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Inulin, a dietary fibre, is widely used as a prebiotic, sugar replacer, and texture modifier in the food industry. In this study, we have shown that irradiation affects the physicochemical properties of inulin, which in turn improves its biological functionality and feasibility as a functional ingredient in synbiotic foods. The biological functionality of 25 kGy-irradiated inulin (IRI) was assessed in terms of antioxidant capacity, protective action against intracellular ROS, and prebiotic activity. Antioxidant assays revealed that irradiated inulin had improved antioxidant activity, which was even greater than that of fructooligosaccharides. Furthermore, IRI was found to be comparatively more effective in maintaining low intracellular ROS levels. The in vitro fermentation studies showed that IRI had higher bifidogenic efficacy than fructooligosaccharides and unirradiated inulin. A synbiotic low-fat yogurt containing IRI (8.5 %) was prepared. In terms of sensory attributes, the developed product was comparable to a commercially available non-synbiotic and high-fat containing product.

M1 metallopeptidases regulate many important biological processes such as angiogenesis, tumour growth, hormone regulation, and immune cell development. Knowledge of substrate specificity mechanism in this family is valuable. An M1 peptidase from Deinococcus radiodurans (M1dr) with preference for bulky hydrophobic residues at N-terminus of peptide substrates was recently reported. In contrast to Escherichia coli aminopeptidase N, a previously characterized M1 peptidase, M1dr exhibits reduced activity towards peptides with N-terminal Arg or Ala residue. In order to illuminate structural basis of substrate specificity, we report several crystal structures of M1dr with different amino acids bound to the active site. Structural analysis indicated that the enzyme makes subtle adjustments to multiple residues leading to significant volume change of the active site cavity to accommodate residues of varying sizes (Leu to Trp). This study further reveals that the low preference for Arg at N-terminus of peptide substrate arises from a non-productive conformation in which many of the Arg molecules bind where they block the proton donor essential for the peptidase reaction. Hence, this study illuminates the substrate-binding mechanism and also reveals the structural basis for the substrate specificity of M1dr enzyme.

Peptidase E (PepE) is a nonclassical serine peptidase with a Ser-His-Glu catalytic triad. It is specific for dipeptides with an N-terminal aspartate residue (Asp-X dipeptidase activity). Its homolog from Listeria monocytogenes (PepElm) has a Ser-His-Asn "catalytic triad." Based on sequence alignment we predicted that the PepE homolog from Deinococcus radiodurans (PepEdr) would have a Ser-His-Asp "catalytic triad." We confirmed this by solving the crystal structure of PepEdr to 2.7 Å resolution. We show that PepElm and PepEdr lack the Asp-X dipeptidase activity. Our analyses suggest that absence of P1 pocket in the active site could be the main reason for this lack of typical activity. Sequence and structural data reveal that the PepE homologs can be divided into long and short PepEs based on presence or absence of a C-terminal tail which adopts a β-hairpin conformation in the canonical PepE from Salmonella enterica. A long PepE from Bacillus subtilis with Ser-His-Asp catalytic triad exhibits Asp-X dipeptidase activity. Whereas the three long PepEs enzymatically characterized till date have been found to possess the Asp-X dipeptidase activity, the three enzymatically characterized short PepEs lack this activity irrespective of the nature of their catalytic triads. This study illuminates the structural and functional heterogeneity in the S51 family and also provides structural basis for the functional variability among PepE homologs.

The present study identified at least two novel angiotensin I-converting enzyme (ACE) inhibitory peptides in horse gram flour after hydrolysis with alcalase. The ACE inhibitory peptides from horse gram hydrolysate (HGH) were purified by ultrafiltration and different chromatography techniques followed by identification using nano liquid chromatography-mass spectrometry (nano LC-MS/MS). Further, based on in silico analysis, twelve potential ACE inhibitory peptides from HGH were synthesized and assayed for their ACE inhibitory activity. Two peptides TVGMTAKF and QLLLQQ exhibited the highest ACE inhibitory activity (IC50 values of 30.3 μM and 75.0 μM, respectively) and were characterized kinetically to show Ki values of 0.01 μM and 0.18 μM, respectively. In addition, these two novel peptides exhibited competitive mode of inhibition as well as retained their ACE inhibitory activity after incubation with gastrointestinal proteases (pepsin, trypsin, and chymotrypsin). Thus, the ACE inhibitory peptides identified from HGH shows promise to be used as a functional food ingredient for controlling hypertension.

