Carangoides bartholomaei (Cuvier, 1833) stomach: a source of aspartic proteases for industrial and biotechnological applications / Estômago de Carangoides bartholomaei (Cuvier, 1833): uma fonte de proteases aspárticas para aplicações industriais e biotecnológicas

The viscera and other residues from fish processing are commonly discarded by the fishing industry. These by products can be a source of digestive enzymes with industrial and biotechnological potential. In this study, we aimed at the extraction, characterization, and application of acidic proteases from the stomach of Carangoides bartholomaei (Cuvier, 1833). A crude extract from the stomachs was obtained and submitted to a partial purification process by salting-out, which obtained a Purified Extract (PE) with a specific proteolytic activity of 54.0 U·mg-¹. A purification of 1.9 fold and a yield of 41% were obtained. The PE presents two isoforms of acidic proteases and a maximum proteolytic activity at 45 °C and pH 2.0. The PE acidic proteolytic activity was stable in the pH range of 1.5 to 7.0 and temperature from 25 °C to 50 °C. Purified Extract kept 35% of its proteolytic activity at the presence of NaCl 15% (m/v) but was totally inhibited by pepstatin A. Purified Extract aspartic proteases presented high activity in the presence of heavy metals such as Cd2+, Hg2+, Pb2+, Al3+, and Cu2+. The utilization of PE as an enzymatic addictive in the collagen extraction from Nile tilapia scales has doubled the process yield. The results indicate the potential of these aspartic proteases for industrial and biotechnological applications.

As vísceras e outros resíduos do processamento de peixes são geralmente descartados pela indústria pesqueira. Esses resíduos podem ser uma fonte de enzimas digestivas com potencial industrial e biotecnológico. Neste estudo, objetivamos a extração, caracterização e aplicação de proteases aspárticas do estômago de Carangoides bartholomaei (Cuvier, 1833). Um extrato bruto do estômago foi obtido e submetido a um processo de purificação parcial, que obteve um Extrato Purificado (EP) com uma atividade proteolítica específica de 54,0 U·mg-¹. Foi obtida uma purificação de 1,9 vezes e um rendimento de 41%. O EP apresenta duas isoformas de proteases ácidas e atividade proteolítica máxima a 45 °C e pH 2,0. A atividade proteolítica do EP foi estável na faixa de pH de 1,5 a 7,0 e temperatura de 25 °C a 50 °C. O EP manteve 35% de sua atividade proteolítica na presença de NaCl a 15% (m/v), mas foi totalmente inibida pela pepstatina A. As proteases ácidas do EP apresentaram alta atividade na presença de metais pesados como o Cd2+, Hg2+, Pb2+, Al3+ e Cu2+. A utilização de EP como aditivo enzimático na extração de colágeno a partir de escamas de tilápia do Nilo dobrou o rendimento do processo. Os resultados indicam um potencial dessas proteases para aplicações industriais e biotecnológicas.

Carangoides bartholomaei (Cuvier, 1833) stomach: a source of aspartic proteases for industrial and biotechnological applications / Estômago de Carangoides bartholomaei (Cuvier, 1833): uma fonte de proteases aspárticas para aplicações industriais e biotecnológicas

The viscera and other residues from fish processing are commonly discarded by the fishing industry. These by products can be a source of digestive enzymes with industrial and biotechnological potential. In this study, we aimed at the extraction, characterization, and application of acidic proteases from the stomach of Carangoides bartholomaei (Cuvier, 1833). A crude extract from the stomachs was obtained and submitted to a partial purification process by salting-out, which obtained a Purified Extract (PE) with a specific proteolytic activity of 54.0 U·mg-¹. A purification of 1.9 fold and a yield of 41% were obtained. The PE presents two isoforms of acidic proteases and a maximum proteolytic activity at 45 °C and pH 2.0. The PE acidic proteolytic activity was stable in the pH range of 1.5 to 7.0 and temperature from 25 °C to 50 °C. Purified Extract kept 35% of its proteolytic activity at the presence of NaCl 15% (m/v) but was totally inhibited by pepstatin A. Purified Extract aspartic proteases presented high activity in the presence of heavy metals such as Cd2+, Hg2+, Pb2+, Al3+, and Cu2+. The utilization of PE as an enzymatic addictive in the collagen extraction from Nile tilapia scales has doubled the process yield. The results indicate the potential of these aspartic proteases for industrial and biotechnological applications.(AU)

