Displaying publications 1 - 20 of 41 in total

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  1. Study of an inhibitory effect of plant polyphenolic compounds against digestive enzymes using bench-working experimental evidence predicted by molecular docking and dynamics
    Vyas K, Prabaker S, Prabhu D, Sakthivelu M, Rajamanikandan S, Velusamy P, et al.
    Int J Biol Macromol, 2024 Feb;259(Pt 1):129222.
    PMID: 38185307 DOI: 10.1016/j.ijbiomac.2024.129222
    The substantial nutritional content and diversified biological activity of plant-based nutraceuticals are due to polyphenolic chemicals. These chemicals are important and well-studied plant secondary metabolites. Their protein interactions are extensively studied. This relationship is crucial for the logical development of functional food and for enhancing the availability and usefulness of polyphenols. This study highlights the influence of protein types and polyphenols on the interaction, where the chemical bindings predominantly consist of hydrophobic interactions and hydrogen bonds. The interaction between polyphenolic compounds (PCs) and digestive enzymes concerning their inhibitory activity has not been fully studied. Therefore, we have examined the interaction of four digestive enzymes (α-amylase, pepsin, trypsin, and α-chymotrypsin) with four PCs (curcumin, diosmin, morin, and 2',3',4'-trihydroxychalcone) through in silico and in vitro approaches. In vitro plate assays, enzyme kinetics, spectroscopic assays, molecular docking, and simulations were performed. We observed all these PCs have significant docking scores and preferable interaction with the active site of the digestive enzymes, resulting in the reduction of enzyme activity. The enzyme-substrate binding mechanism was determined using the Lineweaver Burk plot, indicating that the inhibition occurred competitively. Among four PCs diosmin and morin has the highest interaction energy over digestive enzymes with IC50 value of 1.13 ± 0.0047 and 1.086 ± 0.0131 μM. Kinetic studies show that selected PCs inhibited pepsin, trypsin, and chymotrypsin competitively and inhibited amylase in a non-competitive manner, especially by 2',3',4'-trihydroxychalcone. This study offers insights into the mechanisms by which the selected PCs inhibit the enzymes and has the potential to enhance the application of curcumin, diosmin, morin, and 2',3',4'-trihydroxychalcone as natural inhibitors of digestive enzymes.
    Matched MeSH terms: Trypsin/metabolism
  2. Potential protein hydrolysates from the white and purple flower varieties of Orthosiphon aristatus leaves
    Chua LS, Abdullah FI, Lim TK, Lin Q
    Food Chem, 2024 Jan 30;432:137261.
    PMID: 37651783 DOI: 10.1016/j.foodchem.2023.137261
    This study was aimed to extract bioactive peptides from the white and purple flower varieties of Orthosiphon aristatus leaves. The herb is well known for its pharmacological importance, possibly attributed to its plant proteins. Phenol based extraction was used to extract plant proteins, and then hydrolysed by proteolytic enzymes such as trypsin (serine protease) and pepsin (aspartic protease). MS/MS analysis revealed that 145 and 125 proteins were detected from the white and purple flower varieties, respectively. Trypsin hydrolysates were showed to have a higher degree of hydrolysis (24-33%), resulting in higher antioxidant and antibacterial activities. The white flower of trypsin hydrolysates showed a higher radical scavenging activity which could be attributed to its higher content of stress proteins (19%). However, trypsin hydrolysates from the purple flower showed higher ferric reducing power and bacterial growth inhibition. The performance of hydrolysates was better than ampicillin in inhibiting Acinetobacter baumanni and Staphylococcus aureus.
    Matched MeSH terms: Trypsin
  3. Fulltext An alternative peptone preparation using Hermetia illucens (Black soldier fly) hydrolysis: process optimization and performance evaluation
    Liu G, Tiang MF, Ma S, Wei Z, Liang X, Sajab MS, et al.
    PeerJ, 2024;12:e16995.
    PMID: 38426145 DOI: 10.7717/peerj.16995
    BACKGROUND: Hermetia illucens (HI), commonly known as the black soldier fly, has been recognized for its prowess in resource utilization and environmental protection because of its ability to transform organic waste into animal feed for livestock, poultry, and aquaculture. However, the potential of the black soldier fly's high protein content for more than cheap feedstock is still largely unexplored.