The antioxidant properties and ACE inhibitory activity of green gram, horse gram, lentil, chickpea, cowpea, black pea and white pea were evaluated before and after different processing conditions and in vitro protein digestion (IVPD). Chemical assays indicated that unprocessed horse gram had the highest antioxidant activity. The process of soaking decreased radical scavenging activity (RSA), total antioxidant capacity (TAC), ferric ion reducing power (FRAP) and metal ion chelation activity (MICA) in all the legumes. Cooking of the soaked seeds led to further decreases in these activities. Germination improved MICA in chickpea and lentil as well as TAC in all the legumes except for white pea. IVPD increased RSA and TAC of all the legumes by 2–4 folds, while FRAP and MICA in selective legumes showed a significant contribution from the peptides released due to hydrolysis of proteins. The scavenging and reducing activities in this legume as well as other legumes were correlated with free phenolics content. However, in contrast to the chemical assay, biological assays showed no such correlation. Significant ACE inhibitory activity was observed only after IVPD of the legumes.

Xaa-Pro peptidases (XPP) are dinuclear peptidases of MEROPS M24B family that hydrolyze Xaa-Pro iminopeptide bond with a trans-proline at the second position of the peptide substrate. XPPs specific towards dipeptides are called prolidases while those that prefer longer oligopeptides are called aminopeptidases P. Though XPPs are strictly conserved in bacterial and archaeal species, the structural and sequence features that distinguish between prolidases and aminopeptidases P are not always clear. Here, we report 1.4 Å resolution crystal structure of a novel XPP from Deinococcus radiodurans (XPPdr). XPPdr forms a novel dimeric structure via unique dimer stabilization loops of N-terminal domains such that their C-terminal domains are placed far apart from each other. This novel dimerization is also the consequence of a different orientation of N-terminal domain in XPPdr monomer than those in other known prolidases. The enzymatic assays show that it is a prolidase with broad substrate specificity. Our structural, mutational, and molecular dynamics simulation analyses show that the conserved Arg46 of N-terminal domain is important for the dipeptide selectivity. Our BLAST search found XPPdr orthologs with conserved sequence motifs which correspond to unique structural features of XPPdr, thus identify a new subfamily of bacterial prolidases.

The human aminopeptidase XPNPEP3 is associated with cystic kidney disease and TNF-TNFR2 cellular signaling. Its yeast and plant homolog Icp55 processes several imported mitochondrial matrix proteins leading to their stabilization. However, the molecular basis for the diverse roles of these enzymes in the cell is unknown. Here, we report the crystal structure of human XPNPEP3 with bound apstatin product at 1.65 A resolution, and we compare its in vitro substrate specificity with those of fungal Icp55 enzymes. In contrast to the suggestions by earlier in vivo studies of mitochondrial processing, we found that these enzymes are genuine Xaa-Pro aminopeptidases, which hydrolyze peptides with proline at the second position (P1′). The mitochondrial processing activity involving cleavage of peptides lacking P1′ proline was also detected in the purified enzymes. A wide proline pocket as well as molecular complementarity and capping at the S1 substrate site of XPNPEP3 provide the necessary structural features for processing the mitochondrial substrates. However, this activity was found to be significantly lower as compared with Xaa-Pro aminopeptidase activity. Because of similar activity profiles of Icp55 and XPNPEP3, we propose that XPNPEP3 plays the same mitochondrial role in humans as Icp55 does in yeast. Both Xaa-Pro aminopeptidase and mitochondrial processing activities of XPNPEP3 have implications toward mitochondrial fitness and cystic kidney disease. Furthermore, the presence of both these activities in Icp55 elucidates the unexplained processing of the mitochondrial cysteine desulfurase Nfs1 in yeast. The enzymatic and structural analyses reported here provide a valuable molecular framework for understanding the diverse cellular roles of XPNPEP3.

Idli , a steam-cooked breakfast food item consumed in India, is famous as a staple food for its spongy texture and unique fermented taste. Idli preparation is a time consuming process; although instant Idli pre-mixes as powder or batter are available in the market, they do not have the distinctive taste and aroma similar to the Idli prepared at home. Hence ready-to-eat (RTE) form of this food is in demand. Therefore, an attempt was made to prepare RTE Idli bearing similar taste as home-cooked Idli with an extended shelf-life of up to two months at an ambient temperature using Electron Beam Irradiation (EBI) at dosages 2.5 kGy, 5 kGy and 7.5 kGy and combination processing comprised of EBI dosage at 2.5 kGy and thermal treatment (80 °C for 20 min). The treated Idli 's were microbiologically and sensorially evaluated at storage periods of zero day, 14 days, 30 days and 60 days. Idli's irradiated at 7.5 kGy and subjected to combination processing at 2.5 kGy and thermal treatment were shelf-stable for 60 days. 2.5 kGy and 5 kGy radiation dosages alone were not sufficient to preserve Idli samples for more than 14 days. Undesirable change in sensory properties of Idli was observed at an EBI dosage of 7.5 kGy. Sensory properties of combination processed Idli 's were found to undergo minor change over the storage period. The present work suggests that lowest radiation dosage in combination with thermal treatment could be useful to achieve the extended shelf-life without considerably impairing the organoleptic quality of Ready-to-Eat Idli .

