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ARS Home » Pacific West Area » Albany, California » Western Regional Research Center » Bioproducts Research » Research » Publications at this Location » Publication #326796

Research Project: Technologies for Improving Industrial Biorefineries that Produce Marketable Biobased Products

Location: Bioproducts Research

Title: Expression and characterization of hyperthermostable exo-polygalacturonase TtGH28 from Thermotoga thermophilus

item Wagschal, Kurt
item Stoller, Jeanette
item Chan, Victor
item Lee, Charles
item GRIGORESCU, ARABELA - Northwestern University
item Jordan, Douglas

Submitted to: Molecular Biotechnology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 5/5/2016
Publication Date: 5/21/2016
Citation: Wagschal, K.C., Stoller, J.R., Chan, V.J., Lee, C.C., Grigorescu, A.A., Jordan, D.B. 2016. Expression and characterization of hyperthermostable exo-polygalacturonase TtGH28 from Thermotoga thermophilus. Molecular Biotechnology. 58(7):509-519. doi: 10.1007/s12033-016-9948-8.

Interpretive Summary: Pectin rich biomass is an underutilized waste stream from the sugar and juice industry that can be converted to value added products. Pectin from citrus peels is mainly a polymer termed homogalacturonan that consists of esterified galacturonic acid, and once de-esterified and depolymerized, galacturonic acid can be converted to diacids and to ascorbic acid. One of the main enzymes responsible for the depolymerization of pectin is exo-polygalacturonase, which removes galacturonic acid one residue at a time from the chain. We describe here the biophysical and kinetic characterization of a hyper-thermostable polygalacturonase termed TtGH28, and these kinetic parameters can be used to model how fast the enzyme can depolymerize pectin. A great advantage of this enzyme is that it is hyper-thermostable, thus able to withstand temperatures of 86 'C for 1 hour and lose only ½ of the initial activity, indicating the enzyme would be stable for extended periods of time at elevated reactor temperatures, thereby increasing the rate of reaction, lowering the viscosity, and lowering the contamination risk, all of which potentially increase the economic viability of the process.

Technical Abstract: The gene TtGH28 encoding a putative GH28 polygalacturonase from Pseudothermotoga thermarum DSM 5069 (Theth_0397, NCBI# AEH50492.1) was synthesized, expressed in E. 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 Kt0.5 = 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 Ksi values for the oligogalacturonides were in the low mM range, and Ki for product galacturonic acid was in the low 'M range. Kinetic modeling of the progress of reaction showed that the enzyme is both fully exo- and fully non-processional.