    METHODS: This study innovatively explores the potential of H. illucens larvae (HIL) protein as a peptone substitute for microbial culture media. Four commercial proteases (alkaline protease, trypsin, trypsase, and papain) were explored to hydrolyze the defatted HIL, and the experimental conditions were optimized via response surface methodology experimental design. The hydrolysate of the defatted HIL was subsequently vacuum freeze-dried and deployed as a growth medium for three bacterial strains (Staphylococcus aureus, Bacillus subtilis, and Escherichia coli) to determine the growth kinetics between the HIL peptone and commercial peptone.

    RESULTS: The optimal conditions were 1.70% w/w complex enzyme (alkaline protease: trypsin at 1:1 ratio) at pH 7.0 and 54 °C for a duration of 4 h. Under these conditions, the hydrolysis of defatted HIL yielded 19.25% ±0.49%. A growth kinetic analysis showed no significant difference in growth parameters (μmax, Xmax, and λ) between the HIL peptone and commercial peptone, demonstrating that the HIL hydrolysate could serve as an effective, low-cost alternative to commercial peptone. This study introduces an innovative approach to HIL protein resource utilization, broadening its application beyond its current use in animal feed.

    Matched MeSH terms: Trypsin
  4. Electrochemical biosensor for trypsin activity assay based on cleavage of immobilized tyrosine-containing peptide
    Filippova TA, Masamrekh RA, Shumyantseva VV, Latsis IA, Farafonova TE, Ilina IY, et al.
    Talanta, 2023 May 15;257:124341.
    PMID: 36821964 DOI: 10.1016/j.talanta.2023.124341
    In this work, we proposed a biosensor for trypsin proteolytic activity assay using immobilization of model peptides on screen-printed electrodes (SPE) modified with gold nanoparticles (AuNPs) prepared by electrosynthetic method. Sensing of proteolytic activity was based on electrochemical oxidation of tyrosine residues of peptides. We designed peptides containing N-terminal cysteine residue for immobilization on an SPE, modified with gold nanoparticles, trypsin-specific cleavage site and tyrosine residue as a redox label. The peptides were immobilized on SPE by formation of chemical bonds between mercapto groups of the N-terminal cysteine residues and AuNPs. After the incubation with trypsin, time-dependent cleavage of the immobilized peptides was observed by decline in tyrosine electrochemical oxidation signal. The kinetic parameters of trypsin, such as the catalytic constant (kcat), the Michaelis constant (KM) and the catalytic efficiency (kcat/KM), toward the CGGGRYR peptide were determined as 0.33 ± 0.01 min-1, 198 ± 24 nM and 0.0016 min-1 nM-1, respectively. Using the developed biosensor, we demonstrated the possibility of analysis of trypsin specificity toward the peptides with amino acid residues disrupting proteolysis. Further, we designed the peptides with proline or glutamic acid residues after the cleavage site (CGGRPYR and CGGREYR), and trypsin had reduced activity toward both of them according to the existing knowledge of the enzyme specificity. The developed biosensor system allows one to perform a comparative analysis of the protease steady-state kinetic parameters and specificity toward model peptides with different amino acid sequences.
    Matched MeSH terms: Trypsin/metabolism
  5. Fulltext Spectrum of PRSS1, SPINK1, CTRC, CFTR, and CPA1 Gene Variants in Chronic Pancreatitis Patients in Russia
    Litvinova MM, Khafizov KF, Speranskaya AS, Matsvay AD, Asanov AY, Nikolskaya KA, et al.
    Sovrem Tekhnologii Med, 2023;15(2):60-70.
    PMID: 37389024 DOI: 10.17691/stm2023.15.2.06
    The aim of the study was to define the spectrum of genetic risk factors of chronic pancreatitis (CP) development in patients living in the European part of the Russian Federation.

    MATERIALS AND METHODS: The study group included 105 patients with CP, with the age of the disease onset under 40 years old (the average age of onset was 26.9 years). The control group consisted of 76 persons without clinical signs of pancreatitis. The diagnosis of chronic pancreatitis in patients was made on the basis of clinical manifestations and the results of laboratory and instrumental investigations. Genetic examination of patients was conducted using the next-generation sequencing (NGS) technology and included targeted sequencing of all exons and exon-intron boundaries of the PRSS1, SPINK1, CTRC, CFTR, and CPA1 genes. The genotyping of the rs61734659 locus of the PRSS2 gene was also conducted.

