Enzyme immobilization by multipoint covalent attachment on supports activated with aliphatic aldehyde groups (e.g., glyoxyl agarose) has proven to be an excellent immobilization technique for enzyme ...stabilization. Borohydride reduction of immobilized enzymes is necessary to convert enzyme–support linkages into stable secondary amino groups and to convert the remaining aldehyde groups on the support into hydroxy groups. However, the use of borohydride can adversely affect the structure–activity of some immobilized enzymes. For this reason, 2-picoline borane is proposed here as an alternative milder reducing agent, especially, for those enzymes sensitive to borohydride reduction. The immobilization-stabilization parameters of five enzymes from different sources and nature (from monomeric to multimeric enzymes) were compared with those obtained by conventional methodology. The most interesting results were obtained for bacterial (R)-mandelate dehydrogenase (ManDH). Immobilized ManDH reduced with borohydride almost completely lost its catalytic activity (1.5% of expressed activity). In contrast, using 2-picoline borane and blocking the remaining aldehyde groups on the support with glycine allowed for a conjugate with a significant activity of 19.5%. This improved biocatalyst was 357-fold more stable than the soluble enzyme at 50 °C and pH 7. The results show that this alternative methodology can lead to more stable and active biocatalysts.
Limited chickpea protein hydrolysates ranging from 1% to 10% degree of hydrolysis were produced from chickpea protein isolate (CPI) using Alcalase immobilised on glyoxyl-agarose gels. ...Alcalase-glyoxyl derivative produced after 24
h of immobilisation at room temperature was 24 times more stable than soluble enzyme and presented approximately 51% of the activity of Alcalase. The chemical composition of chickpea hydrolysates were very close to that of CPI. Solubility, oil absorption, emulsifying activity and stability, and foaming capacity and stability were determined. All protein hydrolysates showed higher solubility than intact proteins, especially at pHs near isoelectric point of native chickpea proteins. Moreover, all hydrolysates had better functional properties, except emulsifying activity, than the original protein isolate.
Although cell-free systems and immortalized cell lines have been used to demonstrate the potential health benefits of lupine proteins and peptides, no study has examined the effects of lupine protein ...hydrolysates (LPHs) on the immune and oxidative responses of non-immortalized human cells. Therefore, the aims of this study were to evaluate the effects of the in vitro administration of LPHs from Lupinus angustifolius on the immunological and oxidative statuses of human peripheral blood mononuclear cells (PBMCs) from 53 healthy donors. LPHs reduced PBMCs proliferation and the levels of Th1, Th9 and Th17 pro-inflammatory cytokines without being cytotoxic. LPHs also skewed the pro-/anti-inflammatory balance towards a Th2 protective response. Additionally, LPHs increased superoxide dismutase and catalase activities, and the total antioxidant capacity (TAC). This study is the first to show that LPHs reduce T cell inflammatory responses and improve the anti-inflammatory/pro-inflammatory cytokine balance and the TAC by PBMCs. Thus, LPHs may represent an effective option for developing nutritional strategies to prevent pathologies with underlying inflammation and oxidative stress.
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•LPHs reduce human PBMCs proliferation.•Th1, Th9 and Th17 inflammatory responses are decreased in LPHs treated PBMCs.•LPHs skew the pro-/anti-inflammatory balance towards a protective phenotype.•The total antioxidant capacity is improved in LPHs-treated PBMCs.
•Lupine protein hydrolysates attenuate expression of proinflammatory cytokines.•Lupine protein hydrolysates decrease migration capability of macrophages.•Lupine hydrolysates may help to prevent ...diseases related to chronic inflammation.
