Biochemical and kinetic characterization of the recombinant GH28 Stereum purpureum endopolygalacturonase and its biotechnological application

Author(s)

Carli, Sibeli ^[1] ; Meleiro, Luana Parras ^[1] ; Ward, Richard John ^[1]

Affiliation

^[1] Univ Sao Paulo, Dept Chem, Fac Filosofia Ciencias & Letras Ribeirao Preto, Ribeirao Preto, SP - Brazil

• Document type: Journal article
• Source: International Journal of Biological Macromolecules; v. 137, p. 469-474, SEP 15 2019.
• Web of Science Citations: 0

Abstract

Endopolygalacturonase (EndoPG) from Stereum purpureum was expressed as a soluble protein in Pichia pastoris GS115, where after 3 days methanol induction the enzyme activity in the culture supernatant was 40 U mL(-1). After purification by IMAC, SDS-PAGE analysis showed that the molecular weight of EndoPG was approximately 60.0 kDa. The carbohydrate content of the recombinant enzyme was estimated to be 67.0% (w/w). The optimum temperature and pH of catalysis were 60-70 degrees C and pH of 4.5, respectively. The enzyme was highly stable over the pH range 6.0-8.0 and retained approximately 60% of its initial activity after incubation at 70 degrees C for 30 min. The enzyme showed a specific activity of 5040.0 +/- 217 U mg(-1) and hydrolyzed citrus pectin with V-max and a K-M of 4947.10 +/- 393.63 U mg(-1) and 2.45 +/- 0.23 mg mL(-1), respectively, and showed a catalytic efficiency of 2052.90 +/- 193.54 mL mg(-1) s(-1). EndoPG alone reduced the viscosity of papaya juice by 20% after 30 min, and increased its transmittance about 50% with a concomitant reduction of the color by about 55% after 5 h of enzymatic treatment. For apple juice, the relative reduction of viscosity was 30% after 5 h, and the reduction of the color was 30% with a 12% increase in transmittance. Supplementation of a commercial enzymatic cocktail for lignocellulose saccharification with EndoPG increased total reducing sugar release by 8.6 +/- 2.1% against sugar cane bagasse, indicating improved access of the cellulolytic enzymes to the biomass polysaccharides. (C) 2019 Published by Elsevier B.V. (AU)

• FAPESP's process: 17/13734-3 - Engineering of an endopolygalacturonase by insertion of different Carbohydrate Binding Modules (CBMs): chimerogenesis and proximity effects
• Grantee: Sibeli de Carli
• Support Opportunities: Scholarships in Brazil - Doctorate
• FAPESP's process: 16/17582-0 - Thermostability modulation of a ß-glucosidase stimulated by glucose and xylose using site directed glycosylation
• Grantee: Luana Parras Meleiro Garcia
• Support Opportunities: Scholarships in Brazil - Post-Doctoral