    RESULTS: Genetic risk factors of the CP development were found in 61% of patients. Pathogenic and likely-pathogenic variants associated with the risk of CP development were identified in the following genes: CTRC (37.1% of patients), CFTR (18.1%), SPINK1 (8.6%), PRSS1 (8.6%), and CPA1 (6.7%). The frequent gene variants in Russian patients with CP were as follows: CTRC gene - c.180C>T (rs497078), c.760C>T (rs121909293), c.738_761del24 (rs746224507); cumulative odds ratio (OR) for all risk alleles was 1.848 (95% CI: 1.054-3.243); CFTR gene - c.3485G>T (rs1800120), c.1521_1523delCTT (p.Phe508del, rs113993960), and c.650A>G (rs121909046); OR=2.432 (95% CI: 1.066-5.553). In the SPINK1, PRSS1, and CPA1 genes, pathogenic variants were found only in the group of patients with CP. The frequent variants of the SPINK1 gene include c.101A>G (p.Asn34Ser, rs17107315) and c.194+2T>C (rs148954387); of the PRSS1 gene - c.86A>T (p.Asn29Ile, rs111033566); of the CPA1 gene - c.586-30C>T (rs782335525) and c.696+23_696+24delGG. The OR for the CP development for the c.180TT genotype (rs497078) CTRC according to the recessive model (TT vs. CT+CC) was 7.05 (95% CI: 0.86-263, p=0.011). In the CTRC gene, the variant c.493+49G>C (rs6679763) appeared to be benign, the c.493+51C>A (rs10803384) variant was frequently detected among both the diseased and healthy persons and did not demonstrate a protective effect. The protective factor c.571G>A (p.Gly191Arg, rs61734659) of the PRSS2 gene was detected only in the group of healthy individuals and confirmed its protective role. 12.4% of the patients with CP had risk factors in 2 or 3 genes.

    CONCLUSION: Sequencing of the coding regions of the PRSS1, SPINK1, CTRC, CFTR, and CPA1 genes allowed to identify genetic risk factors of the CP development in 61% of cases. Determining the genetic cause of CP helps to predict the disease course, perform preventive measures in the proband's relatives, and facilitate a personalized treatment of the patient in future.

    Matched MeSH terms: Trypsin/genetics; Trypsinogen
  6. Identification of antioxidant peptides derived from tropical jackfruit seed and investigation of the stability profiles
    Chai TT, Xiao J, Mohana Dass S, Teoh JY, Ee KY, Ng WJ, et al.
    Food Chem, 2021 Mar 15;340:127876.
    PMID: 32871354 DOI: 10.1016/j.foodchem.2020.127876
    Jackfruit is a sweet tropical fruit with very pleasant aroma, and the ripe seeds are edible. In this study, jackfruit seed proteins were isolated and subjected to trypsin digestion. The resultant protein hydrolysate was then subjected to antioxidant assay-guided purification, using centrifugal filtration, C18 reverse-phase and strong cation exchange (SCX) fractionations. The purified SCX fraction was further analyzed by de novo peptide sequencing, and two peptide sequences were identified and synthesized. Peptide JFS-2 (VGPWQK) was detected with antioxidant potential, with EC50 value comparable to that of commercial GSH antioxidant peptide. Additionally, the identified peptides were tested with protein protection potential, in an albumin protein denaturation inhibitory assay. Concurrently, we also investigated the pH, temperature, and gastrointestinal-digestion stability profiles for the identified peptide. With further research efforts, the identified peptides could potentially be developed into preservative agent for protein-rich food systems or as health-promoting diet supplements.
    Matched MeSH terms: Trypsin
  7. Protein decomplexation and proteomics: A complementary assessment method of the physicochemical purity of antivenom
    Tan CH, Liew JL, Chong HP, Tan NH
    Biologicals, 2021 Jan;69:22-29.
    PMID: 33431232 DOI: 10.1016/j.biologicals.2020.12.004
    The quality of antivenom is governed by its safety and efficacy profiles. These quality characteristics are much influenced by the purity of antivenom content. Rigorous assessment and meticulous monitoring of antivenom purity at the preclinical setting is hence crucial. This study aimed to explore an integrative proteomic method to assess the physicochemical purity of four commercially available antivenoms in the region. The antivenoms were subjected to Superdex 200 HR 10/30 size-exclusion fast-protein liquid chromatography (SE-FPLC). The proteins in each fraction were trypsin-digested and analyzed by nano-ESI-liquid chromatography-tandem mass spectrometry (LC-MS/MS). SE-FPLC resolved the antivenom proteins into three major protein components of very high (>200 kDa), high (100-120 kDa) and medium (<60 kDa) molecular weights. The major components (80-95% of total proteins) in the antivenoms were proteins of 100-120 kDa consisting of mainly the light and partially digested heavy immunoglobulin chains, consistent with F(ab')2 as the active principle of the antivenoms. However, LC-MS/MS also detected substantial quantity of large proteins (e.g. alpha-2-macroglobulins), immunoglobulin aggregates and impurities e.g. albumins in some products. The method is practical and able to unveil the quantitative and qualitative aspects of antivenom protein compositions. It is therefore a potentially useful preclinical assessment tool of antivenom purity.