As vísceras e outros resíduos do processamento de peixes são geralmente descartados pela indústria pesqueira. Esses resíduos podem ser uma fonte de enzimas digestivas com potencial industrial e biotecnológico. Neste estudo, objetivamos a extração, caracterização e aplicação de proteases aspárticas do estômago de Carangoides bartholomaei (Cuvier, 1833). Um extrato bruto do estômago foi obtido e submetido a um processo de purificação parcial, que obteve um Extrato Purificado (EP) com uma atividade proteolítica específica de 54,0 U·mg-¹. Foi obtida uma purificação de 1,9 vezes e um rendimento de 41%. O EP apresenta duas isoformas de proteases ácidas e atividade proteolítica máxima a 45 °C e pH 2,0. A atividade proteolítica do EP foi estável na faixa de pH de 1,5 a 7,0 e temperatura de 25 °C a 50 °C. O EP manteve 35% de sua atividade proteolítica na presença de NaCl a 15% (m/v), mas foi totalmente inibida pela pepstatina A. As proteases ácidas do EP apresentaram alta atividade na presença de metais pesados como o Cd2+, Hg2+, Pb2+, Al3+ e Cu2+. A utilização de EP como aditivo enzimático na extração de colágeno a partir de escamas de tilápia do Nilo dobrou o rendimento do processo. Os resultados indicam um potencial dessas proteases para aplicações industriais e biotecnológicas.(AU)

Effects of Enzymatic Hydrolysis Conditions on the Antioxidant Activity of Red Tilapia (Oreochromis spp.) Viscera Hydrolysates.

BACKGROUND: Fish is an essential source of nutrients for human nutrition due to the composition of proteins, vitamins, and minerals, among other nutrients. Enzymatic hydrolysis represents an alternative for the use of by-products of the aquaculture industry. OBJECTIVE: We propose to evaluate the effect of stirring speed, temperature, and initial protein concentration on the degree of hydrolysis of proteins and antioxidant activity of red tilapia (Oreochromis spp.) viscera hydrolysates. METHODS: The effect of stirring speed, temperature, and initial protein concentration on the degree of hydrolysis of proteins and antioxidant activity was evaluated using an experimental design that was adjusted to a polynomial equation. The hydrolysate was fractioned to determine the antioxidant activity of the fractions, and functional properties were also measured. RESULTS: Stirring speed and protein concentration presented a statistically significant effect (p <0.05) on all the response variables. However, the temperature did not present a statistically significant effect on the degree of hydrolysis. DISCUSSION: The best conditions of hydrolysis were stirring speed of 51.44 rpm, a temperature of 59.15°C, and the protein concentration of 10 g L-1. The solubility of the hydrolysate protein was high at different pH, and the hydrolysate fraction with the highest antioxidant activity has a molecular weight <1 kDa. CONCLUSION: The degree of hydrolysis and the biological activity of red tilapia viscera hydrolysates (Oreochromis spp.) are affected by temperature, substrate concentration, and stirring speed. The optimal conditions of hydrolysis allowed to obtain a hydrolysate with antioxidant activity are due to the peptides with low molecular weight.

Purification and characterization of a protease from the visceral mass of Mytella charruana and its evaluation to obtain antimicrobial peptides.

This work describes purification of a protease from the visceral mass of the mussel Mytella charruana as well as evaluation of its ability to hydrolyze milk casein to generate antimicrobial peptides. The enzyme showed pI of 4.1 and a single polypeptide band of 83.1 kDa after SDS-PAGE. Sequence similarities with tropomyosin and myosin from mollusks were detected. The protease showed a trypsin-like activity with optimal temperature of 40 °C and stability in a wide pH range (3.0-9.0). Km was 4.28 ±â€¯0.34 mM of the synthetic substrate N-benzoyl-dl-arginyl-ρ-nitroanilide, whereas Vmax was 0.056 ±â€¯0.001 nmol min-1. The enzyme hydrolyzed casein, and the hydrolysate inhibited the growth of Escherichia coli, Micrococcus luteus, Bacillus subtilis, and Klebsiella pneumoniae at a minimal inhibitory concentration of 5.0 µg mL-1. In conclusion, the visceral mass of M. charruana contains a trypsin-like protease that can generate peptides from casein that have a bacteriostatic effect.

Trypsin from viscera of vermiculated sailfin catfish, Pterygoplichthys disjunctivus, Weber, 1991: its purification and characterization.

