Construction of a Tandem Repeat Peptide Sequence with Pepsin Cutting Sites to Produce Recombinant α-Melanocyte-Stimulating Hormone

The production of α-melanocyte-stimulating hormone (α-MSH), a peptide hormone composed of 13 amino acids, is attempted by recombinant expression using E. coli as the host. To achieve this aim, a synthetic gene containing eight tandem repeats of msh gene (8msh) was designed for ribosomal synthesis of 8 α-MSH. The merit of the strategy is to diminish the peptide toxicity against the host cell and to achieve a higher production yield. Pepsin cleavage sites are introduced between the peptides for enzymatic proteolysis to obtain the monomeric peptide of α-MSH. The constructed plasmid was transformed into different strains of E. coli hosts, and E. coli XL1-Blue with gene 8msh revealed the highest yield of 8 α-MSH. Although 8 α-MSH was fractionalized in the insoluble pellets after cell lysis, pepsin cleavage was able to produce soluble α-MSH peptide, as analyzed and confirmed by mass spectrometry and peptide activity assays. The production of α-MSH was quantified using HPLC with a yield of 42.9 mg/L of LB culture. This study demonstrates the feasibility of producing α-MSH using recombinant expression of tandem repeat gene. The production procedure involves minimal post-treatment and processing and can be scaled up for industrial application.

Sensopeptidomic Kinetic Approach Combined with Decision Trees and Random Forests to Study the Bitterness during Enzymatic Hydrolysis Kinetics of Micellar Caseins

Protein hydrolysates are, in general, mixtures of amino acids and small peptides able to supply the body with the constituent elements of proteins in a directly assimilable form. They are therefore characterised as products with high nutritional value. However, hydrolysed proteins display an unpleasant bitter taste and possible off-flavours which limit the field of their nutrition applications. The successful identification and characterisation of bitter protein hydrolysates and, more precisely, the peptides responsible for this unpleasant taste are essential for nutritional research. Due to the large number of peptides generated during hydrolysis, there is an urgent need to develop methods in order to rapidly characterise the bitterness of protein hydrolysates. In this article, two enzymatic hydrolysis kinetics of micellar milk caseins were performed for 9 h. For both kinetics, the optimal time to obtain a hydrolysate with appreciable organoleptic qualities is 5 h. Then, the influence of the presence or absence of peptides and their intensity over time compared to the different sensory characteristics of hydrolysates was studied using heat maps, random forests and regression trees. A total of 22 peptides formed during the enzymatic proteolysis of micellar caseins and influencing the bitterness the most were identified. These methods represent simple and efficient tools to identify the peptides susceptibly responsible for bitterness intensity and predict the main sensory feature of micellar casein enzymatic hydrolysates.

A comparative study of synthetic winged peptides for absolute protein quantification

A proper internal standard choice is critical for accurate, precise, and reproducible mass spectrometry-based proteomics assays. Synthetic isotopically labeled (SIL) proteins are currently considered the gold standard. However, they are costly and challenging to obtain. An alternative approach uses SIL peptides or SIL "winged" peptides extended at C- or/and N-terminus with an amino acid sequence or a tag cleaved during enzymatic proteolysis. However, a consensus on the design of a winged peptide for absolute quantification is missing. In this study, we used human serum albumin as a model system to compare the quantitative performance of reference SIL protein with four different designs of SIL winged peptides: (i) commercially available SIL peptides with a proprietary trypsin cleavable tag at C-terminus, (ii) SIL peptides extended with five amino acid residues at C-terminus, (iii) SIL peptides extended with three and (iv) with five amino acid residues at both C- and N-termini. Our results demonstrate properties of various SIL extended peptides designs, e.g., water solubility and efficiency of trypsin enzymatic cleavage with primary influence on quantitative performance. SIL winged peptides extended with three amino acids at both C- and N-termini demonstrated optimal quantitative performance, equivalent to the SIL protein.

Changes in functional and technological indicators of african ostrich meat under the action of vegetable enzymes in the composition of salting brine

The paper considers such tasks as the selection of a promising enzyme preparation and giving it characteristics; substantiation of rational doses of enzyme introduction into whole muscle products from ostrich meat; study of the influence of fermentation, salting and machining on the functional and technological properties of ostrich meat. On the basis of complex researches the expediency and practically proved expediency of use of the enzyme papain as a part of brines for injection molding at production of ham products from ostrich meat is scientifically proved and practically proved. The functional and technological properties of ham made from African ostrich meat made in Ukraine have been studied. The results of studies of the effect of enzymatic proteolysis of ostrich meat with papain on the technological parameters of raw materials are presented. The introduction of the optimal amount of brine in the meat, which allows to increase the yield and improve the totality of the finished product. Under the influence of papain, the meat acquires a tender soft texture and pleasant taste, due to the hydrolytic changes of proteins and the accumulation of their breakdown products, which are precursors of substances that subsequently give high flavoring properties to the finished product. The use of modern biotechnological methods that enhance technological processes and improve such quality indicators as tenderness, juiciness, taste and aroma, open up opportunities to expand the range and increase the production of ostrich meat products.

