Controlled Enzymatic Hydrolysis: A New Strategy for the Discovery of Antimicrobial Peptides
publication date:2013 publication description: Probiotics and Antimicrobial Proteins
publication description: The use of antimicrobial peptides (AMPs) is an alternative to traditional antibiotics. AMPs are obtained using different methods such as bacterial synthesis, chemical synthesis and controlled enzymatic hydrolysis. The later is an interesting approach that deserves our attention because of the yields gathered and peptides engineered. Usually, activities of AMPs obtained in such a way are tightly dependent on the hydrolysis mechanism used. This paper deals with the hydrolysis of hemoglobin mechanism as a potential source of AMPs. Production of AMPs from hemoglobin using enzymatic controlled system is linked to hemoglobin structure. Further, we show that bovine hemoglobin, which is sensitive to peptic hydrolysis, results upon enzymatic digestion as a great source of AMPs. The hemoglobin in native and denatured states was hydrolyzed by “one-by-one” and “zipper” mechanisms, respectively. Nevertheless, a new mechanism named “semi-zipper” mechanism is obtained when protein is in molten globule structural state, constituting an original strategy for AMPs production. Seventy seven percentage of the peptides obtained by this new strategy showed antibacterial activity against nine strains
See publication Controlled Enzymatic Hydrolysis: A New Strategy for the Discovery of Antimicrobial PeptidesSee publication
publication title: Obtaining antimicrobial peptides by controlled peptic hydrolysis of bovine hemoglobin.
publication dateAug 1, 2011 publication descriptionInt J Biol Macromol.
publication descriptionUnder standard conditions, the peptides and specially the active peptides were obtained from either the denatured hemoglobin that all structures are completely modified or either the native hemoglobin where all structures are intact. In these conditions, antibacterial peptides were isolated from a very complex peptidic hydrolysate which contains more than one hundred peptides having various sizes and characteristics, involving a complex purification process. The new hydrolysis conditions were obtained by using 40% methanol, 30% ethanol, 20% propanol or 10% butanol. These conditions, where only the secondary structure of hemoglobin retains intact, were followed in order to enrich the hydrolyzed hemoglobin by active peptides or obtain new antibacterial peptides. In these controlled peptic hydrolysis of hemoglobin, a selective and restrictive hydrolysate contained only 29 peptides was obtained. 26 peptides have an antibacterial activity against Micrococcus luteus, Listeria innocua, and Escherichia coli with MIC from 187.1 to 1 μM. Among these peptides, 13 new antibacterial peptides are obtained only in these new hydrolysis conditions.
See publication Obtaining antimicrobial peptides by controlled peptic hydrolysis of bovine hemoglobin.See publication
publication title: α 67-106 of bovine hemoglobin: a new family of antimicrobial and angiotensin I-converting enzyme inhibitory peptides
publication dateApr 2011 publication descriptionEuropean Food Research and Technology
publication descriptionProtein hydrolysates are of a significant interest, due to their potential application as a source of bioactive peptides in nutraceutical and pharmaceutical domains. The present study was focused on bovine hemoglobin hydrolysate obtained with pig pepsin in the presence of 30% ethanol. This hydrolysate was fractioned by reversed-phase high-performance liquid chromatography (RP-HPLC) into 12 major fractions (F1–F12). All fractions were analyzed by ESI/MS and ESI/MS/MS, in order to characterize and identify the peptides in these fractions. This hydrolysis permitted to generate a new serial of bioactive peptides with both antimicrobial and ACE inhibitory activities. Identified peptides were TKAVEHLDDLPGALSELSDLHAHKLRVDPVNFKLLSHSLL, LDDLPGALSELSDLHAHKLRVDPVNFKLLSHSL, KLLSHSL, and LLSHSL corresponding respectively to the 67-106, 73-105, 99-105, and 100-105 fragments of the α chain of bovine hemoglobin. They were the first found from bovine hemoglobin. These purified peptides have an antibacterial activity against four bacteria strains: Kocuria luteus A270, Listeria innocua, Escherichia coli, and Staphylococcus aureus with a MIC between 187.1 and 35.2 μM. On the other hand, these peptides displayed at the same time ACE inhibitory activity with an IC50 range from 42.55 to 1,095 μM.