Xaa-Pro dipeptidase (XPD) catalyzes hydrolysis of iminopeptide bond in dipeptides containing trans-proline as a second residue. XPDs are found in all living organisms and are believed to play an essential role in proline metabolism. Here, we report crystal structures and extensive enzymatic studies of XPD from Xanthomonas campestris (XPDxc), the first such comprehensive study of a bacterial XPD. We also report enzymatic activities of its ortholog from Mycobacterium tuberculosis (XPDmt). These enzymes are strictly dipeptidases with broad substrate specificities. They exhibit substrate inhibition and allostericity, as described earlier for XPD from Lactococcus lactis (XPDll). The structural, mutational and comparative data have revealed a novel mechanism of dipeptide selectivity and substrate binding in these enzymes. Moreover, we have identified conserved sequence motifs that distinguish these enzymes from other prolidases, thus defining a new subfamily. This study provides a suitable structural template for explaining unique properties of this XPDxc subfamily. In addition, we report unique structural features of XPDxc protein like an extended N-terminal tail region and absence of a conserved Tyr residue near the active site.

Zinc metallopeptidases of the M1 family (M1 peptidases) with unique metal binding motif HEXXH(X)18E regulate many important biological processes such as tumor growth, angiogenesis, hormone regulation, and immune cell development. Typically, these enzymes exist in three-domain [N-terminal domain (N-domain), catalytic domain, and C-terminal domain (C-domain)] or four-domain (N-domain, catalytic domain, middle domain, and C-domain) format in which N-domain and catalytic domain are more conserved. The C-domain plays important roles in substrate binding and gating. In this study we report the first structure of a two-domain (N-domain and catalytic domain) M1 peptidase at 2.05 A resolution. Despite the lack of C-domain, the enzyme is active and prefers peptide substrates with large hydrophobic N-terminal residues. Its substrate-bound structure was determined at 1.9 A resolution. Structural analyses supported by site directed mutagenesis and molecular dynamics simulations reveal structural features that could compensate for the lack of C-domain. A unique loop insertion (loop A) in the N-domain has important roles in gating and desolvation of active site. Three Arg residues of the catalytic domain are involved in substrate-binding roles typically played by positively charged residues of C-domain in other M1 peptidases. Further, its unique exopeptidase sequence motif, LALET, creates a more hydrophobic environment at the S1 subsite (which binds N-terminal residue of the substrate in aminopeptidases) than the more common GXMEN motif in the family. This leads to high affinity for large hydrophobic residues in the S1 subsite, which contributes towards efficient substrate binding in absence of C-domain.

M24B peptidases cleaving Xaa-Pro bond in dipeptides are prolidases whereas those cleaving this bond in longer peptides are aminopeptidases-P. Bacteria have small aminopeptidases-P (36-39 kDa), which are diverged from canonical aminopeptidase-P of Escherichia coli (50 kDa). Structure-function studies of small aminopeptidases-P are lacking. We report crystal structures of small aminopeptidases-P from E. coli and Deinococcus radiodurans, and report substrate-specificities of these proteins and their ortholog from Mycobacterium tuberculosis. These are aminopeptidases-P, structurally close to small prolidases except for absence of dipeptide-selectivity loop. We noticed absence of this loop and conserved arginine in canonical archaeal prolidase (Maher et al., Biochemistry. 43, 2004, 2771-2783) and questioned its classification. Our enzymatic assays show that this enzyme is an aminopeptidase-P. Further, our mutagenesis studies illuminate importance of DXRY sequence motif in bacterial small aminopeptidases-P and suggest common evolutionary origin with human XPNPEP1/XPNPEP2. Our analyses reveal sequence/structural features distinguishing small aminopeptidases-P from other M24B peptidases.

