Xylose isomerase from Piromyces sp. E2 is a promiscuous enzyme with epimerase activity

Author(s)

Barreto, Matheus Quintana ; Garbelotti, Carolina Victal ; Soares, Jessica de Moura ; Grandis, Adriana ; Buckeridge, Marcos Silveira ; Leone, Francisco Assis ; Ward, Richard John

• Document type: Journal article
• Source: Enzyme and Microbial Technology; v. 166, p. 9-pg., 2023-03-24.

Abstract

Xylose isomerase catalyzes the isomerization of D-xylose to D-xylulose with promiscuous activity for other saccharides including D-glucose, D-allose, and L-arabinose. The xylose isomerase from the fungus Piromyces sp. E2 (PirE2_XI) is used to engineer xylose usage by the fermenting yeast Saccharomyces cerevisiae, but its biochemical characterization is poorly understood with divergent catalytic parameters reported. We have measured the kinetic parameters of the PirE2_XI and analyzed its thermostability and pH-dependence towards different substrates. The PirE2_XI shows promiscuous activity towards D-xylose, D-glucose, D-ribose and Larabinose with variable effects depending on different divalent ions and epimerizes D-xylose at C3 to produce Dribulose in a substrate/product dependent ratio. The enzyme follows Michaelis-Menten kinetics for the substrates used and although KM values for D-xylose are comparable at 30 and 60 degrees C, the kcat/KM is three-fold greater at 60 degrees C. The purified PirE2_XI shows maximal activity at 65 degrees C in the pH range of 6.5-7.5 and is a thermostable enzyme, maintaining full activity over 48 h at 30 degrees C or 12 h at 60 degrees C. This is the first report demonstrating epimerase activity of the PirE2_XI and its ability to isomerize D-ribose and L-arabinose, and provides a comprehensive in vitro study of substrate specificity, effect of metal ions and temperature on enzyme activity and these findings advance the knowledge of the mechanism of action of this enzyme. (AU)

• FAPESP's process: 16/24139-6 - Design and Characterization of Multifunctional Xylanolytic Enzymes
• Grantee: Richard John Ward
• Support Opportunities: Regular Research Grants
• FAPESP's process: 14/50884-5 - INCT 2014: National Institute of Science and Technology of Bioethanol
• Grantee: Marcos Silveira Buckeridge
• Support Opportunities: Research Projects - Thematic Grants
• FAPESP's process: 18/25664-2 - Expression and purification of recombinants xylanolitics enzymes and study of their activities against sugar cane extracted arabinoxylans
• Grantee: Carolina Victal Garbelotti
• Support Opportunities: Scholarships in Brazil - Doctorate (Direct)