Meat Waste Valorization through Protein Hydrolysis using Different Types of Proteases

Five different commercial proteases (Alcalase 2.4L, Flavourzyme 1000L, Neutrase, Protamex, and PTN) were evaluated for the simultaneous recovery of protein and lipids through hydrolysis. The hydrolysis reaction was monitored using the pH-stat procedure, in which samples were collected after 240 min of hydrolysis using each enzyme. The samples were analyzed for the degree of hydrolysis, protein hydrolysate, collagen, lipids, and fatty acids. A clear relationship was observed between the degree of hydrolysis and the amounts of recovered products. Serine endopeptidases from the microbial source (Alcalase) resulted in the maximum degree of hydrolysis (27.5%), lipid recovery (82.6%), and protein hydrolysates quality (average molecular weight of the hydrolysates = 472 Dalton), followed by formulations of serine protease and metalloprotease from the microbial source (Protamex). Metalloproteases from the microbial source (Neutrase) resulted in maximum collagen recovery (87.1%). Serine endopeptidases from the animal source (PTN) and endo/exopeptidases from the fungal source (Flavourzyme) exhibited an intermediate efficacy between Alcalase and Neutrase. In the case of all proteases, the product fatty acid profile matched well with that of the meat waste, which suggests that no chemical changes occurred in the lipids after the hydrolysis with the evaluated proteases. These results suggest that hydrolysis using proteases could serve as an ecofriendly and viable alternative for obtaining additional value from meat waste.

Aminobenzosuberone Scaffold as a Modular Chemical Tool for the Inhibition of Therapeutically Relevant M1 Aminopeptidases

The synthesis of racemic substituted 7-amino-5,7,8,9-tetrahydrobenzocyclohepten-6-one hydrochlorides was optimized to enhance reproducibility and increase the overall yield. In order to investigate their specificity, series of enzyme inhibition assays were carried out against a diversity of proteases, covering representative members of aspartic, cysteine, metallo and serine endopeptidases and including eight members of the monometallic M1 family of aminopeptidases as well as two members of the bimetallic M17 and M28 aminopeptidase families. This aminobenzosuberone scaffold indeed demonstrated selective inhibition of M1 aminopeptidases to the exclusion of other tested protease families; it was particularly potent against mammalian APN and its bacterial/parasitic orthologues EcPepN and PfAM1.

mRNA overexpression of kallikrein-related peptidase 14 (KLK14) is an independent predictor of poor overall survival in chronic lymphocytic leukemia patients

Tissue kallikrein and kallikrein-related peptidases (KLKs) compose a family of serine endopeptidases with much clinical interest in oncology, as their potential as diagnostic and/or prognostic molecular biomarkers in several human malignancies has already been evidenced. However, none of the members of this family has ever been studied in hematological malignancies. Based on our preliminary results regarding the differential mRNA expression of severalTotal RNA was isolated from 69 CLL patients and 31 non-leukemic blood donors. After reverse transcription of poly(A)-RNA,According to ROC analysis,

Abstract 1456: Proteomic Analysis Of Proteins Bound By D-4F In Plasma From C57bl/6 And ApoA-I KO Mice

D-4F, an 18 amino acid apolipoprotein A-I (ApoA-I) mimetic, has been shown to be highly effective at improving vascular function in murine models of atherosclerosis. It has been hypothesized that D-4F inhibits atherosclerosis and increases vasodilation by interacting with ApoA-I to improve HDL function. However, the mechanisms by which D-4F protects vascular function remain unclear. We hypothesize that D-4F binds proteins involved in atherosclerosis. To test this hypothesis, we incubated HA-tagged D-4F with plasma from C57bl/6 and ApoA-I knockout mice (ApoA-I KO) on a C57bl/6 background and identified the proteins that were bound. Plasma from C57bl/6 mice or ApoA-I KO mice (n=6/group) was incubated with HA-GG-D-4F (Ac-YPYDVPDYA-GG-DWFKAFYDKVAEKFKEAF-NH2) or HA-GG-scramble-D-4F (Ac-YPYDVPDYA-GG-DWFAKDYFKKAFVEEFAK-NH2) at 10μg/ml. Next an anti-HA antibody was used to immunoprecipitate the HA-tagged peptides and associated proteins. The proteins were then analyzed by ESI-nano-LC/MS/MS. Analysis revealed that D-4F bound several proteins involved in lipid metabolism (ApoA-I, -II, -IV, ApoE, ApoC-I, -II and -III, -IV); complement regulatory protein (Complement C3); several kinases (PKC and Serine/threonine-protein kinase); and Serpins (Serine protease inhibitor 1 and A3K) which inhibit serine endopeptidases in C57bl/6 mice plasma. Although ApoA-I KO mice do not express ApoA-I, D-4F bound several lipoproteins associated with lipid metabolism (ApoE, ApoA-II, -IV, and ApoC-II, -III, -IV) in plasma from ApoA-I KO mice. D-4F also associated with the complement regulatory protein, Complement C3 in plasma of ApoA-I KO mice. Interestingly, D-4F bound serum paraoxonase and bradykinin only in the plasma of ApoA-I KO mice. These data show that D-4F associates, either directly or indirectly through lipoproteins, with a unique array of proteins involved in lipid metabolism; and second; that D-4F’s ability to associate with Complement C3, serum paraoxonase, bradykinin and Serpins suggest that D-4F may be able to modify the atherogenic properties of proteins involved in inflammation.