In the present study dry red kidney beans ( Phaseolus vulgaris ), irradiated in the dose range of 0.25–10.0 kGy were evaluated for proximate composition, functional, sensory and antioxidant properties. Radiation processing up to 10 kGy did not affect proximate composition, hydration capacity and free fatty acid value. All the sensory attributes were unaffected at 1.0 kGy dose. The dose of 10 kGy, showed lower values for odor and taste, however, they were in acceptable range. Significant improvement in textural quality and reduction in cooking time was observed at dose of 10 kGy. Antioxidant activity of radiation processed samples was also assessed after normal processing such as soaking and pressure cooking. Both phenolic content and antioxidant activity evaluated in terms of DPPH free radical scavenging assay and inhibition in lipid peroxidation using rabbit erythrocyte ghost system, were marginally improved (5–10%) at the dose of 10 kGy in dry and cooked samples. During storage of samples for six months, no significant change was observed in sensory, cooking and antioxidant properties. Thus, radiation treatment of 1 kGy can be applied to get extended shelf life of kidney beans with improved functional properties without impairing bioactivity; nutritional quality and sensory property.

Chicken intestine, an underutilized by-product of poultry industry is a rich source of tissue proteases, especially aspartic proteases. Two of the major aspartic proteases of the tissue were purified from lysosomal and post lysosomal fractions and identified by N-terminal amino acid sequencing as cathepsin D and pepsin, respectively. The enzymes differed in their molecular and biochemical properties like molecular weight, optimum conditions for protein degradation and stability in different conditions of pH and temperature. Both enzymes were completely inhibited by pepstatin. The kinetic constant, Km for chicken intestinal pepsin and cathepsin D (for haemoglobin substrate) were estimated to be 6.45 μM and 22.7 μM, respectively. The interaction of both enzymes with different food proteins suggests pepsin could be used in applications demanding extensive hydrolysis of proteins, while cathepsin D could be used in those needing controlled degradation of proteins. Practical Applications Chicken intestine is a by-product of poultry industry. The tissue could serve as a major source of proteases especially aspartic proteases, which can be used as an alternative for proteases currently used in food industries. Aspartic proteases (pepsin and cathepsin D) from this tissue can be recovered and used for preparation of protein hydrolysates in a more economical way. Furthermore, the byproduct can be exploited to increase revenues for poultry processors.

Three members of peptidase family M20D from Burkholderia cepacia (BcepM20D; Uniprot accession no. A0A0F7GQ23), Deinococcus radiodurans R1 (DradM20D; Uniprot accession no. Q9RTP6) and Staphylococcus aureus (HmrA; Uniprot accession no. Q99Q45) were characterized in terms of their preference for various substrates. The results thus reveal that all the enzymes including HmrA lack endopeptidase as well as aminopeptidase activities and possess strong carboxypeptidase activity. Further, the amidohydrolase activity exerted on other substrates like N-Acetyl-Amino acids, N-Carbobenzoxyl-Amino acids and Indole acetic acid (IAA)-Amino acids is due to the ability of these enzymes to accommodate different types of chemical groups other than the amino acid at the S1 pocket. Further, data on peptide hydrolysis strongly suggests that all the three enzymes are primarily carboxydipeptidases exhibiting highest catalytic efficiency (kcat/Km 5-36 × 10(5) M(-1) s(-1)) for Met-X substrates, where -X could be Ala/Gly/Ser/Tyr/Phe/Leu depending on the source organism. The supportive evidence for the substrate specificities was also provided with the molecular docking studies carried out using structure of SACOL0085 and homology modelled structure of BcepM20D. The preference for different substrates, their binding at active site of the enzyme and possible role of these enzymes in recycling of methionine are discussed in this study.

Horse gram hydrolysate (HGH) with different degree of hydrolysis (DH) (20, 25, 35, 40, and 45%) was prepared from whole horse gram flour using alcalase. The amino acid composition of HGH showed the presence of essential amino acids. The alcalase hydrolysis (DH ≥ 20%) increased protein solubility with a notable difference in the pH range of 3–5 (p 0.05) in preference between control soup and soup mixed with HGH. Thus, these results suggest the possibility of HGH to be used as an appropriate functional ingredient with different food applications including in management of oxidative stress as well as in controlling hypertension .

