Extracción y caracterización de proteasas de pepino de mar Isostichopus fuscus recolectado en el Golfo de California, México

Extraction and characterization of sea cucumber Isostichopus fuscus proteases, collected at the Gulf of California, Mexico

A.C. Hernández-Sámano¹, X. Guzmán-García¹, R. García-Barrientos², F. Ascencio-Valle³, A. Sierra-Beltrán³, B. Vallejo-Córdoba⁴, A.F. González-Córdova⁴, M.J. Torres-Llanez⁴ y I. Guerrero-Legarreta¹*

¹ Universidad Autónoma Metropolitana, Unidad Iztapalapa. San Rafael Atlixco 186. C.P. 09340. México D.F., México. *Autora para la correspondencia. E-mail: isabel_guerrero_legarreta@yahoo.com

² Universidad Politécnica de Tlaxcala. Av. Universidad Politécnica 1. C.P. 90180. Tlaxcala, México.

⁴ Centro de Investigación en Alimentación y Desarrollo, A.C. Carretera a la Victoria Km. 0.6. C.P. 83304. Hermosillo, Sonora.

Recibido 9 de Julio de 2014
Aceptado 22 de Febrero de 2015

Resumen

Se estudiaron extractos enzimáticos del músculo ventral de Isostichopus fuscus. Se observó máxima actividad proteolítica a pH 2, 6 y 8, y 50-60°C. La mayor estabilidad enzimática fue a pH 2-6 (excepto pH 3), y a 0-40°C. Se detectó una actividad residual mayor a 74% después del calentamiento a 70°C y 80°C por 1 h, confirmándose la presencia de enzimas termo resistentes. Asimismo, los extractos retuvieron 59% de la actividad inicial después de almacenarse a 4-5°C por 21 días. Hg²⁺ aumentó la actividad proteolítica 16%, mientras Cu²⁺ y Mn²⁺ la inhibieron parcialmente, lo que sugirió la presencia de cisteín y metalo proteasas. El extracto enzimático se inhibió por pepstatina A, β-mercaptoetanol y EDTA, confirmando la predominancia de aspartil, cisteín y metalo proteasas. Por SDS-PAGE se identificaron cuatro proteínas (180.6, 114.5, 91.7 y 52.9 kDa) y estudios de zimografía corroboraron la presencia de proteasas. Después de purificar el extracto por intercambio aniónico se detectó una proteasa de 49 kDa, posiblemente metalo proteasa. Por espectrometría de masas se encontró homología con tripsina y con la cadena E del complejo inhibidor de triptasa (LDTI). Debido a que no se confirmó homología con ninguna metalo proteasa previamente descrita, se sugirió que esta proteasa es una enzima novedosa.

Palabras clave: pepino de mar, Isostichopus fuscus, músculo ventral, proteasas, caracterización enzimática.

Enzyme extracts obtained from I. fuscus ventral muscle were studied. Peak activity was observed at pH 2, 6, and 8, and 50-60°C, whereas maximum enzymatic stability occurred at pH 2 to 6 (except pH 3) and 0 to 40°C. More than 74% residual activity was retained after heating at 70°C and 80°C for 1 h, confirming the presence of heat-resistant enzymes. The extracts also retained 59% initial activity after 21-day storage at 4-5°C. Hg²⁺ increased the proteolytic activity in 16%, whereas Cu²⁺ and Mn²⁺ caused partial inhibition, suggesting the presence of cysteine- and metallo- proteases. Extracts were inhibited by pepstatin A, β-mercaptoethanol and EDTA, confirming the predominance of aspartyl-, cystein-, and metallo-, proteases. Four proteins were identified by SDS-PAGE (180.6, 114.5, 91.7 and 52.9 kDa). Zymograms on casein confirmed the presence of proteolytic enzymes. After purification by anion exchange chromatography a 49 kDa protease, possible metalloprotease, was detected. Mass spectrometry of the partially purified protein showed homology with trypsin and chain E, leech-derived tryptase inhibitor trypsin complex (LDTI). Due that no homology was found with any previously described metalloproteases, the presence of a novel enzyme is suggested.

Key words: sea cucumber, Isostichopus fuscus, ventral muscle, proteases, enzyme characterization.

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