Angiotensin I‐converting enzyme inhibitory and Ca‐binding activities of peptides prepared from tuna cooking juice and spleen proteases

Tuna cooking juice is a by‐product from the tuna canning industry. In this study, tuna cooking juice was hydrolysed by proteases extracted from the spleen. Tuna cooking juice showed the highest ACE inhibitory and Ca‐binding activities after hydrolysis for 270 and 180 min, respectively. The hydrolysate was further fractionated by ultrafiltration. The permeate exhibited highest ACE inhibitory and Ca‐binding activities when passed through 1 and 5 kDa cut‐off membranes, respectively. Gel filtration chromatography was used to determine the MW of bioactive peptides that exhibited highest ACE inhibitory and Ca‐binding activities. Those peptides that exhibited highest ACE inhibitory and Ca‐binding activities were the MW range of 238–829 Da and 1355–1880 Da, respectively. These results suggest that the tuna cooking juice and the spleen protease extract are a potential source of bioactive peptides that can be utilised as bioactive ingredients in functional food and nutraceuticals.     

Identification of Host Cellular Protein Substrates of SARS-COV-2 Main Protease

The novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of coronavirus disease-19 (COVID-19) being associated with severe pneumonia. Like with other viruses, the interaction of SARS-CoV-2 with host cell proteins is necessary for successful replication, and cleavage of cellular targets by the viral protease also may contribute to the pathogenesis, but knowledge about the human proteins that are processed by the main protease (3CLpro) of SARS-CoV-2 is still limited. We tested the prediction potentials of two different in silico methods for the identification of SARS-CoV-2 3CLpro cleavage sites in human proteins. Short stretches of homologous host-pathogen protein sequences (SSHHPS) that are present in SARS-CoV-2 polyprotein and human proteins were identified using BLAST analysis, and the NetCorona 1.0 webserver was used to successfully predict cleavage sites, although this method was primarily developed for SARS-CoV. Human C-terminal-binding protein 1 (CTBP1) was found to be cleaved in vitro by SARS-CoV-2 3CLpro, the existence of the cleavage site was proved experimentally by using a His₆-MBP-mEYFP recombinant substrate containing the predicted target sequence. Our results highlight both potentials and limitations of the tested algorithms. The identification of candidate host substrates of 3CLpro may help better develop an understanding of the molecular mechanisms behind the replication and pathogenesis of SARS-CoV-2.     

Facile optical resolution of amino acid esters via hydrolysis by an industrial enzyme in organic solvents

Racemic amino acid esters were optically resolved via hydrolysis in organic solvents by the catalysis of an industrial alkaline protease, “Alcalase”. The products which were composed mainly of L-amino acids were insoluble and easily separated by filtration. The activity of the enzyme and enantiomeric excess of the products were significantly dependent on the nature of solvent and the water content in the reaction media. Generally, high values of enantiomeric excess were obtained at low water contents. Many natural and unnatural amino acids were resolved by this method.     

Further characterisation of protease inhibitors from pigeon pea (cajanus cajan (l) millsp) seeds

Cajanus trypsin inhibitor (CTI) and Cajanus trypsin-chymotrypsin inhibitor (CTCI) previously purified from cv TAT-10 were further characterised. The modification of the inhibitors revealed the presence of lysine at the trypsin reactive site in both CTI and CTCI. Modification of tyrosine at the reactive site of CTCI did not abolish chymotrypsin inhibition suggesting the presence of leucine or phenylalanine as reported in other chymotrypsin inhibitors. CTCI did not contain tryptophan. Dissociation constants for the inhibitors with bovine trypsin were in the region of 0.69 nmol (CTCI) and 0.029 nmol (CTI). Although the protease inhibitors lost their inhibitory activity on exposure to 2-mercaptoethanol they remained attached to the enzyme. The inhibitors were not very effective against the protease from Helicoverpa armigera which is a serious field pest of Cajanus. Dissociation constants for the inhibitors with the larval enzyme were in the region of 100 nmol.     

Comparison among feature extraction methods for HIV-1 protease cleavage site prediction

Recently, several works have approached the HIV-1 protease specificity problem by applying a number of methods from the field of machine learning. However, it is still difficult for researchers to choose the best method due to the lack of an effective comparison. For the first time we have made an extensive study on methods for feature extraction for the problem of HIV-1 protease. We show that a fusion of classifiers trained in different feature spaces permits to obtain a drastically error reduction with respect to the performance of the state-of-the-art.     