Serine peptidases of the prolyl oligopeptidase (POP) family are of substantial therapeutic importance because of their involvement in diseases such as diabetes, cancer, neurological diseases, and autoimmune disorders. Proper annotation and knowledge of substrate specificity mechanisms in this family are highly valuable. Although endopeptidase, dipeptidyl peptidase, tripeptidyl peptidase, and acylaminoacyl peptidase activities have been reported previously, here we report the first instance of carboxypeptidase activity in a POP family member. We determined the crystal structures of this carboxypeptidase, an S9C subfamily member from Deinococcus radiodurans, in its active and inactive states at 2.3-A resolution, providing an unprecedented view of assembly and disassembly of the active site mediated by an arginine residue. We observed that this residue is poised to bind substrate in the active structure and disrupts the catalytic triad in the inactive structure. The assembly of the active site is accompanied by the ordering of gating loops, which reduces the effective size of the oligomeric pore. This prevents the entry of larger peptides and constitutes a novel mechanism for substrate screening. Furthermore, we observed structural adaptations that enable its carboxypeptidase activity, with a unique loop and two arginine residues in the active site cavity orienting the peptide substrate for catalysis. Using these structural features, we identified homologs of this enzyme in the POP family and confirmed the presence of carboxypeptidase activity in one of them. In conclusion, we have identified a new type within POP enzymes that exhibits not only unique activity but also a novel substrate-screening mechanism.

Unprocessed, malted, gamma irradiated (2 kGy and 10 kGy) and enzymatically treated [α-amylase + glucoamylase (E1)/α-amylase + glucoamylase + alcalase (E2)] finger millet (ragi) – soybean blends (7:3) were assessed for their use as substrates for the growth of a mixed culture of lactic acid bacteria (LAB) [ Lactobacillus rhamnosus MTCC 1423, Lactobacillus plantarum MTCC 2621 and Lactobacillus fermentum MTCC 903]. Results showed comparable growth profile in all the media to that of standard (MRS) media used for growth of these organisms achieving 3–4 log cycle increase in cell counts in 16 h. However, the blend of these grains could be used more efficiently, if subjected to malting, gamma irradiation or enzymatic processing. The attributes like lowering of pH, accumulation of organic acids and reduction in viscosity were better achieved with the use of processed commodities. Changes in the profile of sugars and free amino nitrogen (FAN) due to processing were in agreement with the rate of fermentation. Results suggest that radiation processing or enzymatic treatment could be used as faster alternatives to the malting process for improving the quality of cereals and legumes with regard to supporting the growth of a mixed culture LAB.

The human aminopeptidase XPNPEP3 is associated with cystic kidney disease and TNF-TNFR2 cellular signaling. Its yeast and plant homolog Icp55 processes several imported mitochondrial matrix proteins leading to their stabilization. However, the molecular basis for the diverse roles of these enzymes in the cell is unknown. Here, we report the crystal structure of human XPNPEP3 with bound apstatin product at 1.65 Å resolution, and we compare its in vitro substrate specificity with those of fungal Icp55 enzymes. In contrast to the suggestions by earlier in vivo studies of mitochondrial processing, we found that these enzymes are genuine Xaa-Pro aminopeptidases, which hydrolyze peptides with proline at the second position (P1′). The mitochondrial processing activity involving cleavage of peptides lacking P1′ proline was also detected in the purified enzymes. A wide proline pocket as well as molecular complementarity and capping at the S1 substrate site of XPNPEP3 provide the necessary structural features for processing the mitochondrial substrates. However, this activity was found to be significantly lower as compared with Xaa-Pro aminopeptidase activity. Because of similar activity profiles of Icp55 and XPNPEP3, we propose that XPNPEP3 plays the same mitochondrial role in humans as Icp55 does in yeast. Both Xaa-Pro aminopeptidase and mitochondrial processing activities of XPNPEP3 have implications toward mitochondrial fitness and cystic kidney disease. Furthermore, the presence of both these activities in Icp55 elucidates the unexplained processing of the mitochondrial cysteine desulfurase Nfs1 in yeast. The enzymatic and structural analyses reported here provide a valuable molecular framework for understanding the diverse cellular roles of XPNPEP3.

Enzyme gates are important dynamic features that regulate function. Study of these features is critical for understanding of enzyme mechanism. In this study, the active-site gate of M32 carboxypeptidases (M32CP) is illuminated. Only a handful of members of this family have been structurally and functionally characterized and various aspects of their activity and mechanism are yet not clarified. Here, crystal structure of putative M32CP from Deinococcus radiodurans (M32dr) was solved to 2.4 A resolution. Enzymatic assays confirmed its identity as a carboxypeptidase. Open and relatively closed conformations observed in the structure provided supporting evidence for previously hypothesized hinge motion in this family of enzymes. Molecular dynamics simulations of 1.5 μs displayed distinct open and closed conformations revealing amplitude of the motion to be beyond what was observed in the crystal structure. Hinge region and anchoring region of this shell-type gate were identified. A small displacement of 3 A and a helical tilt of 9° propagated by the hinge region translates into a 10 A motion at the top of the gate. The dynamics of the gate was supported by our mutagenesis experiment involving formation of disulphide bond across helices of the gate. The nearly inactive mutant enzyme showed 65-fold increase in the enzymatic activity in presence of reducing agent. Further, while a previously proposed structural basis would have led to its classification in subfamily II, experimentally observed substrate length restriction places M32dr in subfamily I of M32CPs.

