Expression and characterization of hyperthermostable exo-polygalacturonase RmGH28 from Rhodothermus marinus

Authors: Wagschal, Kurt; Stoller, Jeanette; Chan, Victor; Jordan, Douglas

Submitted to: Applied Biochemistry and Biotechnology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 5/19/2017
Publication Date: 5/29/2017
Citation: Wagschal, K.C., Stoller, J.R., Chan, V.J., Jordan, D.B. 2017. Expression and characterization of hyperthermostable exo-polygalacturonase RmGH28 from Rhodothermus marinus. Applied Biochemistry and Biotechnology. 183(4):1503-1515. https://doi.org/10.1007/s12010-017-2518-0.
DOI: https://doi.org/10.1007/s12010-017-2518-0

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 RmGH28, 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 93.9 degrees Celsius 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 RmGH28 from the organism Rhodothermus marinus putatively encoding a glycosyl hydrolase family 28 polygalacturonase was expressed in E. coli, and the enzyme purified and biochemically characterized. The gene was found to encode an exo- polygalacturonase, with galacturonic acid monomer and the polymer substrate (n-1) as the products released when acting on de-esterified polygalacturonic acid from citrus pectin. The enzyme at 25 degrees Celsius had kcat ~6 sec-1 when acting on polygalacturonic acid, with Km in the low 'M range and a substrate inhibition constant Ksi ~70 'M. The enzyme was hyperthermophilic, with ½ initial enzyme activity remaining after 1 hr incubation at 93.9 degrees Celcius. The amino acid sequence of RmGH28 is highly homologous to other known hyperthermophilic exo-polygalacturonases, which together can serve as starting points for structure-function studies and molecular breeding enzyme engineering approaches.