Abstract

ESPEJO-MOJICA, Angela J. et al. Characterization of recombinant human lysosomal beta-hexosaminidases produced in the methylotrophic yeast Pichia pastoris. Univ. Sci. [online]. 2016, vol.21, n.3, pp.195-217. ISSN 0122-7483.  https://doi.org/10.11144/Javeriana.SC21-3.corh.

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 α-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.

Keywords : β-N-acetylhexosaminidases; characterization; Pichia pastoris, recombinant hexosaminidases; Sandhoff disease; Tay Sachs disease..

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