Gamma radiation inactivation of phytohemagglutinin (PHA) of red kidney bean in purified form as well as in seeds in different moisture conditions was demonstrated at different doses viz. 1, 10, 30, 50, 100 and 200 kGy. Irradiation of PHA in dry state (99.5% dry) or in 50% moisture condition showed structural intactness as studied by SDS–PAGE, size exclusion chromatography (SEC), fluorescence measurement, CD spectroscopy below 30 kGy, while a 50% reduction in hemagglutinin and mitogenic activity was observed in the dose range of 30–50 kGy. Radiation inactivation of purified PHA was more pronounced when irradiated in aqueous solution form resulting in complete destruction of secondary and tertiary structure as well as function at a dose of 10 kGy. Radiation processing of dry and soaked seeds of kidney bean exhibited 50% loss in functional activity of PHA at the doses of 50 and 30 kGy, respectively. Practicality of implication of the radiation processing for inactivation of this antinutrient in legume seeds is discussed.

Xaa-Pro dipeptidase (XPD; prolidase; EC 3.4.13.9) specifically hydrolyzes dipeptides with a prolyl residue at the carboxy-terminus.Xanthomonasspp. possess two different isoforms of XPD (48 and 43 kDa) which share ∼24% sequence identity. The XPD of 43 kDa in size (XPD43) fromXanthomonasspp. is unusual as it lacks the strictly conserved tyrosine residue (equivalent to Tyr387 inEscherichia coliaminopeptidase P) that is suggested to be important in the proton-shuttle transfer required for catalysis in the M24B (MEROPS) family. Here, the crystallization and preliminary X-ray analysis of XPD43 fromX. campestris(GenBank accession No. NP_637763) are reported. Recombinant XPD43 was crystallized using the microbatch-under-oil technique. Diffraction data were collected on the recently commissioned protein crystallography beamline (PX-BL21) at the Indian synchrotron (Indus-2, 2.5 GeV) to 1.83 Å resolution with 100% completeness. The crystal belonged to space groupP212121, with unit-cell parametersa= 84.32,b= 105.51,c= 111.35 Å. Two monomers are expected to be present in the asymmetric unit of the crystal, corresponding to a solvent content of 58%. Structural analysis of XPD43 will provide new insights into the role of the conserved residues in catalysis in the M24B family.

Acylpeptide hydrolase (APH; EC 3.4.19.1), which belongs to the S9 family of serine peptidases (MEROPS), catalyzes the removal of anN-acylated amino acid from a blocked peptide. The role of this enzyme in mammalian cells has been suggested to be in the clearance of oxidatively damaged proteins as well as in the degradation of the β-amyloid peptides implicated in Alzheimer's disease. Detailed structural information for the enzyme has been reported from two thermophilic archaea; both of the archaeal APHs share a similar monomeric structure. However, the mechanisms of substrate selectivity and active-site accessibility are totally different and are determined by inter-domain flexibility or the oligomeric structure. An APH homologue from a bacterium,Deinococcus radiodurans(APHdr), has been crystallized using microbatch-under-oil employing the random microseed matrix screening method. The protein crystallized in space groupP21, with unit-cell parametersa= 77.6,b= 189.6,c= 120.4 Å, β = 108.4°. A Matthews coefficient of 2.89 Å3 Da−1corresponds to four monomers, each with a molecular mass of ∼73 kDa, in the asymmetric unit. The APHdr structure will reveal the mechanisms of substrate selectivity and active-site accessibility in the bacterial enzyme. It will also be helpful in elucidating the functional role of this enzyme inD. radiodurans.

The physicochemical and functional characteristics of irradiated locust bean gum (LBG) were investigated for their use as a source of dietary fibre in enteral food. Radiation processing mainly affected the molecular weight of the polymer in dose-dependent manner. The attributes like lowering of viscosity, increase in solubility and swelling index were better achieved with the use of irradiated polymer. A 17% reduction in glucose dialysis retardation index (GDRI) and decrease in bile acid dialysis retardation index (BDRI) due to irradiation were in agreement with the rate of depolymerization. LBG was also shown to protect probiotic L. plantarum cells during their passage in gastrointestinal conditions. Radiation processing of the gum further improved this property exhibiting only 3 log cycle decrease in count as compared to unirradiated polymer, exhibiting nearly 4 log cycle decrease from initial cell count of 9 log CFU mL−1. In view of the changes in the rheological properties of LBG due to irradiation, an enteral feed supplemented with irradiated LBG as source of dietary fiber was prepared without affecting the product viscosity.