Hydrolysis of human horny cells by alkaline protease: Morphological observation of the process

The interaction of highly purified alkaline protease fromBacillus sp. KSM-K16 with the horny cells of human skin contained in skin grime was directly visualized by electron microscopy. It became clear that the protease first penetrates the horny cells and then adsorbs, mainly onto the internal structure of the cells at the initial stage of hydrolysis, and directly hydrolyzes the keratin filaments, though the marginal band surrounding them retains its original shape. Then, hydrolysate produced from the keratin filaments flows out of the cell, and early in the hydrolysis process keratin filaments decrease and then disappear, leaving a marginal band, i.e., the cell turns to a hollow state. As a result, the remaining marginal band loses support from inside the cell, thus promoting cleavage and dispersion. Until this stage in the protease reaction, the remarkable liberation of hydrolysis products as water-soluble protein does not occur.     

Flaking and extrusion as mechanical treatments for enzyme-assisted aqueous extraction of oil from soybeans

Flaking and extruding dehulled soybeans were evaluated as a means of enhancing oil extraction efficiency during enzyme-assisted aqueous processing of soybeans. Cellulase, protease, and their combination were evaluated for effectiveness in achieving high oil extraction recovery from extruded flakes. Aqueous extraction of extruded full-fat soy flakes gave 68% recovery of the total available oil without using enzymes. A 0.5% wt/wt protease treatment after flaking and extruding dehulled soybeans increased oil extraction recovery to 88% of the total available oil. Flaking and extruding enhanced protease hydrolysis of proteins freeing more oil. Treating extruded flakes with cellulase, however, did not enhance oil extraction either alone or in combination with protease. Discrepancies in oil extraction recoveries were encountered when merely considering crude free fat because some oil became bound to denatured protein during extrusion and/or sample drying. Bound fat was unavailable for determination by using the hexane extraction method, but was accounted for by using the acid hydrolysis method for total oil determination. Oil extraction recovery from extruded soybean flakes was affected by oil determination methods, which was not the case for unextruded full-fat soy flour.     

Effects of amino acid replacements around the reactive site of chicken ovomucoid domain 3 on the inhibitory activity toward chymotrypsin and trypsin

We have previously shown that replacing the P1-site residue(Ala) of chicken ovomucoid domain 3 (OMCHI3) with a Met or Lysresults in the acquisition of inhibitory activity toward chymotrypsinor trypsin, respectively. However, the inhibitory activitiesthus induced are not strong. In the present study, we introducedadditional amino acid replacements around the reactive siteto try to make the P1-site mutants more effective inhibitorsof chymotrypsin or trypsin. The amino acid replacement AspTyrat the P2' site of OMCHI3(P1Met) resulted in conversion to a35000-fold more effective inhibitor of chymotrypsin with aninhibitor constant (K_i) of 1.17x10^–11 M. The K_i valueof OMCHI3(P1Met, P2'Ala) indicated that the effect on the interactionwith chymotrypsin of removing a negative charge from the P2'site was greater than that of introducing an aromatic ring.Similarly, enhanced inhibition of trypsin was observed whenthe AspTyr replacement was introduced into the P2' site of OMCHI3(P1Lys).Two additional replacements, AspAla at the P4 site and ArgAlaat the P3' site, made the mutant a more effective inhibitorof trypsin with a K_i value of 1.44x10^–9 M. By contrast,ArgAla replacement at the P3' site of OMCHI3(P1Met, P2'Tyr)resulted in a greatly reduced inhibition of chymotrypsin, andAspAla replacement at the P4 site produced only a small changewhen compared with a natural variant of OMCHI3. These resultsclearly indicate that not only the P1-site residue but alsothe characteristics, particularly the electrostatic properties,of the amino acid residues around the reactive site of the proteaseinhibitor determine the strength of its interactions with proteases.Furthermore, amino acids with different characteristics arerequired around the reactive site for strong inhibition of chymotrypsinand trypsin.     

Free energy perturbation studies on binding of A-74704 and its diester analog to HIV-1 protease

Free energy simulations have been employed to rationalize thebinding differences between A-74704, a pseudo C₂- symmetricinhibitor of HIV-1 protease and its diester analog. The diesteranalog inhibitor, which misses two hydrogen bonds with the enzymeactive site, is surprisingly only 10-fold weaker. The calculatedfree energy difference of 1.7 ± 0.6 kcal/mol is in agreementwith the experimental result. Further, the simulations showthat such a small difference in binding free energies is dueto (1) weaker hydrogen bond interactions between the two (P₁and P₁) NH groups of A-74704 with Gly27/Gly27' carbonyls ofthe enzyme and (2) the higher desolvation free energy of A-74704compared with its ester analog. The results of these calculationsand their implications for design of HIV-1 protease inhibitorsare discussed.