Search results for: proteases

Authors: Doostishoar Farzad, Forootanfar Hamid, Hasan-Bikdashti Morvarid, Faramarzi Mohammad Ali, Ameri Atefe

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Introduction: Chili peppers and related plants in the family of capsaicum produce a mixture of capsaicins represent anticarcinogenic, antimutagenic, and chemopreventive properties. Vanillylamine, the main product of capsaicin hydrolysis is applied as a precursor for manufacturing of natural vanillin (a famous flavor). It is also used in the production of synthetic capsaicins harboring a wide variety of physiological and biological activities such as antibacterial and anti-inflammatory effects as well as enhancing of adrenal catecholamine secretion, analgesic, and antioxidative activities. The ability of some lipases, such as Novozym 677 BG and Novozym 435 and also some proteases e.g. trypsine and penicillin acylase, in capsaicin hydrolysis and green synthesis of vanillylamine has been investigated. In the present study the optimized culture broth of a newly isolated lipase-producing bacterial strain (Stenotrophomonas maltophilia) applied for the hydrolysis of capsaicin. Materials and methods: In order to compare hydrolytic activity of optimized and basal culture broth through capsaicin 2 mL of each culture broth (as sources of lipase) was introduced to capsaicin solution (500 mg/L) and then the reaction mixture (total volume of 3 mL) was incubated at 40 °C and 120 rpm. Samples were taken every 2 h and analyzed for vanillylamine formation using HPLC. Same reaction mixture containing boiled supernatant (to inactivate lipase) designed as blank and each experiment was done in triplicate. Results: 215 mg/L of vanillylamine was produced after the treatment of capsaicin using the optimized medium for 18 h, while only 61 mg/L of vanillylamine was detected in presence of the basal medium under the same conditions. No capsaicin conversion was observed in the blank sample, in which lipase activity was suppressed by boiling of the sample for 10 min. Conclusion: The application of optimized broth culture for the hydrolysis of capsaicin led to a 43% conversion of that pungent compound to vanillylamine.

Keywords: Capsaicin, green synthesis, lipase, stenotrophomonas maltophilia

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Authors: Tooba N. Shamsi, Romana Perveen, Sadaf Fatima

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Protease Inhibitors (PIs) are widespread in nature, produced by animals, plants and microorganisms. They play vital role in various biological activities by keeping a check on activity of proteases. Present study aims to investigate antioxidant and anti-inflammatory properties of PPI from Cajanus cajan (CCTI) and Phaseolus limensis (LBTI). PPI was purified from C. cajan (PUSA-992) by ammonium sulfate precipitation followed by ion exchange chromatography. The anti-oxidant activity was analyzed by two most common radical scavenging assays of FRAP (ferric reducing antioxidant power) and DPPH (1,1- diphenyl-2-picrylhydrazyl). Also, in-vitro anti-inflammatory activity was evaluated using albumin denaturation assay and membrane stabilization assay at different concentrations. Ascorbic acid and aspirin were used as a standards for antioxidant and anti-inflammatory assays respectively. The PPIs were also checked for antimicrobial activity against a number of bacterial strains. The CCTI and LBTI showed DPPH radical scavenging activity in a concentration–dependent manner with IC50 values 544 µg/ml and 506 µg/ml respectively comparative to ascorbic acid which was 258 µg/ml. Following FRAP assay, it was evaluated that LBTI had 87.5% and CCTI showed 84.4% antioxidant activity, taking value of standard ascorbic acid to be 100%. The PPIs also showed in-vitro anti‐inflammatory activity by inhibiting the heat induced albumin denaturation with IC50 values of 686 µg/ml and 615 µg/ml for CCTI and LBTI respectively compared to the standard (aspirin) which was 70.8 µg/ml. Red blood cells membrane stabilization with IC50 values of 641 µg/ml and 587 µg/ml for CCTI and LBTI respectively against aspirin which showed IC50 value of 70.4 µg/ml. PPIs showed antibacterial activity against 7 known strains while there was apparently no action against fungi.

