Obtainment and characterization of digestive aspartic proteases from the fish Caranx hippos (Linnaeus, 1766) / Obtenção e caracterização de proteases aspárticas digestórias do peixe Caranx hippos (Linnaeus, 1766)

This work aimed to obtain aspartic proteases of industrial and biotechnological interest from the stomach of the crevalle jack fish (Caranx hippos). In order to do so, a crude extract (CE) of the stomach was obtained and subjected to a partial purification by salting-out, which resulted in the enzyme extract (EE) obtainment. EE proteases were characterized physicochemically and by means of zymogram. In addition, the effect of chemical agents on their activity was also assessed. By means of salting-out it was possible to obtain a purification of 1.6 times with a yield of 49.4%. Two acid proteases present in the EE were observed in zymogram. The optimum temperature and thermal stability for EE acidic proteases were 55 ºC and 45 °C, respectively. The optimum pH and pH stability found for these enzymes were pH 1.5 and 7.0, respectively. Total inhibition of EE acid proteolytic activity was observed in the presence of pepstatin A. dithiothreitol (DTT) and Ca2+ did not promote a significant effect on enzyme activity. In the presence of heavy metals, such as Al3+, Cd2+ and Hg2+, EE acidic proteases showed more than 70% of their enzymatic activity. The results show that it is possible to obtain, from the stomach of C. hippos, aspartic proteases with high proteolytic activity and characteristics that demonstrate potential for industrial and biotechnological applications.

Este trabalho objetivou obter proteases aspárticas de interesse industrial e biotecnológico a partir do estômago do peixe xaréu (Caranx hippos). Para isso, foi obtido um extrato bruto do estômago, o qual foi submetido a uma purificação parcial por salting-out onde se obteve o extrato enzimático (EE). As proteases do EE foram caracterizadas físico-quimicamente e através de zimograma. Além disso, o efeito de agentes químicos sobre sua atividade também foi avaliado. Através de salting-out foi possível obter uma purificação de 1,6 vezes com rendimento de 49,4%. Foram observadas duas proteases ácidas presentes no EE através de zimograma. A temperatura ótima e a estabilidade térmica para as proteases ácidas do EE foram de 55 ºC e 45 °C, respectivamente. O pH ótimo e a estabilidade ao pH encontrados para estas enzimas foram o pH 1,5 e 7,0, respectivamente. Observou-se a inibição total da atividade proteolítica ácida do EE na presença de pepstatina A. O ditiotreitol (DTT) e o Ca2+ não promoveram efeito significativo na atividade enzimática. Na presença de metais pesados, como Al3+, Cd2+ e Hg2+, o EE manteve mais de 70% de atividade enzimática do EE. Os resultados mostram que é possível obter, a partir do estômago de C. hippos, proteases aspárticas com alta atividade proteolítica e características que demonstram potencial 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.

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.

Determination of aspartic protease gene dosage in the Onchocerca volvulus genome

Aspartic proteases are a relatively small group of enzymes which express in various nematodes including Onchocerca volvulus. An estimation of the gene copy number corresponding to the OV7A clone, which contains a cDNA insert encoding approximately two-thirds of the entire coding sequence of aspartic protease of O. volvulus, was made by slot blot analysis in a closely related species O. gibsoni genome. Nylon membrane was loaded with serial dilutions of genomic DNA alongside the OV7A plasmid DNA before hybridizing the membrane to that [32]P-labeled cDNA insert. To prepare the initial probe, OV7A cDNA insert was amplified using gene-specific primers. By comparing the signal intensity of slot blot hybridization of known amounts of genomic DNA and plasmid DNA containing the cDNA insert under similar conditions, the abundance of sequence homologues to the [32]P-labeled cDNA insert in the genome was calculated. For confirmation, southern blot analysis was performed by digesting genomic DNA with a panel of different restriction enzymes. Hybridizing patterns of the same probe revealed a single band except when predicted internal restriction sites were affected. It was confirmed that Onchocerca contains a single copy of the gene corresponding to this cDNA insert per haploid genome