A Novel Fibrinogen-clotting Enzyme, TL-BJ, from the Venom of the Snake Bothrops jararaca: Purification and Characterization

SummaryThree chromatographically distinct forms of a novel fibrinogenclotting serine endopeptidase, TL-BJ 1, 2 and 3, were purified from the venom of Bothrops jararaca by a combination of ammonium sulfate precipitation and chromatographic steps. The three forms of TL-BJ have similar amidolytic and plasma coagulating activities. TL-BJ 1, TL-BJ 2 and TL-BJ 3 cause the specific clotting of fibrinogen with release of fibrinopeptide A, the specific activities are 16.8 NIH U/mg (TL-BJ 1), 16.7 NIH U/mg (TL-BJ 2) and 20.8 NIH U/mg (TL-BJ 3). The most sensitive chromogenic substrates for measuring the amidolytic activity of TL-BJ 3 were D-Pro-Phe-Arg-pNA, D-Phe-pipecolyl-ArgpNA and Z-D-Arg-Gly-Arg-pNA. The amidolytic and coagulant activities of TL-BJ were inhibited by phenylmethylsulfonyl fluoride but not by hirudin. Benzamidine derivatives, which are competitive inhibitors of trypsin-like serine endopeptidases, also inhibited the amidolytic activity of TL-BJ. In SDS/PAGE the main bands of TL-BJ 1, 2 and 3 showed molecular masses of 30 kDa, 31 kDa and 32 kDa. Upon incubation with N-glycosidase F only TL-BJ 3 remained unchanged, whereas TL-BJ 1 and TL-BJ 2 showed products with molecular masses around 23 kDa. Thus, TL-BJ 3 does not seem to be N-glycosylated. The N-terminal amino acid sequences of TL-BJ 2 and TL-BJ 3 are identical while TL-BJ 1 has five substitutions.

The yeast aminopeptidase Y

A metal-dependent aminopeptidase (EC 3.4.11.-), designated APase Y, has been purified to homogeneity by conventional methods. The enzyme is composed of a single polypeptide chain with molecular mass of 102 kilodaltons, estimated by sodium dodecyl sulphate – polyacrylamide gel electrophoresis, with a blocked N-terminal amino acid. It possesses neither endopeptidase nor carboxypeptidase activity and is strongly inhibited by metal-chelating agents, Zn2+, and the protein inhibitor from Neurospora crassa. APase Y is insensitive to Cl anions, S—S reducing reagents, serine protease inhibitors, and the peptidase inhibitor benzamidine. Co2+, Hg2+, and p-chloromercuribenzoate can activate the enzyme up to 22, 20, and 55%, respectively. The holoenzyme is resistant to yeast endopeptidases A, B, and Y, whereas the apoenzyme (obtained after treatment with chelators) is susceptible to the serine endopeptidases B and Y. The enzyme catalyzes hydrolysis of most L peptides possessing free α-amino (or imino) group by stepwise removal of N-terminal residue. Peptides with L-leucine at the N terminus are cleaved preferentially. The enzyme is unable to catalyze hydrolysis of X—Pro type peptide bonds, and inefficiently hydrolyzes bonds between Asp—X and Glu—X. L-leucine p-nitroanilide hydrolyzes optimally at pH 8.2 with a Km value of 1 mM. The purified enzyme is stable during storage in 0.05 M phosphate buffer, pH 6.7, containing 40–50% glycerol, at −20 °C.