Keywords: Cajanus cajan, Phaseolus limensis, Lima beans, protein protease inhibitor, antioxidant, anti-inflammatory, antimicrobial activity

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Authors: Madalin Enache, Simona Neagu, Roxana Cojoc, Ioana Gomoiu, Delia Ionela Dobre, Ancuta Roxana Trifoi

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Various types of halophilic and halotolerant microorganisms able to be cultivated in laboratory on culture media with a wide range of sodium chloride content are isolated from several salted environments. The extracellular enzymes of these microorganisms showed the enzymatic activity in these spectrums of salinity thus being attractive for several biotechnological processes developed at high ionic strength. In present work, a number of amylase, protease, esterase, lipase, cellulase, pectinase, xilanases and innulinase were identified for more than 50th bacterial strains isolated from water samples and sapropelic mud from four saline and hypersaline lakes located in Romanian plain. On the other hand, the cellulase and pectinase activity were also detected in some halotolerant microorganisms isolated from secondary agricultural flow of grapes processing. The preliminary data revealed that from totally tested strains seven harbor proteases activity, eight amylase activity, four for esterase and another four for lipase, three for pectinase and for one strain were identified either cellulase or pectinase activity. There were no identified enzymes able to hydrolase innulin added to culture media. Several strains isolated from sapropelic mud showed multiple extracellular enzymatic activities, namely three strains harbor three activities and another seven harbor two activities. The data revealed that amylase and protease activities were frequently detected if compare with other tested enzymes. In the case of pectinase were investigated, their ability to be used for increasing resveratrol recovery from material resulted after grapes processing. In this way, the resulted material from grapes processing was treated with microbial supernatant for several times (two, four and 24 hours) and the content of resveratrol was detected by High Performance Liquid Chromatography method (HPLC). The preliminary data revealed some positive results of this treatment.

Keywords: halophilic microorganisms, enzymes, pectinase, salinity

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Authors: Carol N. Flores-Fernández, Guadalupe Espilco, Cynthia Esquerre, Amparo I. Zavaleta

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Saline environments represent a valuable source of enzymes with novel properties and particular features for application in food, pharmaceutical and chemical industry. This study focuses on the isolation and screening of hydrolase-producing bacteria from Chilca salterns and the evaluation of their biotechnological potential. Soil samples were collected from Chilca salterns in Peru. For the isolation, medium containing 0.2 % of yeast extract, 5 % of NaCl and 10 % of the soil sample was used. After 72 h of incubation at 37 °C, serial dilutions were made up to 10−12 dilutions, spread on agar plates with 0.5 % of yeast extract and 5 % of NaCl, and incubated at 37 °C for 48 h. Screening of hydrolase-producing bacteria was carried out for cellulases, amylases, lipases, DNase, and proteases on specific media. Moreover, protease-producing bacteria were tested using protein extracted from the following legumes as substrate: Glycine max, Lupinus mutabilis, Pisum sativum, Erythrina edulis, Cicer arietinum, Phaseolus vulgaris and Vicia faba. A total of 16 strains were isolated from soil samples. On the screening media; 75, 44, 81 and 50 % were cellulase, amylase, DNase and protease producers, respectively. Also, 19 % of the isolates produced all the hydrolytic enzymes above mentioned. Lipase producers were not found. The 37 % and 12 % of the strains grew at 20 % and 30 % of salt concentration, respectively. In addition, 75 % of the strains grew at pH range between 5 and 10. From the total of protease-producing bacteria, 100 % hydrolyzed Glycine max, Lupinus mutabilis, and Pisum sativum protein, while 87 % hydrolyzed Erythrina edulis and Cicer arietinum protein. Finally, 75 % and 50 % of the strains hydrolyzed Phaseolus vulgaris and Vicia faba protein, respectively. Hydrolase-producing bacteria isolated from Chilca salterns in Peru grew at high salt concentrations and wide range of pH. In addition, protease-producing bacteria hydrolyzed protein from different sources such as leguminous. These enzymes have great biotechnological potential and could be used for different industrial processes and applications.

Keywords: bacteria, extracellular, hydrolases, Peru, salterns

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Authors: Abtehal Y. Anaas, Mohd. Nazmi Bin Abd. Manap

