a template (A), Ramachandaran plot by PDBsum (B), Superimposition of predicted DPP-II with HPBP (C), Secondary structure analysis (D), Ligand binding analysis by COFACTOR (E).

1

2

3

40

4

9(A)

(1)

(2)

(3)

(4)

(5)

(6)

Fig 9 7.5% SDS-PAGE: Chicken myfibril proteins, lane 1-Control, lane 2- myofibrils treated with Neutral protease (NP), lane 3- treated with DPP-II, lane 4- treated with NP+DPP-II (A). Microscopic images of enzyme treated meat under plane polarized light. 1- Control, 2- sample treated with Neutral

41

endoprotease (NP) , 3- sample treated with DPP-II, 4- sample treated with DPP-II and NP, 5- sample with extracellular, 6- sample with whole cells (B)

64.

42