Sahayog N. Jamdar

Influence of γ-Radiation on the Structure and Function of Soybean Trypsin Inhibitor

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.

Crystallization and preliminary X-ray diffraction analysis of Xaa-Pro dipeptidase from Xanthomonas campestris

Xaa-Pro dipeptidase (XPD; prolidase; EC 3.4.13.9) specifically hydrolyzes dipeptides with a prolyl residue at the carboxy-terminus. Xanthomonas spp. possess two different isoforms of XPD (48 and 43 kDa) which share ∼24% sequence identity. The XPD of 43 kDa in size (XPD43) from Xanthomonas spp. is unusual as it lacks the strictly conserved tyrosine residue (equivalent to Tyr387 in Escherichia coli aminopeptidase 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 from X. 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 group P212121, with unit-cell parameters a = 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.

A novel aminopeptidase from Burkholderia cepacia specific for acidic amino acids

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.

Crystal structure and biochemical investigations reveal novel mode of substrate selectivity and illuminate substrate inhibition and allostericity in a subfamily of Xaa-Pro dipeptidases

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.

Structure of the human aminopeptidase XPNPEP3 and comparison of its in vitro activity with Icp55 orthologs: Insights into diverse cellular processes

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.

Active site gate of M32 carboxypeptidases illuminated by crystal structure and molecular dynamics simulations

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 2.4Å 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 1.5μs 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 3Å and a helical tilt of 9° propagated by the hinge region translates into a 10Å 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.

Expression, purification, crystallization and preliminary X-ray diffraction analysis of acylpeptide hydrolase from Deinococcus radiodurans

Acylpeptide hydrolase (APH; EC 3.4.19.1), which belongs to the S9 family of serine peptidases (MEROPS), catalyzes the removal of an N-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 Alzheimers 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 group P21, with unit-cell parameters a = 77.6, b = 189.6, c = 120.4 Å, β = 108.4°. A Matthews coefficient of 2.89 Å(3) Da(-1) corresponds 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 in D. radiodurans.

Effect of enzymatic hydrolysis on the functional, antioxidant, and angiotensin I-converting enzyme (ACE) inhibitory properties of whole horse gram flour

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). The emulsifying activity and stability of HGH improved with increase in pH, especially at DH ≥ 25% (p < 0.05). With increase in DH, the foaming properties reduced while the antioxidant and angiotensin I-converting enzyme inhibitory activities increased. Sensory evaluation showed no significant difference (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 .

Crystal structure of a novel prolidase from Deinococcus radiodurans identifies new subfamily of bacterial prolidases

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.

Influence of a ragi-soyabean combination on the viability of probiotic organisms during storage and under simulated gastrointestinal conditions