See publication α 67-106 of bovine hemoglobin: a new family of antimicrobial and angiotensin I-converting enzyme inhibitory peptidesSee publication
publication title: Evaluation of angiotensin I-converting enzyme (ACE) inhibitory activities of smooth hound (Mustelus mustelus) muscle protein hydrolysates generated by gastrointestinal proteases: identification of the most potent active peptide
publication date:May 2010 European Food Research and Technology
publication description: In this study, smooth hound protein hydrolysates (SHPHs), obtained by treatment with various gastrointestinal proteases, were analyzed for their angiotensin I-converting enzyme (ACE) inhibitory activities. Protein hydrolysates were obtained by treatment with crude alkaline enzyme extract, low molecular weight (LMW) alkaline protease, trypsin-like protease and pepsin from Mustelus mustelus, and bovine trypsin. All hydrolysates exhibited inhibitory activity toward ACE. Hydrolysate generated with alkaline protease extract displayed the highest ACE inhibitory activity, and the higher inhibition activity (82.6% at 2 mg/mL) was obtained with a hydrolysis degree of 18.8%. This hydrolysate was then fractionated by size exclusion chromatography on a Sephadex G-25 into five major fractions (P1–P5). ACE inhibitory activities of all fractions were assayed, and P3 was found to display a high ACE inhibitory activity (62.24% at 1 mg/mL). P3 was then fractionated by reversed-phase high-performance liquid chromatography (RP-HPLC) and ten fractions of ACE inhibitors were found (F1–F10). Sub-fraction F3 showed the strongest ACE inhibitory activity, being able to suppress more than 60% of initial enzyme activity at a concentration of 100 μg/mL. The amino acid sequence of peptide F3 was determined by ESI/MS and ESI–MS/MS as Ala-Gly-Ser, and the IC50 value for ACE inhibitory activity was 0.13 ± 0.03 mg/mL. Further, purified peptide F3 maintained inhibitory activity even after in vitro digestion with gastrointestinal proteases in order to demonstrate gastrointestinal stability digestion to enable oral application. These results indicate that smooth hound protein hydrolysate possesses potent antihypertensive activity.
See publication Evaluation of angiotensin I-converting enzyme (ACE) inhibitory activities of smooth hound (Mustelus mustelus) muscle protein hydrolysates generated by gastrointestinal proteases: identification of the most potent active peptideSee publication
publication title: Analysis of novel angiotensin I-converting enzyme inhibitory peptides from enzymatic hydrolysates of cuttlefish (Sepia officinalis) muscle proteins.
publication date:Mar 2010 publication description: J Agric Food Chem.
publication description: The angiotensin I-converting enzyme (ACE) inhibitory activities of protein hydrolysates prepared from cuttlefish (Sepia officinalis) proteins by treatment with various bacterial proteases were investigated. The hydrolysate generated by the crude enzyme from Bacillus mojavensis A21 displayed the highest ACE inhibitory activity, and the higher inhibition activity (87.11 +/- 0.92% at 2 mg/mL) was obtained with hydrolysis degree of 16%. This hydrolysate was fractionated by size exclusion chromatography on a Sephadex G-25 into eight major fractions (P(1)-P(8)). Fraction P(6), which exhibited the highest ACE inhibitory activity, was then fractionated by reversed-phase high performance liquid chromatography (RP-HPLC). Eleven ACE inhibitory peptides were isolated, and their molecular masses and amino acids sequences were determined using ESI-MS and ESI-MS/MS, respectively. The structures of the most potent peptides were identified as Ala-His-Ser-Tyr, Gly-Asp-Ala-Pro, Ala-Gly-Ser-Pro and Asp-Phe-Gly. The first peptide displayed the highest ACE inhibitory activity with an IC(50) of 11.6 microM. The results of this study suggest that cuttlefish protein hydrolysates are a good source of ACE inhibitory peptides.