    Matched MeSH terms: Trypsin
  8. Fulltext Crustacean Proteases and Their Application in Debridement
    Perera E, Rodriguez-Viera L, Montero-Alejo V, Perdomo-Morales R
    Trop Life Sci Res, 2020 Jul;31(2):187-209.
    PMID: 32922675 DOI: 10.21315/tlsr2020.31.2.10
    Digestive proteases from marine organisms have been poorly applied to biomedicine. Exceptions are trypsin and other digestive proteases from a few cold-adapted or temperate fish and crustacean species. These enzymes are more efficient than enzymes from microorganism and higher vertebrates that have been used traditionally. However, the biomedical potential of digestive proteases from warm environment species has received less research attention. This review aims to provide an overview of this unrealised biomedical potential, using the debridement application as a paradigm. Debridement is intended to remove nonviable, necrotic and contaminated tissue, as well as fibrin clots, and is a key step in wound treatment. We discuss the physiological role of enzymes in wound healing, the use of exogenous enzymes in debridement, and the limitations of cold-adapted enzymes such as their poor thermal stability. We show that digestive proteases from tropical crustaceans may have advantages over their cold-adapted counterparts for this and similar uses. Differences in thermal stability, auto-proteolytic stability, and susceptibility to proteinase inhibitors are discussed. Furthermore, it is proposed that the feeding behaviour of the source organism may direct the evaluation of enzymes for particular applications, as digestive proteases have evolved to fill a wide variety of feeding habitats, natural substrates, and environmental conditions. We encourage more research on the biomedical application of digestive enzymes from tropical marine crustaceans.
    Matched MeSH terms: Trypsin
  9. Fulltext Purification and identification of novel cytotoxic oligopeptides from soft coral Sarcophyton glaucum
    Quah Y, Mohd Ismail NI, Ooi JLS, Affendi YA, Abd Manan F, Teh LK, et al.
    J Zhejiang Univ Sci B, 2019 1 8;20(1):59-70.
    PMID: 30614230 DOI: 10.1631/jzus.B1700586
    Globally, peptide-based anticancer therapies have drawn much attention. Marine organisms are a reservoir of anticancer peptides that await discovery. In this study, we aimed to identify cytotoxic oligopeptides from Sarcophyton glaucum. Peptides were purified from among the S. glaucum hydrolysates produced by alcalase, chymotrypsin, papain, and trypsin, guided by a methylthiazolyldiphenyl-tetrazolium bromide (MTT) assay on the human cervical cancer (HeLa) cell line for cytotoxicity evaluation. Purification techniques adopted were membrane ultrafiltration, gel filtration chromatography, solid phase extraction (SPE), and reversed-phase high-performance liquid chromatography (RP-HPLC). Purified peptides were identified by de novo peptide sequencing. From papain hydrolysate, three peptide sequences were identified: AGAPGG, AERQ, and RDTQ (428.45, 502.53, and 518.53 Da, respectively). Peptides synthesized from these sequences exhibited cytotoxicity on HeLa cells with median effect concentration (EC50) values of 8.6, 4.9, and 5.6 mmol/L, respectively, up to 5.8-fold stronger than the anticancer drug 5-fluorouracil. When tested at their respective EC50, AGAPGG, AERQ, and RDTQ showed only 16%, 25%, and 11% cytotoxicity to non-cancerous Hek293 cells, respectively. In conclusion, AERQ, AGAPGG, and RDTQ are promising candidates for future development as peptide-based anticancer drugs.
    Matched MeSH terms: Chymotrypsin; Trypsin
  10. Fulltext The impact of four processing methods on trypsin-, chymotrypsin- and alpha-amylase inhibitors present in underutilised legumes
    Choi WC, Parr T, Lim YS
    J Food Sci Technol, 2019 Jan;56(1):281-289.