C disease is a major lifestyle discover and claim nearly about 5 to 6 million. Indian population According to who it accounts 64 percent of all death in every year in India. The incidence of this disease increase with age having peak at middle age around 50 to 60 years .Only comprehensive risk reduction programme with appropriate changes in life style will result in the successful treatment and prevention of cardiovascular disease. Present investigation has been under taken to assess the correlation in the prevalence of cardiovascular disease with the specified risk factor in particular food in take pattern among cardiovascular diseases patients . Selected rural areas of Beed District of Maharashtra State. Five hundred cardiovascular disease male patients. Between 40 to60year in age group from parali & patoda districts .Were selected purpositive random sampling method .Food intake was assessed with the help of 24 recall method. It is concluded that mostly non vegetarians except fish eaters consumption of milk and milk products animal fact groundnut oil and lifestyle like dinks and smoking cigarette were found to strongly associated with prevalence of cardiovascular disease.Overweight and obesity in childhood show a negative impact on both physical and psychological health. The mechanism of obesity development is not fully understood and it is believed to be a disorder with multiple causes. Environmental factors, lifestyle preferences, and cultural environment play pivotal roles in the rising prevalence of obesity worldwide. Obesity is an excess proportion of total body fat. A person is considered obese when his or her weight is 20% or more above normal weight. The most common measure of obesity is the body mass index or BMI. A person is considered overweight if his or her BMI is between 25 and 29.9; a person is considered obese if his or her BMI is over 30.On the other hand, there are supporting evidences that excessive sugar intake by soft drink, increased portion size, and steady decline in physical activity have been playing major roles in the rising rates of obesity all around the world. Consequently, both over-consumption of calories and reduced physical activity are involved in childhood obesity. Almost all researchers agree that prevention could be the key strategy for controlling the current epidemic of obesity. Prevention may include primary prevention of overweight or obesity, secondary prevention or prevention of weight regains following weight loss, and avoidance of more weight increase in obese persons unable to lose weight. Until now, most approaches have focused on changing the behavior of individuals in diet and exercise. Prevention may be achieved through a variety of interventions like targeting built envir onment, physical activity, and diet. The increased risk of chronic diseases requires effective strategies to promote health, facilitating the adoption of proper life styles from childhood.Genetically modified foods are the foods obtained from plants, animals and microorganisms which have their genetic code modified by selective introduction of specific DNA segments by the help of gene splicing technique. These techniques are much more precise than mutagenesis where an organism is exposed to radiation or chemicals to create a non-specific but stable change. With the help of genetic engineering, a selective trait can be introduced into plants, animals and microbes to get desired results, for example, increased production yield and much better insect, pest and disease resistance, improvement in nutritional value and appearance, taste, drought resistant.Introduction: Omega-3 fatty acids are considered essential fatty acids. Also known as poly unsaturated fatty acids (PUFA’S). Omega-3 fatty acids can be found in fishes and other sea foods, nut oils. Omega-3 fatty acids play crucial role in brain function as well as normal growth and development, they reduce risk of heart diseases. Research shows that omega-3 fatty acids reduce inflammation and may help lower risk of chronic diseases such as heart disease, cancer, arthritis. Omega-3 fatty acids are appear to be important for cognitive (brain memory and performance) and behavioral function. Symptoms of omega-3 fatty acids include fatigue, poor memory, dry skin, heart problems, depression, and poor circulation. Methodology: To reach the demand for omega-3 fatty acids we opted to develop noodles i.e. hand made and machine made with two different flours i.e. wheat and Maida flour based noodles with flax seeds, pumpkin seeds, water melon seeds, walnuts, multi grain (flax, watermelon, pumpkin seeds), egg based and non egg based and fish varieties were developed. Thus fifteen varieties of noodles were developed. Panel members were selected. A questionnaire with five point hedonic scale was taken where the maximum score is 5 and minimum score was 1 basing on the attributes appearance, flavor, texture, taste, overall acceptance. Result: The results were positive and very good compliments were received by the panel members. A handmade multi grain noodle which is very nutritious as well as scored excellent was a successful experiment. Conclusion: There is considerable change in the day by day life of an average Indian due to various reasons like urbanization, changing life styles, increased in working population etc. In view of all these aspects it can be assumed that there will be an increase in consumption of processed foods because these products were convenient in use.Thus industrial level and house hold purposes such type of extruded products or convenience products can be developed for our convenience but salubriously.Abstract Abstract Abstract Abstract Synbiotic food products in matrices other than dairy foods are needed to address the problem of lactose intolerance in certain segments of the population. In this study an attempt was made to develop a synbiotic food based on cereals, millets and legumes which comprise dietary staples in India. A nutritious combination of ragi-soybean (7:3) was found to be very effective in supporting growth of probiotic microorganisms. A 10% slurry was inoculated with a mixed culture of approx. 5 log cfu mL -1 of L. casei (MTCC 1423), L. plantarum (MTCC 2621) and L. fermentum (MTCC 0903) and fermented at 37°C for 16 h. An increase in approx. 4 log cfu mL -1 was achieved at the end of the fermentation period with an increased production of lactic acid (0.393 g %) and a decrease in pH (4.8) and viscosity (240 cP). The mixture was lyophilised with trehalose (1%) added as a cryoprotectant. The viability of the organisms during storage was studied at 5, 10, 15, 25 ± 2°C for a period of 8 weeks. The results showed that the viability of the organisms was best at 5°C. At other temperatures the viability was reduced as temperature of storage increased. The efficacy of the formulation in maintaining stability of the organisms during exposure to simulated gastrointestinal juices was studied. The results showed for the first time that ragi-soybean combination could provide a good medium for enhanced survival of the probiotic organisms and potential for development of a synbiotic product.A study was carried out to prepare synbiotic ice cream incorporating Lactobacillus acidophilus and inulin and viability of L. acidophilus was analyzed on storage. Whey protein concentrate (WPC) was incorporated in the ice cream mix to improve the textural and nutritional quality of ice cream. A faster melting rate was noticed in the probiotic and synbiotic ice cream samples. Incorporation of inulin in ice cream mix significantly (P<0.01) improved the growth of Lactobacillus acidophilus. Freezing of the ice cream mix caused a reduction of 0.61 to 0.77 log counts of L. acidophilus count. A significant reduction (P<0.01) in the count of L. acidophilus was observed during storage. It is concluded that incorporation of inulin increases count of L. acidophilus and the organism could survive at therapeutic minimum probiotic level of 106 cells /ml for 15 days of storage at -18 to -23°C in ice cream.