Poultry viscera protein hydrolysate (PVPH) was fractionated using ultrafiltration membrane into three peptide fractions viz. Fractions I (>10 kDa), II (3–10 kDa) and III (

Legumes are rich source of proteins, dietary fiber, micronutrients and bioactive phytochemicals. Thirty different varieties of commonly consumed legumes in India, were screened for phenolic content and antioxidant activity using, radical scavenging [(1,1-diphenyl-2-picryl-hydrazyl (DPPH·) and 2,2'-azino-bis (3-ethylbenz-thiazoline-6-sulfonic acid, (ABTS·⁺], Ferric Reducing Antioxidant Power (FRAP) and metal ion (Fe²⁺) chelation assays. Legumes varied largely in their antioxidant activity. Horse gram, common beans, cowpea (brown and red) and fenugreek showed high DPPH· radical scavenging activity (400 units/g), while lablab bean (cream and white), chickpea (cream and green), butter bean and pea (white and green) showed low antioxidant activity (125 units/g). Green gram, black gram, pigeon pea, lentils, cowpea (white) and common bean (maroon) showed intermediate activity. Similar trend was observed when the activity was assessed with ABTS·⁺ and FRAP assays. Thus most of the varieties having light color seed coat, except soybean exhibited low antioxidant activity. While legumes having dark color seed coat did not always possessed high antioxidant activity (e.g. moth bean, black pea, black gram, lentils). Antioxidant activity showed positive correlation (r²0.95) with phenolic contents, in DPPH·, ABTS·⁺ and FRAP assays, whereas poor correlation (r²=0.297) was observed between Fe²⁺ chelating activity of the legumes and phenolic contents.

An aminopeptidase with relatively higher specificity towards N-terminal Pro residue was purified to homogeneity from chicken intestine. Peptide Mass Finger printing studies identified the enzyme as Leukotriene A 4 hydrolase (LTA 4 H). The molecular weight of the enzyme was found to be 66 kDa. The enzyme exhibited optimum activity at pH 7.5 and 55 °C and showed activation individually by albumin, deoxycholate and anions like SCN − , Cl − , Br − and I − . Unlike its mammalian counterpart, chicken intestinal LTA 4 H exhibited highest activity towards N-terminal Pro followed by Leu and Ala and least towards Arg. The aminopeptidase activity was completely inhibited by 1,10 phenanthroline and bestatin, while partially by EDTA and pefabloc. Potential of this enzyme for its use in debittering of protein hydrolysate is discussed.

Functional properties, antioxidant and Angiotensin-converting enzyme (ACE) inhibitory activities of peanut protein isolate (PPI) and peanut protein hydrolysate (PPH) prepared using Alcalase, at different (10%, 20%, 30% and 40%) degrees of hydrolysis, (DH) were investigated. Hydrolysis (at DH > 10%) significantly (p 80%) of PPI, especially in the pH range of 4–6. However, PPI showed better emulsifying and foaming properties than PPH (p

Enzymatic hydrolysates of casein and soybean were treated with alginate immobilized chicken intestinal mucosa, as an aminopeptidase source, to bring about debittering. The mucosa was hygienised by irradiation (20 kGy) which brought about a complete decontamination of the tissue accompanied by a 20% loss in aminopepidase activity. The effectiveness of the process was demonstrable by a higher acceptability and a marked reduction in bitterness scores for casein (from 4.4 to 2.5) and soybean (from 3.8 to 2.2) in organoleptic analysis. The action of aminopeptidases to bring about this change was corroborated by a concomitant increase in free amino acids and a decrease in average peptide length of the samples after treatment. The RP HPLC profiles of casein and soybean protein hydrolysates before and after treatment showed a higher content of peaks in the hydrophilic region suggesting a decrease in hydrophobic peptides, responsible for bitter taste, in both the samples. Immobilization of the mucosal tissue in alginate afforded an increased pH and temperature tolerance to the enzymes. The possibility of the system for continuous operation over extended time periods is also discussed.