Pterygoplichthys disjunctivus viscera trypsin was purified by fractionation with ammonium sulphate, gel filtration, affinity and ion exchange chromatography (DEAE-Sepharose). Trypsin molecular weight was approximately 27.5kDa according to SDS-PAGE, shown a single band in zymography. It exhibited maximal activity at pH 9.5 and 40°C, using N-benzoyl-dl-arginine-p-nitroanilide (BAPNA) as substrate. Enzyme was effectively inhibited by phenyl methyl sulphonyl fluoride (PMSF) (100%), N-α-p-tosyl-l-lysine chloromethyl ketone (TLCK) (85.4%), benzamidine (80.2%), and soybean trypsin inhibitor (75.6%) and partially inhibited by N-tosyl-l-phenylalanine chloromethyl ketone (TPCK) (10.3%), ethylendiaminetetraacetic acid (EDTA) (8.7%) and pepstatin A (1.2%). Enzyme activity was slightly affected by metal ions (Fe(2+)>Hg(2+)>Mn(2+)>K(+)>Mg(2+)>Li(+)>Cu(2+)). Trypsin activity decreased continuously as NaCl concentration increased (0-30%). Km and kcat values were 0.13mM and 1.46s(-1), respectively. Results suggest the enzyme have a potential application where room processing temperatures (25-35°C) or high salt (30%) concentration are needed, such as in fish sauce production.

Cystyl aminopeptidase activity in the plasma, viscera and brain of the snake Bothrops jararaca.

The relationship between plasma osmolality and cystyl aminopeptidase was characterized in the snake Bothrops jararaca and comparisons were made with the emerging picture of this relationship in rats. The profile of cystyl aminopeptidase activity under basal conditions was determined in the soluble and membrane-bound forms in visceral organs and in the central nervous system in comparison with that of alanyl aminopeptidase. The regional localization of cystyl and alanyl aminopeptidase activities was studied in the central nervous system. The basal level of plasma cystyl aminopeptidase, four- to six-fold higher than in rats, suggests its importance to help regulate circulating levels of neurohypophysial peptides in B. jararaca snake. The osmotic sensitivity of this plasma enzyme, undetectable in male, but about three-fold higher in female snakes than in rats, reveals a sexual dimorphism. In marked contrast to those observed in rats, low levels of soluble and particulate forms in the kidney indicate that cystyl aminopeptidase plays a minor metabolizing role at this anatomical location in B. jararaca. Despite of the regional-specific divergence between the levels of rat and snake enzymes, the bilaterally symmetric pattern of the diencephalic distribution of alanyl aminopeptidase reflects functional homologies between these two distantly related species.

Functional role, cellular source, and tissue distribution of rat elastase-2, an angiotensin II-forming enzyme.

We recently described a chymostatin-sensitive elastase-2 as the major angiotensin (ANG) II-forming enzyme in the perfusate of the rat mesenteric arterial bed (MAB) with the same cDNA sequence as rat pancreatic elastase-2. The role of this enzyme in generating ANG II was examined in the rat isolated and perfused MAB. The vasoconstrictor effect elicited by ANG I and the renin substrate tetradecapeptide was only partially inhibited by captopril but abolished by the combination of captopril and chymostatin or N-acetyl-Ala-Ala-Pro-Leu-chloromethylketone (Ac-AAPL-CK; inhibitor originally developed for human elastase-2). The effect induced by [Pro11,d-Ala12]-ANG I, an ANG I-converting enzyme (ACE)-resistant biologically inactive precursor of ANG II, was blocked by chymostatin or Ac-AAPL-CK. It was also demonstrated that cultured rat mesenteric endothelial cells synthesize elastase-2 and that mRNA for this enzyme can be detected in different rat tissues such as the pancreas, MAB, lung, heart, kidney, liver, and spleen. In conclusion, the demonstration of a functional alternative pathway to ACE for ANG II generation in the rat MAB and the fact that cultured MAB endothelial cells are capable of producing and secreting elastase-2 represent strong evidence of a physiological role for this enzyme in the rat vasculature.

Aminopeptidases in visceral organs during alterations in body fluid volume and osmolality.

Enzymatic cleavage of some peptides in the local environment could be included among the mechanisms related to the regulation of hydrosaline balance. In order to examine this hypothesis, we measured representative aminopeptidase activities in visceral organs of rats after applying certain hydrosaline challenges. Decreased levels (about 30%) of particulate puromycin-insensitive-neutral aminopeptidase in the renal medulla and of soluble acid aminopeptidase in the lung were observed under hyperosmolality and hypovolemia. Decreased levels (more than 45%) of particulate type-I-pyroglutamyl aminopeptidase in the heart were observed under altered volemia. These results indicate that aminopeptidases at these anatomical locations might be involved in the regulation of body fluid volume and osmolality.