Vol. 21 No. 3 (2016), Biotechnology
Vol. 21 No. 3 (2016)

Characterization of recombinant human lysosomal beta-hexosaminidases produced in the methylotrophic yeast Pichia pastoris

Biotechnology
Published 2016-09-29
Angela J. Espejo Mojica
Institute for the Study of Inborn Errors of Metabolism, School of Sciences, Pontificia Universidad Javeriana. Bogotá, Colombia
Angela Mosquera
Institute for the Study of Inborn Errors of Metabolism, School of Sciences, Pontificia Universidad Javeriana. Bogotá, Colombia
Alexander Rodríguez-López
Institute for the Study of Inborn Errors of Metabolism, School of Sciences, Pontificia Universidad Javeriana. Bogotá, ColombiaChemistry Department, School of Science, Pontificia Universidad Javeriana, Bogotá, Colombia
Dennis Díaz
Institute for the Study of Inborn Errors of Metabolism, School of Sciences, Pontificia Universidad Javeriana. Bogotá, Colombia
Laura Beltrán
Institute for the Study of Inborn Errors of Metabolism, School of Sciences, Pontificia Universidad Javeriana. Bogotá, Colombia
Francy Liliana Hernández
Institute for the Study of Inborn Errors of Metabolism, School of Sciences, Pontificia Universidad Javeriana. Bogotá, Colombia
Carlos J. Alméciga Díaz
Institute for the Study of Inborn Errors of Metabolism, School of Sciences, Pontificia Universidad Javeriana. Bogotá, Colombia
Luis A. Barrera
Institute for the Study of Inborn Errors of Metabolism, School of Sciences, Pontificia Universidad Javeriana. Bogotá, ColombiaClínica de Errores Innatos del Metabolismo. Hospital Universitario San Ignacio. Bogotá, Colombia.
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Keywords

β-N-acetylhexosaminidases
characterization
Pichia pastoris
recombinant hexosaminidases
Sandhoff disease
Tay Sachs disease.

How to Cite

Characterization of recombinant human lysosomal beta-hexosaminidases produced in the methylotrophic yeast Pichia pastoris. (2016). Universitas Scientiarum, 21(3), 195-217. https://doi.org/10.11144/Javeriana.SC21-3.corh
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Abstract

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 at
4 °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.

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