β-hexosaminidases (Hex) are dimeric enzymes involved in the lysosomal degradation of glycolipids and glycans. They are formed by α- and/or β-subunits encoded by HEXA and HEXB genes, respectively. Mutations in these genes lead to Tay Sachs or Sandhoff diseases, which are neurodegenerative disorders caused by the accumulation of non-degraded glycolipids. Although tissue-derived Hex have been widely characterized, limited information is available for recombinant β-hexosaminidases. In this study, human lysosomal recombinant Hex (rhHex-A, rhHex-B, and rhHex-S) were produced in the methylotrophic yeast Pichia pastoris GS115. The highest specific enzyme activities were 13,124 for rhHexA; 12,779 for rhHex-B; and 14.606 U.mg-1 for rhHex-S. These results were 25- to 50-fold higher than those obtained from normal human leukocytes. Proteins were purified and characterized at different pH and temperature conditions. All proteins were stable at acidic pH, and at4 °C and 37 °C. At 45 °C rhHex-S was completely inactivated, while rhHex-A and rhHex-B showed high stability. This study demonstrates P. pastoris GS115 potential for polymeric lysosomal enzyme production, and describes the characterization of recombinant β-hexosaminidases produced within the same host.

• Autores: Espejo Mojica, Angela Johana; Institute for the Study of Inborn Errors of Metabolism, School of Sciences, Pontificia Universidad Javeriana. Bogotá, Colombia; Mosquera, Angela Rocío; Institute for the Study of Inborn Errors of Metabolism, School of Sciences, Pontificia Universidad Javeriana. Bogotá, Colombia; Rodríguez-López, Edwin Alexander; Institute for the Study of Inborn Errors of Metabolism, School of Sciences, Pontificia Universidad Javeriana. Bogotá, Colombia Chemistry Department, School of Science, Pontificia Universidad Javeriana, Bogotá, Colombia; Díaz, Dennis Johana; Institute for the Study of Inborn Errors of Metabolism, School of Sciences, Pontificia Universidad Javeriana. Bogotá, Colombia; Betrán, Laura Milena; Institute for the Study of Inborn Errors of Metabolism, School of Sciences, Pontificia Universidad Javeriana. Bogotá, Colombia; Hernández, Francy Liliana; Institute for the Study of Inborn Errors of Metabolism, School of Sciences, Pontificia Universidad Javeriana. Bogotá, Colombia; Alméciga Díaz, Carlos Javier; Institute for the Study of Inborn Errors of Metabolism, School of Sciences, Pontificia Universidad Javeriana. Bogotá, Colombia; Barrera, Luis Alejandro; Institute for the Study of Inborn Errors of Metabolism, School of Sciences, Pontificia Universidad Javeriana. Bogotá, Colombia Clínica de Errores Innatos del Metabolismo. Hospital Universitario San Ignacio. Bogotá, Colombia.
• Tipo de recurso: Article of journal
• Fecha de publicación: 2016
• Institución: Pontificia Universidad Javeriana
• Repositorio: Repositorio Universidad Javeriana
• Idioma: eng
• OAI Identifier: oai:repository.javeriana.edu.co:10554/31211
• Acceso en línea: http://revistas.javeriana.edu.co/index.php/scientarium/article/view/15736; http://hdl.handle.net/10554/31211
• Palabra clave: Biotechnology; β-N-acetylhexosaminidases; characterization; Pichia pastoris; recombinant hexosaminidases; Sandhoff disease; Tay Sachs disease.
• Rights: openAccess
• License: Atribución-NoComercial-SinDerivadas 4.0 Internacional