    PMID: 30728570 DOI: 10.1007/s13197-018-3488-0
    The global trend in increasing plant-based protein diets due to health and ideological reasons, has created an increased demand for food legumes that exceeds current production. To meet this demand, it is timely to reduce relying solely on soybean, and explore the potential of the underutilised legumes that are cultivated regionally. Underutilised legumes are rich in protein, carbohydrates and other nutrients that are essential for consumer. However, relatively little is known about their anti-nutritional properties and processing methods. Anti-nutritional factors (ANFs) such as enzyme inhibitors are prevalent in legumes and may interfere with digestibility and nutrient absorption. Nevertheless, an optimised food processing method will overcome this challenge and warrant a safe inclusion of legume in plant-based protein diets. Hence current study aimed to optimise the food processing methods (soaking, wet heating, autoclaving and freezing) and evaluate their efficiency in eliminating the enzyme inhibitors [trypsin, chymotrypsin (CIA) and α-amylase (AIA) inhibitors] present in seven underutilised legumes. Current study showed that autoclaving at 121 °C for 15 min reduced the AIA in all underutilised legumes tested. The AIA and CIA of bambara groundnut were successfully inactivated by wet heating at 50 °C for 60 min, and by autoclaving at 121 °C for 15 min. While the CIA of chickpea was successfully inactivated by freezing at - 80 °C for 24 h.
    Matched MeSH terms: Chymotrypsin; Trypsin
  11. Fulltext Cell surface proteins of cryptococcus neoformans mediate adherence and internalisation into mammalian cells
    Choo, K.K., Chin, V.K., Chong, P.P., Ho, S.H., Yong, V.C.
    JUMMEC, 2019;22(2):24-30.
    MyJurnal
    Cryptococcus neoformans is an encapsulated fungal pathogen that causes severe disease primarily in
    immunocompromised patients. Adherence and internalisation of microbial pathogens into host cells often
    begin with engagement of microbes to the surface receptors of host. However, the mechanisms involved
    remain poorly understood. In this study, we investigated the association of cell surface determinants of C.
    neoformans with mammalian cells. Our results showed that treatment with trypsin, but not paraformaldehyde
    or heat killing, could reduce host-cryptococci interaction, suggesting the involvement of cell surface proteins
    (CSPs) of C. neoformans in the interaction. We extended our investigations to determine the roles of CSPs
    during cryptococci-host cells interaction by extracting and conjugating CSPs of C. neoformans to latex beads.
    Conjugation of CSPs with both encapsulated and acapsular C. neoformans increased the association of latex
    beads with mammalian alveolar epithelial cells, alveolar macrophages and monocyte-derived macrophages.
    Further examination on the actin organisation of the host cells implied the involvement of actin-dependent
    phagocytosis in the internalisation of C. neoformans in CSP-conjugated latex beads. We hypothesised that
    CSPs present on the cell wall of C. neoformans mediate the adherence and actin-dependent phagocytosis
    of cryptococci by mammalian cells. Our results warrant further studies on the exact role of CSPs in the
    pathogenesis of cryptococcosis.
    Matched MeSH terms: Trypsin
  12. Fulltext Do pancreatic cancer and chronic pancreatitis share the same genetic risk factors? A PANcreatic Disease ReseArch (PANDoRA) consortium investigation
    Campa D, Pastore M, Capurso G, Hackert T, Di Leo M, Izbicki JR, et al.
    Int J Cancer, 2018 01 15;142(2):290-296.
    PMID: 28913878 DOI: 10.1002/ijc.31047
    Pancreatic ductal adenocarcinoma (PDAC) is a very aggressive tumor with a five-year survival of less than 6%. Chronic pancreatitis (CP), an inflammatory process in of the pancreas, is a strong risk factor for PDAC. Several genetic polymorphisms have been discovered as susceptibility loci for both CP and PDAC. Since CP and PDAC share a consistent number of epidemiologic risk factors, the aim of this study was to investigate whether specific CP risk loci also contribute to PDAC susceptibility. We selected five common SNPs (rs11988997, rs379742, rs10273639, rs2995271 and rs12688220) that were identified as susceptibility markers for CP and analyzed them in 2,914 PDAC cases, 356 CP cases and 5,596 controls retrospectively collected in the context of the international PANDoRA consortium. We found a weak association between the minor allele of the PRSS1-PRSS2-rs10273639 and an increased risk of developing PDAC (ORhomozygous  = 1.19, 95% CI 1.02-1.38, p = 0.023). Additionally all the SNPs confirmed statistically significant associations with risk of developing CP, the strongest being PRSS1-PRSS2-rs10273639 (ORheterozygous  = 0.51, 95% CI 0.39-0.67, p = 1.10 × 10-6 ) and MORC4-rs 12837024 (ORhomozygous  = 2.07 (1.55-2.77, ptrend  = 0.7 × 10-11 ). Taken together, the results from our study do not support variants rs11988997, rs379742, rs10273639, rs2995271 and rs12688220 as strong predictors of PDAC risk, but further support the role of these SNPs in CP susceptibility. Our study suggests that CP and PDAC probably do not share genetic susceptibility, at least in terms of high frequency variants.