The effect of two different lupine protein hydrolysates (LPHs) on in vitro macrophage activation in a THP-1-derived macrophage model was investigated. THP-1-derived macrophages were exposed to RPMI medium containing two LPHs obtained by enzymatic hydrolysis using two different proteases: Izyme AL and Alcalase 2.4 L. Cytokine's expression was measured by quantitative PCR. THP-1-derived macrophages exhibited attenuated expression of proinflammatory cytokines (tumor necrosis factor (TNF), IL-6, IL-1β) and increased expression of anti-inflammatory marker genes (chemokine (C-C motif) ligand 18 (CCL18)) relative to control without LPH. The anti-inflammatory effect of both hydrolysates favored M2 polarization by quenching C-C chemokine receptor type 2 (CCR2) expression and migratory capacity. Furthermore, LPHs significantly decreased nitric oxide production. Moreover, LPHs promoted the survival of human THP-1-derived macrophages. Therefore, inclusion of LPHs in foods may help to prevent chronic diseases associated with chronic inflammation.
The immobilization of trypsin, chymotrypsin and carboxypeptidase A using 4 and 10% glyoxyl-agarose beads at different times of incubation was investigated. Enzyme loadings of 30
mg/mL gel for trypsin ...and chymotrypsin, and 2
mg/mL gel for carboxypetidase A were used. Immobilization rates were very rapid in both supports and reactions were completed after 1
h of reaction. Final residual activities at these concentrations were around 60% for trypsin and chymotrypsin, and 50% for carboxypeptidase A. Comparison of the thermal stability of the soluble and immobilized enzymes revealed that immobilization by binding to 10% glyoxyl-agarose yielded the most stable enzymatic activities. Reaction with this support yielded immobilized trypsin, chymotrypsin, and carboxypeptidase A that were 4700, 10,000, and 1000 times more stable than the soluble enzymes, respectively. It was observed that the number of lysine residues that took part in the immobilization process was a consequence of the type of support and reaction time of the experimental conditions, and that the increasing of the thermal stability of the derivatives was correlated with a increasing number of lysines residues involved in a multipoint covalent attachment.
•Lupine protein isolate was hydrolysed using Alcalase 2.4L and/or Izyme AL.•Protein hydrolysates inhibited some enzymes involved in the inflammatory pathway.•Lupine protein hydrolysates may act as ...potential anti-inflammatory agents.
Lupine protein hydrolysates (LPHs) were obtained from a lupine protein isolate (LPI) by enzymatic hydrolysis using two proteases, Izyme AL and Alcalase 2.4L, and their potential anti-inflammatory capacities were studied by determining their in vitro inhibition of the following enzymes that are involved in the inflammatory process: phospholipase A2 (PLA2), cyclooxygenase 2 (COX-2), thrombin, and transglutaminase (TG). The strongest inhibitory activities toward PLA2 and TG were found in the hydrolysates obtained by hydrolysis with Izyme and subsequently with Alcalase, with more than 70% inhibition obtained in some cases. All of the hydrolysates tested inhibited more than 60% of the COX-2 activity. In no case did the percentage of thrombin activity inhibition exceed 40%. The best inhibitory activities were found in the LPH obtained after 15min of hydrolysis with Alcalase and in the LPH obtained after 60min of hydrolysis with Izyme followed by 15min of hydrolysis with Alcalase. Enzyme kinetic analyses were conducted to determine the Km and Vmax parameters of these two hydrolysates using the Lineweaver–Burk equation. Both hydrolysates competitively inhibited the thrombin and PLA2 activities. In the case of COX-2 and TG, the inhibition appeared to be the mixed type.
Divinyl sulfone (DVS) has been used to activate agarose beads. The DVS activated agarose resulted quite stable in the pH range 5-10 at 25 °C under wet conditions, and can react rapidly with α-amides ...of Cys and His, at pH 5-10, with Lys mainly at pH 10 and with Tyr in a much slower fashion. After blocking with different nucleophiles, the support lost all reactivity, confirming that this protocol could be useful as an enzyme-support reaction end point. Then, chymotrypsin was immobilized on this support at pH 5, 7 and 10. Even though the enzyme was immobilized at all pH values, the immobilization rate decreased with the pH value. The effect of the immobilization on the activity depended on the immobilization pH, at pH 7 the activity decreased (to 50%) more than at pH 10 (by a 25%), while at pH 5 the immobilization has no effect. Then, the effect of blocking with different reagents was analyzed. It was found that blocking with ethylenediamine improved the enzyme activity by 70% and gave the best stability. The stability of all enzyme preparations improved when 24 h incubation was performed at pH 10, but the qualitative stabilization depended on the inactivation conditions. The analysis of the amino acids of the preparation immobilized at pH 10 showed that Lys, Tyr and Cys residues were involved in the immobilization, involving a minimum of 10 residues (glyoxyl agarose gave 4 Lys involved in the immobilization). The new preparation was 4-5 fold more stable than glyoxyl agarose preparation, considered a very stable one, and in some instances was more active than the free enzyme (170% for the enzyme immobilized at pH 10). Thus, DVS activated supports are very promising to permit the multipoint covalent attachment of enzymes, and that way to improve their stability.
