Kinetics and Thermodynamics of Thermal Inactivation of Recombinant E Coli-Lignocelluloses Cel8c, Cel12b, and Peh28, on Cellulose and Pectin: Implications for Lignocellulose Biomass Conversion

Eman Ibrahim¹, Kim D Jones², Keith E Taylor³, Ebtesam N Hosseney⁴, Patrick L Mills⁵,Jean Escudero⁶

^1,2Department of Environmental Engineering,
⁵Department of Chemical Engineering, Texas A&M University-Kingsville, Texas 78363, USA
³Department of Chemistry and Biochemistry, University of Windsor, Windsor, Canada N9B3P4
⁴Department of Botany and Microbiology, Al-Azhar University, Nasr City, Cairo 11884, Egypt
⁶Department of Basic Science, St. Louis College of Pharmacy, St. Louis, Missouri 63110-1088, USA

Abstract

Molecular and biochemical characterization of lignocelluloses cel12B, cel8C and peh28 from Pectobacterium carotovorum subsp. carotovorum (Pcc) expressed in E coli, were characterized in previous work (Ibrahim et al., 2013 and 2017). Cel12B and cel8C were assigned to glycoside-hydrolyase (GH) families 12 and 8, respectively, whereas peh28 was assigned to the GH-28 family. The peh28 sequence was examined for the presence of Arg96 a known processivity determinant in the closely homologous sequence of endo-polygalacturonase I from Aspergillus niger, while the presence of carbohydrate binding domain-II conferring known non-catalytic binding on crystalline-cellulose planar surfaces was found in the cel12B sequence. The industrial relevance of the galacturonic acid arising from peh28-catalyzed hydrolysis of pectin and the glucose and cellobiose formed by cel12B and cel8C, has been investigated. Synergies were observed between cel8C and cel12B on CMC but not Avicel. Kinetic and thermodynamic properties of thermal inactivation of cel12B, cel8C and peh28 at 37, 50 and 60°C were identified using the respective activities on CMC and polygalacturonic acid. Kinetic constants and half-lives of thermal inactivation (kd, t1/2), inactivation energy (Ed), plus inactivation entropies (ΔS), enthalpies (ΔH) and Gibbs free energies (ΔG), revealed high stability, less conformational change and/or protein unfolding characteristics of cel12B and peh28 due to thermal denaturation, compared to cel8C. The apparent thermal stability of peh28 and cel12B and the processivity of peh28, combined with their respective activity on pectin/polygalacturonic and Avicel/CMC, makes these two enzymes very good candidates for lignocellulosic biomass conversions.

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Eman Ibrahim, Kim D Jones, Keith E Taylor, Ebtesam N Hosseney, Patrick L Mills, Jean Escudero. Kinetics and Thermodynamics of Thermal Inactivation of Recombinant E Coli-Lignocelluloses Cel8c, Cel12b, and Peh28, on Cellulose and Pectin: Implications for Lignocellulose Biomass Conversion. International Conference on Technology and Environmental Science (2017): 17. Print.