    Matched MeSH terms: Trypsin/genetics; Trypsinogen/genetics
  13. Fulltext Separation and identification of bioactive peptides from stem of Tinospora cordifolia (Willd.) Miers
    Pachaiappan R, Tamboli E, Acharya A, Su CH, Gopinath SCB, Chen Y, et al.
    PLoS One, 2018;13(3):e0193717.
    PMID: 29494663 DOI: 10.1371/journal.pone.0193717
    Enzyme hydrolysates (trypsin, papain, pepsin, α-chymotrypsin, and pepsin-pancreatin) of Tinospora cordifolia stem proteins were analyzed for antioxidant efficacy by measuring (1) 1,1-diphenyl-2-picrylhydrazyl (DPPH•) radical scavenging activity, (2) 2,20-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS+) radical scavenging capacity, and (3) Fe2+ chelation. Trypsin hydrolysate showed the strongest DPPH• scavenging, while α-chymotrypsin hydrolysate exhibited the highest ABTS+ scavenging and Fe2+ chelation. Undigested protein strongly inhibited the gastrointestinal enzymes, trypsin (50% inhibition at enzyme/substrate ratio = 1:6.9) and α-chymotrypsin (50% inhibition at enzyme/substrate ratio = 1:1.82), indicating the prolonged antioxidant effect after ingestion. Furthermore, gel filtration purified peptide fractions of papain hydrolysates exhibited a significantly higher ABTS+ and superoxide radical scavenging as compared to non-purified digests. Active fraction 9 showing the highest radical scavenging ability was further purified and confirmed by MALDI-TOF MS followed by MS/MS with probable dominant peptide sequences identified are VLYSTPVKMWEPGR, VITVVATAGSETMR, and HIGININSR. The obtained results revealed that free radical scavenging capacity of papain hydrolysates might be related to its consistently low molecular weight hydrophobic peptides.
    Matched MeSH terms: Chymotrypsin/metabolism; Trypsin/metabolism
  14. Fulltext Extraction of gelatin from salmon (Salmo salar) fish skin using trypsin-aided process: optimization by Plackett-Burman and response surface methodological approaches
    Fan H, Dumont MJ, Simpson BK
    J Food Sci Technol, 2017 Nov;54(12):4000-4008.
    PMID: 29085142 DOI: 10.1007/s13197-017-2864-5
    Gelatin from salmon (Salmo salar) skin with high molecular weight protein chains (α-chains) was extracted using trypsin-aided process. Response surface methodology was used to optimise the extraction parameters. Yield, hydroxyproline content and protein electrophoretic profile via sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of gelatin were used as responses in the optimization study. The optimum conditions were determined as: trypsin concentration at 1.49 U/g; extraction temperature at 45 °C; and extraction time at 6 h 16 min. This response surface optimized model was significant and produced an experimental value (202.04 ± 8.64%) in good agreement with the predicted value (204.19%). Twofold higher yields of gelatin with high molecular weight protein chains were achieved in the optimized process with trypsin treatment when compared to the process without trypsin.
    Matched MeSH terms: Trypsin
  15. Fulltext Alpha-1 antitrypsin augmentation therapy decreases miR-199a-5p, miR-598 and miR-320a expression in monocytes via inhibition of NFκB
    Hassan T, de Santi C, Mooney C, McElvaney NG, Greene CM
    Sci Rep, 2017 10 23;7(1):13803.
    PMID: 29062067 DOI: 10.1038/s41598-017-14310-2
    Alpha-1 antitrypsin (AAT) augmentation therapy involves infusion of plasma-purified AAT to AAT deficient individuals. Whether treatment affects microRNA expression has not been investigated. This study's objectives were to evaluate the effect of AAT augmentation therapy on altered miRNA expression in monocytes and investigate the mechanism. Monocytes were isolated from non-AAT deficient (MM) and AAT deficient (ZZ) individuals, and ZZs receiving AAT. mRNA (qRT-PCR, microarray), miRNA (miRNA profiling, qRT-PCR), and protein (western blotting) analyses were performed. Twenty one miRNAs were differentially expressed 3-fold between ZZs and MMs. miRNA validation studies demonstrated that in ZZ monocytes receiving AAT levels of miR-199a-5p, miR-598 and miR-320a, which are predicted to be regulated by NFκB, were restored to levels similar to MMs. Validated targets co-regulated by these miRNAs were reciprocally increased in ZZs receiving AAT in vivo and in vitro. Expression of these miRNAs could be increased in ZZ monocytes treated ex vivo with an NFκB agonist and decreased by NFκB inhibition. p50 and p65 mRNA and protein were significantly lower in ZZs receiving AAT than untreated ZZs. AAT augmentation therapy inhibits NFκB and decreases miR-199a-5p, miR-598 and miR-320a in ZZ monocytes. These NFκB-inhibitory properties may contribute to the anti-inflammatory effects of AAT augmentation therapy.