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Meat Tenderness is one of the most important factors affecting consumers' assessment of meat quality. Variation in meat tenderness is genetically controlled and varies among breeds, and it is also influenced by environmental factors that can affect its creation during rigor mortis and postmortem. The final postmortem meat tenderization relies on the extent of proteolysis of myofibrillar proteins caused by the endogenous activity of the proteolytic calpain system. This calpain system includes different calcium-dependent cysteine proteases, and an inhibitor, calpastatin. It is widely accepted that in farm animals including chickens, the μ-calpain gene (CAPN1) is a physiological candidate gene for meat tenderness. This study aimed to identify the association of single nucleotide polymorphism (SNP) markers in the CAPN1 gene with the tenderness of chicken breast meat from two Malaysian native and commercial broiler breed crosses. Ten, five months old native chickens and ten, 42 days commercial broilers were collected from the local market and breast muscles were removed two hours after slaughter, packed separately in plastic bags and kept at -20ºC for 24 h. The tenderness phenotype for all chickens’ breast meats was determined by Warner-Bratzler Shear Force (WBSF). Thawing and cooking losses were also measured in the same breast samples before using in WBSF determination. Polymerase chain reaction (PCR) was used to identify the previously reported C7198A and G9950A SNPs in the CAPN1 gene and assess their associations with meat tenderness in the two breeds. The broiler breast meat showed lower shear force values and lower thawing loss rates than the native chickens (p<0.05), whereas there were similar in the rates of cooking loss. The study confirms some previous results that the markers CAPN1 C7198A and G9950A were not significantly associated with the variation in meat tenderness in chickens. Therefore, further study is needed to confirm the functional molecular mechanism of these SNPs and evaluate their associations in different chicken populations.

Keywords: CAPNl, chicken, meat tenderness, meat quality, SNPs

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Authors: José Maria, Vânia Laranjo, Luís Abrunhosa, António Inês

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The occurrence of mycotoxigenic moulds such as Aspergillus, Penicillium and Fusarium in food and feed has an important impact on public health, by the appearance of acute and chronic mycotoxicoses in humans and animals, which is more severe in the developing countries due to lack of food security, poverty and malnutrition. This mould contamination also constitutes a major economic problem due the lost of crop production. A great variety of filamentous fungi is able to produce highly toxic secondary metabolites known as mycotoxins. Most of the mycotoxins are carcinogenic, mutagenic, neurotoxic and immunosuppressive, being ochratoxin A (OTA) one of the most important. OTA is toxic to animals and humans, mainly due to its nephrotoxic properties. Several approaches have been developed for decontamination of mycotoxins in foods, such as, prevention of contamination, biodegradation of mycotoxins-containing food and feed with microorganisms or enzymes and inhibition or absorption of mycotoxin content of consumed food into the digestive tract. Some group of Gram-positive bacteria named lactic acid bacteria (LAB) are able to release some molecules that can influence the mould growth, improving the shelf life of many fermented products and reducing health risks due to exposure to mycotoxins. Some LAB are capable of mycotoxin detoxification. Recently our group was the first to describe the ability of LAB strains to biodegrade OTA, more specifically, Pediococcus parvulus strains isolated from Douro wines. The pathway of this biodegradation was identified previously in other microorganisms. OTA can be degraded through the hydrolysis of the amide bond that links the L-β-phenylalanine molecule to the ochratoxin alpha (OTα) a non toxic compound. It is known that some peptidases from different origins can mediate the hydrolysis reaction like, carboxypeptidase A an enzyme from the bovine pancreas, a commercial lipase and several commercial proteases. So, we wanted to have a better understanding of this OTA degradation process when LAB are involved and identify which molecules where present in this process. For achieving our aim we used some bioinformatics tools (BLAST, CLUSTALX2, CLC Sequence Viewer 7, Finch TV). We also designed specific primers and realized gene specific PCR. The template DNA used came from LAB strains samples of our previous work, and other DNA LAB strains isolated from elderberry fruit, silage, milk and sausages. Through the employment of bioinformatics tools it was possible to identify several proteins belonging to the carboxypeptidase family that participate in the process of OTA degradation, such as serine type D-Ala-D-Ala carboxypeptidase and membrane carboxypeptidase. In conclusions, this work has identified carboxypeptidase proteins being one of the molecules present in the OTA degradation process when LAB are involved.

Keywords: carboxypeptidase, lactic acid bacteria, mycotoxins, ochratoxin a.

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Authors: Sachil Kumar, Anoop Kumar Verma, Wahid Ali, Uma Shankar Singh