DVS supports are very suitable to stabilize enzymes
via
multipoint covalent attachment.
•A potential anti-inflammatory peptide was purified from lupine protein hydrolysate.•The sequence was Gly-Pro-Glu-Thr-Ala-Phe-Leu-Arg.•This octapeptide may help to prevent diseases related to chronic ...inflammation.
An anti-inflammatory peptide was identified in lupine protein hydrolysate (LPH) obtained by enzymatic hydrolysis of lupine protein isolate (LPI) with Alcalase. LPH was separated in two fractions by ultrafiltration, and anti-inflammatory activity was evaluated by analysis of the expression of different cytokines in THP-1-derived macrophages by quantitative PCR (qPCR). Fraction <10 kDa was purified by chromatographic techniques, and the most active subfraction was analysed by mass spectrometry. A novel anti-inflammatory peptide was identified and the sequence was Gly-Pro-Glu-Thr-Ala-Phe-Leu-Arg. This peptide was synthesised, and its anti-inflammatory activity was tested at two concentrations. Expression of TNF, IL-1β, and CCL2 was significantly reduced whereas the expression of the anti-inflammatory cytokine IL-10 was induced. Regarding the production of cytokines, only the lowest concentration of the synthetic peptide showed anti-inflammatory properties. Furthermore, the synthetic peptide decreased nitric oxide production. Thus, the identified peptide may help to prevent chronic inflammation associated with several diseases.
Due to its high protein synthesis and deposition rates, skeletal muscle protein deposition is a major determinant of fish growth. Dietary protein complexity is likely to influence protein utilization ...and deposition in skeletal muscle, possibly affecting fish myogenesis. In this study, three microdiets were formulated with different degree of hydrolysis of dietary protein as the changing factor: one diet contained a mix of intact protein sources targeting a peptide with molecular weight >20 kDa (Intact); a second diet contained a hydrolysate with polypeptides ranging from 5 to 70 kDa (PartH); and a third diet contained a high level of a protein hydrolysate mostly composed of small peptides (<5 kDa) (HighH). A possible effect on the regulation of muscle growth in Senegalese sole larvae was evaluated through white muscle cellularity and the expression of muscle growth-related genes at 16 and 36 DAH. The PartH diet promoted white muscle growth during the metamorphosis climax (16 DAH), which was reflected on increased body weight. At 36 DAH, different diets induced different expression patterns of genes encoding for the myogenic regulatory factors, which affected muscle growth dynamics, ultimately promoting growth potential in the Intact group. A lower recruitment of small-sized fibres in the PartH and HighH groups led to reduced potential for muscle growth, which resulted on further reduced somatic growth. Accordingly, fish fed the Intact diet grew better up to a late juvenile stage (60 DAH) and were still heavier than others even after 30 days of feeding all groups on the same commercial diet, at 90 DAH. The up-regulation in the transcript levels of genes encoding for de novo DNA methyltransferases in the HighH group suggest a potential for nutritional programming in this species.
•Moderately hydrolysed protein promotes muscle growth of pre-metamorphic sole larvae.•Dietary intact protein increased growth potential in sole post-larvae and juveniles.•Changes in dietary protein complexity affected myogenesis in sole larvae.•Changes in dietary protein complexity affected the expression of de novo DNMTs.
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