    Matched MeSH terms: Trypsin Inhibitors/therapeutic use*
  16. Fulltext Data for proteome analysis of Bacillus lehensis G1 in starch-containing medium
    Ling HL, Rahmat Z, Murad AMA, Mahadi NM, Illias RM
    Data Brief, 2017 Oct;14:35-40.
    PMID: 28761915 DOI: 10.1016/j.dib.2017.07.026
    Bacillus lehensis G1 is a cyclodextrin glucanotransferase (CGTase) producer, which can degrade starch into cyclodextrin. Here, we present the proteomics data of B. lehensis cultured in starch-containing medium, which is related to the article "Proteome-based identification of signal peptides for improved secretion of recombinant cyclomaltodextrin glucanotransferase in Escherichia coli" (Ling et. al, in press). This dataset was generated to better understand the secretion of proteins involved in starch utilization for bacterial sustained growth. A 2-DE proteomic technique was used and the proteins were tryptically digested followed by detection using MALDI-TOF/TOF. Proteins were classified into functional groups using the information available in SubtiList webserver (http://genolist.pasteur.fr/SubtiList/).
    Matched MeSH terms: Trypsin
  17. Fulltext Methodological Considerations and Comparisons of Measurement Results for Extracellular Proteolytic Enzyme Activities in Seawater
    Obayashi Y, Wei Bong C, Suzuki S
    Front Microbiol, 2017;8:1952.
    PMID: 29067013 DOI: 10.3389/fmicb.2017.01952
    Microbial extracellular hydrolytic enzymes that degrade organic matter in aquatic ecosystems play key roles in the biogeochemical carbon cycle. To provide linkages between hydrolytic enzyme activities and genomic or metabolomic studies in aquatic environments, reliable measurements are required for many samples at one time. Extracellular proteases are one of the most important classes of enzymes in aquatic microbial ecosystems, and protease activities in seawater are commonly measured using fluorogenic model substrates. Here, we examined several concerns for measurements of extracellular protease activities (aminopeptidases, and trypsin-type, and chymotrypsin-type activities) in seawater. Using a fluorometric microplate reader with low protein binding, 96-well microplates produced reliable enzymatic activity readings, while use of regular polystyrene microplates produced readings that showed significant underestimation, especially for trypsin-type proteases. From the results of kinetic experiments, this underestimation was thought to be attributable to the adsorption of both enzymes and substrates onto the microplate. We also examined solvent type and concentration in the working solution of oligopeptide-analog fluorogenic substrates using dimethyl sulfoxide (DMSO) and 2-methoxyethanol (MTXE). The results showed that both 2% (final concentration of solvent in the mixture of seawater sample and substrate working solution) DMSO and 2% MTXE provide similarly reliable data for most of the tested substrates, except for some substrates which did not dissolve completely in these assay conditions. Sample containers are also important to maintain the level of enzyme activity in natural seawater samples. In a small polypropylene containers (e.g., standard 50-mL centrifugal tube), protease activities in seawater sample rapidly decreased, and it caused underestimation of natural activities, especially for trypsin-type and chymotrypsin-type proteases. In conclusion, the materials and method for measurements should be carefully selected in order to accurately determine the activities of microbial extracellular hydrolytic enzymes in aquatic ecosystems; especially, low protein binding materials should be chosen to use at overall processes of the measurement.
    Matched MeSH terms: Chymotrypsin; Trypsin
  18. Generation, Fractionation, and Characterization of Iron-Chelating Protein Hydrolysate from Palm Kernel Cake Proteins
    Zarei M, Ghanbari R, Tajabadi N, Abdul-Hamid A, Bakar FA, Saari N
    J Food Sci, 2016 Feb;81(2):C341-7.