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Globally approximately 55.3 million people die each year. In the India there were 95 lakh annual deaths in 2013. The number of deaths resulted from homicides, suicides and unintentional injuries in the same period was about 5.7 lakh. The ever-increasing crime rate necessitated the development of methods for determining time since death. An erroneous time of death window can lead investigators down the wrong path or possibly focus a case on an innocent suspect. In this regard a research was carried out by analyzing the temperature dependent degradation of a Cardiac Troponin-T protein (cTnT) in the myocardium postmortem as a marker for time since death. Cardiac tissue samples were collected from (n=6) medico-legal autopsies, (in the Department of Forensic Medicine and Toxicology, King George’s Medical University, Lucknow India) after informed consent from the relatives and studied post-mortem degradation by incubation of the cardiac tissue at room temperature (20±2 OC), 12 0C, 25 0C and 37 0C for different time periods ((~5, 26, 50, 84, 132, 157, 180, 205, and 230 hours). The cases included were the subjects of road traffic accidents (RTA) without any prior history of disease who died in the hospital and their exact time of death was known. The analysis involved extraction of the protein, separation by denaturing gel electrophoresis (SDS-PAGE) and visualization by Western blot using cTnT specific monoclonal antibodies. The area of the bands within a lane was quantified by scanning and digitizing the image using Gel Doc. The data shows a distinct temporal profile corresponding to the degradation of cTnT by proteases found in cardiac muscle. The disappearance of intact cTnT and the appearance of lower molecular weight bands are easily observed. Western blot data clearly showed the intact protein at 42 kDa, two major (27 kDa, 10kDa) fragments, two additional minor fragments (32 kDa) and formation of low molecular weight fragments as time increases. At 12 0C the intensity of band (intact cTnT) decreased steadily as compared to RT, 25 0C and 37 0C. Overall, both PMI and temperature had a statistically significant effect where the greatest amount of protein breakdown was observed within the first 38 h and at the highest temperature, 37 0C. The combination of high temperature (37 0C) and long Postmortem interval (105.15 hrs) had the most drastic effect on the breakdown of cTnT. If the percent intact cTnT is calculated from the total area integrated within a Western blot lane, then the percent intact cTnT shows a pseudo-first order relationship when plotted against the log of the time postmortem. These plots show a good coefficient of correlation of r = 0.95 (p=0.003) for the regression of the human heart at different temperature conditions. The data presented demonstrates that this technique can provide an extended time range during which Postmortem interval can be more accurately estimated.

Keywords: degradation, postmortem interval, proteolysis, temperature, troponin

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Authors: Antonela Matana, Dubravka Brdar, Vesela Torlak, Marijana Popovic, Ivana Gunjaca, Ozren Polasek, Vesna Boraska Perica, Maja Barbalic, Ante Punda, Caroline Hayward, Tatijana Zemunik

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Parathyroid hormone (PTH) plays a critical role in the regulation of bone mineral metabolism and calcium homeostasis. Higher PTH levels are associated with heart failure, hypertension, coronary artery disease, cardiovascular mortality and poorer bone health. A twin study estimated that 60% of the variation in PTH concentrations is genetically determined. Only one GWAS of PTH concentration has been reported to date. Identified loci explained 4.5% of the variance in circulating PTH, suggesting that additional genetic variants remain undiscovered. Therefore, the aim of this study was to identify novel genetic variants associated with PTH levels in a general population. We have performed a GWAS meta-analysis on 2596 individuals originating from three Croatian cohorts: City of Split and the Islands of Korčula and Vis, within a large-scale project of “10,001 Dalmatians”. A total of 7 411 206 variants, imputed using the 1000 Genomes reference panel, with minor allele frequency ≥ 1% and Rsq ≥ 0.5 were analyzed for the association. GWAS within each data set was performed under an additive model, controlling for age, gender and relatedness. Meta-analysis was conducted using the inverse-variance fixed-effects method. Furthermore, to identify sex-specific effects, we have conducted GWAS meta-analyses analyzing males and females separately. In addition, we have performed biological pathway analysis. Four SNPs, representing one locus, reached genome-wide significance. The most significant SNP was rs11099476 on chromosome 4 (P=1.15x10-8), which explained 1.14 % of the variance in PTH. The SNP is located near the protein-coding gene RASGEF1B. Additionally, we detected suggestive association with SNPs, rs77178854 located on chromosome 2 in the DPP10 gene (P=2.46x10-7) and rs481121 located on chromosome 1 (P=3.58x10-7) near the GRIK1 gene. One of the top hits detected in the main meta-analysis, intron variant rs77178854 located within DPP10 gene, reached genome-wide significance in females (P=2.21x10-9). No single locus was identified in the meta-analysis in males. Fifteen biological pathways were functionally enriched at a P<0.01, including muscle contraction, ion homeostasis and cardiac conduction as the most significant pathways. RASGEF1B is the guanine nucleotide exchange factor, known to be associated with height, bone density, and hip. DPP10 encodes a membrane protein that is a member of the serine proteases family, which binds specific voltage-gated potassium channels and alters their expression and biophysical properties. In conclusion, we identified 2 novel loci associated with PTH levels in a general population, providing us with further insights into the genetics of this complex trait.

Keywords: general population, genome-wide association analysis, parathyroid hormone, single nucleotide polymorphisms.

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