Assessment of Proteolysis by Pyrylium and Other Fluorogenic Reagents

Aims: We aim to evaluate the potential application of amine reactive fluorogenic reagents for estimating enzymatic proteolysis. Background: Proteolytic enzymes play important roles in regulating many physiological processes in living organisms. Objectives: Assessment of protein degradation by using reagents for protein assay techniques. Methods: We have assayed samples at the start and after 30-60 minutes incubation with trypsin by Chromeo P503 (Py 1 pyrylium compound) and CBQCA (3-(4-carboxybenzoyl) quinoline-2-carboxaldehyde) as amine reactive reagents and NanoOrange as non-amine reactive dye. Results: All BSA prepared samples with trypsin have shown significantly higher fluorescence intensity (FI) versus controls (which reflects proteolysis) when assayed by Chromeo P503 (Py 1 pyrylium compound) and CBQCA (3-(4-carboxybenzoyl) quinoline-2-carboxaldehyde) as amine reactive reagents. However, same samples assayed with NanoOrange as non-amine reactive reagent did not show any significant variation between samples containing trypsin and controls. Conclusion: These results are confirming reliability of highly sensitive protein assays utilizing amine reactive fluorogenic reagents for general estimation of proteolysis.

Anti-Hyperglycemic Effects of Green Crab Hydrolysates Derived by Commercially Available Enzymes

The predation and burrowing activity of invasive green crabs have had detrimental effects on important marine resources and habitats. Our objective is to develop bioactive hydrolysates by enzymatic proteolysis of underutilized green crab. Mechanically separated mince was hydrolyzed with Alcalase, Protamex, Flavourzyme, and Papain (1%) for 60 min. Subsequently, the hydrolysates were introduced to a simulated gastrointestinal digestion model. Selected samples were fractionated by ultrafiltration, and their anti-hyperglycemic effects including α-glucosidase, α-amylase, and dipeptidyl peptidase-IV (DPP-IV) inhibitory activities and glucagon-like 1 (GLP-1) secretory activity were evaluated. The Protamex treatment showed the highest α-glucosidase inhibitory activity (IC50 1.38 ± 0.19 mg/mL) compared to other enzyme treatments and the crab mince control, and its α-amylase inhibitory activity (IC50 11.02 ± 0.69 mg/mL) was lower than its α-glucosidase inhibitory activity. Its GLP-1 secretory activity was approximately four times higher than the positive control (10 mM glutamine). The <3 kD fraction contributed significantly to the anti-hyperglycemic activity of Protamex-derived hydrolysates, and this activity was stable after simulated digestion. Our results suggest that green crab hydrolysates obtained by Protamex treatment have the potential for type 2 diabetes management and could be incorporated in food products as a health-promoting ingredient.

Cerebrolysin – mechanism of action and application in psychiatry and neurology

Cerebrolysin is a medication, which has been used in psychiatry and neurology for over 50 years. The aim of this paper is to present the current state of knowledge on cerebrolysin. It is a non-lipid mixture of free L-amino acids and neuropeptides of low molecular weight that are purified via enzymatic proteolysis from brains of young pigs. It also contains magnesium, phosphorus, potassium and selenium. Cerebrolysin passes the blood-brain barrier. It was proved that it supports endogenous regeneration processes, mimics the behaviour of neurotrophic factors and modifies their level, affects the Sonic Hedgehog protein’s signalling pathway, protects from the pathological events and cascades which stem from an injury or a neurodegenerative disease, reduces amounts of free radicals as well as pro-apoptotic enzymes, modulates inflammatory response and also affects neuroplasticity and neurogenesis. Because of these characteristics, cerebrolysin is used in treatment of patients suffering from strokes, traumatic brain injuries, patients with cognitive disorders, including Alzheimer’s dementia and vascular dementia. The standard route of administration is via injections; however, some research was conducted on oral administration as well. The commonly reported adverse effects of cerebrolysin are transient and of mild severity. Results of research on the effectiveness and safety of cerebrolysin seem to be encouraging. They confirm high safety level and theoretical legitimacy of its use. The treatment of patients with organic, metabolic and neurodegenerative syndromes is difficult and often ineffective. Therefore, every available method of treatment needs to be taken under consideration.