Expression and Characterization of Hyperthermostable Exo-polygalacturonase TtGH28 from Thermotoga thermophilus

Abstract

D-galacturonic acid is a potential platform chemical comprising the principal component of pectin in the citrus processing waste stream. Several enzyme activities are required for the enzymatic production of galacturonic acid from pectin, including exo- and endo-polygalacturonases. The gene TtGH28 encoding a putative GH28 polygalacturonase from Pseudothermotoga thermarum DSM 5069 (Theth_0397, NCBI# AEH50492.1) was synthesized, expressed in Escherichia coli, and characterized. Alignment of the amino acid sequence of gene product TtGH28 with other GH28 proteins whose structures and details of their catalytic mechanism have been elucidated shows that three catalytic Asp residues and several other key active site residues are strictly conserved. Purified TtGH28 was dimeric and hyperthermostable, with K t (0.5) = 86.3 °C. Kinetic parameters for activity on digalacturonic acid, trigalacturonic acid, and polygalacturonic acid were obtained. No substrate inhibition was observed for polygalacturonate, while the K si values for the oligogalacturonides were in the low mM range, and K i for product galacturonic acid was in the low μM range. Kinetic modeling of the progress of reaction showed that the enzyme is both fully exo- and fully non-processional.

Synergistic action of thermophilic pectinases for pectin bioconversion into D-galacturonic acid

“…Error bars represent one standard deviation from the mean (n = 2). exhibited a Michaelis-Menten kinetics [27,29,38,39]. Additionally, we can see in Table 2 that exo-PGs from Thermotoga maritima presented the highest Vmax values.…”

Improved secretory expression and characterization of thermostable xylanase and β-xylosidase from Pseudothermotoga thermarum and their application in synergistic degradation of lignocellulose

“…The bacterium possesses a series of thermostable cellulosic degrading enzymes ( Dipasquale et al, 2018 ). Previously characterized enzymes from Pseudothermotoga thermarum exhibited excellent thermostability at high temperature (>80°C) ( Wagschal et al, 2016 ; Almeida et al, 2022 ). In the present work, thermostable xylanase and β -xylosidase genes from Pseudothermotoga thermarum were cloned and secretory expressed in B. subtilis , respectively.…”

Exploration of Two Pectate Lyases from Caldicellulosiruptor bescii Reveals that the CBM66 Module Has a Crucial Role in Pectic Biomass Degradation

“…A pectate lyase, PelS6, from the B. amyloliquefaciens S6 strain displayed highly thermostability and survival at a wide range of pHs, which could be suitable for juice clarity (8). To identify thermostable and highly active enzymes, the number of studies investigating thermophilic microorganisms such as those from the genera Thermotoga and Caldicellulosiruptor has been growing recently (9)(10)(11)(12)(13)(14).…”