    PMID: 26720491 DOI: 10.1111/1750-3841.13200
    Palm kernel cake protein was hydrolyzed with different proteases namely papain, bromelain, subtilisin, flavourzyme, trypsin, chymotrypsin, and pepsin to generate different protein hydrolysates. Peptide content and iron-chelating activity of each hydrolysate were evaluated using O-phthaldialdehyde-based spectrophotometric method and ferrozine-based colorimetric assay, respectively. The results revealed a positive correlation between peptide contents and iron-chelating activities of the protein hydrolysates. Protein hydrolysate generated by papain exhibited the highest peptide content of 10.5 mM and highest iron-chelating activity of 64.8% compared with the other hydrolysates. Profiling of the papain-generated hydrolysate by reverse phase high performance liquid chromatography fractionation indicated a direct association between peptide content and iron-chelating activity in most of the fractions. Further fractionation using isoelectric focusing also revealed that protein hydrolysate with basic and neutral isoelectric point (pI) had the highest iron-chelating activity, although a few fractions in the acidic range also exhibited good metal chelating potential. After identification and synthesis of papain-generated peptides, GGIF and YLLLK showed among the highest iron-chelating activities of 56% and 53%, whereas their IC50 were 1.4 and 0.2 μM, respectively.
    Matched MeSH terms: Trypsin/metabolism
  19. Fulltext Temporal regulation of proteome profile in the fruit fly, Drosophila melanogaster
    Subramanian P, Jayapalan JJ, Abdul-Rahman PS, Arumugam M, Hashim OH
    PeerJ, 2016;4:e2080.
    PMID: 27257555 DOI: 10.7717/peerj.2080
    Background. Diurnal rhythms of protein synthesis controlled by the biological clock underlie the rhythmic physiology in the fruit fly, Drosophila melanogaster. In this study, we conducted a proteome-wide investigation of rhythmic protein accumulation in D. melanogaster. Materials and Methods. Total protein collected from fly samples harvested at 4 h intervals over the 24 h period were subjected to two-dimensional gel electrophoresis, trypsin digestion and MS/MS analysis. Protein spots/clusters were identified with MASCOT search engine and Swiss-Prot database. Expression of proteins was documented as percentage of volume contribution using the Image Master 2D Platinum software. Results. A total of 124 protein spots/clusters were identified using MS/MS analysis. Significant variation in the expression of 88 proteins over the 24-h period was observed. A relatively higher number of proteins was upregulated during the night compared to the daytime. The complexity of temporal regulation of the D. melanogaster proteome was further reflected from functional annotations of the differently expressed proteins, with those that were upregulated at night being restricted to the heat shock proteins and proteins involved in metabolism, muscle activity, protein synthesis/folding/degradation and apoptosis, whilst those that were overexpressed in the daytime were apparently involved in metabolism, muscle activity, ion-channel/cellular transport, protein synthesis/folding/degradation, redox homeostasis, development and transcription. Conclusion. Our data suggests that a wide range of proteins synthesized by the fruit fly, D. melanogaster, is under the regulation of the biological clock.
    Matched MeSH terms: Trypsin
  20. Collagen binding activity of bacteria isolated from pig and cow small intestine
    Nur Fadhilah Khairil Mokhtar, Raha Abdul Rahim, Malia Mohd Hashim, Shuhaimi Mustafa
    Sains Malaysiana, 2016;45:411-416.
    Bacterial adherence to connective tissue, especially to collagen has been vastly known for their invasive and infectious activities. However, the ability to exploit the unique and specific interactions between bacteria and collagen as a novel approach in detection of placental collagen has never been explored. This study aimed to determine bacteria with binding specificity to placental collagen (Type IV) derived from human and sheep. In order to do this, total bacteria from small intestines of pig and cow were isolated and their ability to bind to Type IV placental collagen (human and sheep) was determined. Interestingly, three bacterial samples; P5, P9 (pig small intestine origin) and B7 (cow small intestine origin) were found to be able to bind strongly to the placental collagen. The bacterial binding to human placental collagen was however, diminished after the bacteria were treated with trypsin, proteinase K (for removal of surface protein) and guanidine hydrochloride (for S-layer removal), suggesting that the interaction of these bacteria to placental collagen was promoted by protein(s) present at the bacterial surface. In addition, significant reduction of placental collagen-binding ability of the bacteria pre-incubated with soluble human placental collagen showed that there is a specific interaction between the bacteria and collagen. P5, P9 and B7 bacteria were found to share 95-97% 16S rRNA sequence similarity to Enterococcus faecalis ZL, Enterococcus hirae ss33b and Enterococcus faecium M3-1, respectively. The results presented here may facilitate future studies in identifying bacterial surface protein(s) responsible for the specific binding of bacteria to collagen and opens new opportunity to utilize the protein(s) for the detection of placental collagen in nutraceutical and food supplements.
    Matched MeSH terms: Trypsin
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