An aminopeptidase with broad substrate specificity was purified to homogeneity (123.7-fold) with a yield of 3.43% from chicken ( Gallus gallus ) intestine using a combination of chromatographic separation strategies. The enzyme was identified as alanyl aminopeptidase or aminopeptidase N (APN) by Peptide Mass Fingerprinting. The molecular weight of the enzyme was estimated to be ∼180 kDa by SDS-PAGE and gel filtration chromatography. The enzyme was found to be a glycoprotein, having 40% sugar residue and a molecular mass of 108 kDa after deglycosylation. The enzymatic activity was optimal at 60 °C and pH 6.0. The enzyme preferentially hydrolyzed Leu-β-NA ( K m = 0.1 mM) followed by Ala, Phe, Tyr and Gly at N-terminal. The enzyme activity was completely inhibited by 1,10 phenanthroline (1 mM) and bestatin (1 mM) confirming it as a metalloprotease. Potential of this enzyme in combination with other endoproteases for the production of debittered protein hydrolysates has been discussed.

Application of gamma radiation for decontamination of poultry viscera was examined. Exposure to a dose of 20 kGy rendered the viscera sterile (

Ionizing radiation inactivated purified chicken intestinal aminopeptidase in media saturated with gases in the order N 2 O>N 2 >air. The D 37 values in the above conditions were 281, 210 and 198 Gy, respectively. OH radical scavengers such as t -butanol and isopropanol effectively nullified the radiation-induced damage in N 2 O. The radicals (SCN) 2 •− , Br 2 •− and I 2 •− inactivated the enzyme, pointing to the involvement of aromatic amino acids and cysteine in its catalytic activity. The enzyme exhibited fluorescence emission at 340 nm which is characteristic of tryptophan. The radiation-induced loss of activity was accompanied by a decrease in the fluorescence of the enzyme suggesting a predominant influence on tryptophan residues. The enzyme inhibition was associated with a marked increase in the K m and a decrease in the V max and k cat values, suggesting an irreversible alteration in the catalytic site. The above observations were confirmed by pulse radiolysis studies.

Three novel Angiotensin I-converting enzyme (ACE) inhibitory peptides were isolated from poultry viscera protein hydrolysate (PVPH). The purification steps involved ultrafiltration of PVPH to eliminate the high molecular weight peptides and RP-HPLC. The ACE inhibitory activity was resolved into three peaks on C18 column with retention times of 15.0, 18.9 and 20.9 min and amino acid sequences of the three peptides were determined to be ARIYH, LRKGNLE and RVWCP, respectively. The peptides exhibited resistance to hydrolysis by gastrointestinal proteases and extreme temperature (100C) and pH (1–12) at least for 2 h. Two of these peptides were competitive inhibitors, while the third was noncompetitive. The in vitro ACE inhibitory activity of PVPH revealed it could be a promising economic source for nutraceutical applications. Practical Applications Poultry viscera is a protein-rich by-product of poultry processing industry. The tissue protein and its hydrolysate offer huge potential for different bioactive peptides. In the present study three novel ACE inhibitory peptides were identified and characterized from poultry viscera protein hydrolysate (PVPH). These peptides could be tested further for their in vivo antihypertensive effect and as functional ingredient in foods.

Soybean trypsin inhibitor (STI) is a known antinutrient and food allergen present in soybean. γ-Radiation has the potential to inactivate the TI protein. However, a systematic study on the influence of different moisture levels during γ radiation on structure and function of the molecule has not been reported. Pure STI was irradiated up to 200 kGy, in dry state, with 50% moisture and in aqueous solution. The radiation damage in molecular structure was assessed using, SDS–PAGE, size exclusion chromatography, fluorescence measurement, and circular dichroism, while functional damage was assessed by the TI assay. In aqueous solution, both the structure and function of TI were almost destroyed at the 10 kGy dose. While with 50% moisture and in dry state, the loss in functional and structural attributes was discernible only at 30 and 100 kGy, respectively. The TI activity was found to be unaffected in dry and soaked seeds of soybean as well as other legumes up to irradiation doses of 100 and 50 kGy, respectively.

An aminopeptidase specific for the N-terminal acidic residue (BcepAP) was purified from the cell extract of Burkholderia cepacia svr as a homotrimeric (subunit mass 66 kDa) molecule. It was identified as an unassigned peptidase of family M61. The only other member characterized so far from this family is a broad-specificity aminopeptidase of Sphingomonas capsulata (ScapAP) with preference for Gly or Ala residues. However, BcepAP exhibited narrow specificity and the preferred substrate was a peptide with an N-terminal Asp or Glu residue, which is quite unusual. The proteins assigned to this family were grouped separately on the basis of their homology to either BcepAP or ScapAP. It led the conclusion that BcepAP is a prototype of a new PepM61 subfamily, with a representative in other Proteobacteria, and to the prediction that members of the family share the ability to cleave N-terminal acidic residues of peptide substrates.