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Expression and Characterization of Hyperthermostable Exopolygalacturonase RmGH28 from Rhodothermus marinus

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Abstract

The gene RmGH28 from the organism Rhodothermus marinus, a putative glycosyl hydrolase family 28 polygalacturonase, was expressed in Escherichia coli and biochemically characterized. The gene was found to encode an exopolygalacturonase termed RmGH28, 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 °C had k cat ∼6 s−1 when acting on polygalacturonic acid, with K m ∼0.7 μM and a substrate inhibition constant K si ∼70 μM. The enzyme was hyperthermophilic, with one half initial enzyme activity remaining after 1-h incubation at 93.9 °C. Since the enzyme can function at high temperatures where reaction rates are increased and the risk of bacterial contamination is decreased, this indicates that RmGH28 can be useful in industry for generating galacturonic acid from pectin. The amino acid sequence of RmGH28 is highly homologous to the known hyperthermophilic exopolygalacturonases TtGH28 and Tm0437, which together can serve as starting points for structure-function studies and molecular breeding enzyme engineering approaches.

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Abbreviations

RmGH28:

Enzyme in this study

monoGalUA:

Galacturonic acid

diGalUA:

Digalacturonic acid

triGalUA:

Trigalacturonic acid

polyGalUA:

Polygalacturonic acid

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Acknowledgments

This work was supported by US Department of Agriculture (USDA) CRIS 3620-41000-118-00D (D.B.J. and J.R.S.) and USDA CRIS 5325-41000-049-00 (K.C.W. and V.J.C.). This research was also supported in part by an appointment to the Agricultural Research Service (ARS) Research Participation Program administered by the Oak Ridge Institute for Science and Education (ORISE) through an interagency agreement between the US Department of Energy (DOE) and the USDA (J.R.S.). ORISE is managed by Oak Ridge Associated Universities (ORAU) under DOE contract number DE-AC05-06OR23100. All opinions expressed in this paper are the authors’ and do not necessarily reflect the policies and views of USDA, ARS, DOE, or ORAU/ORISE. The mention of firm names or trade products does not imply that they are endorsed or recommended by the USDA over other firms or similar products not mentioned. USDA is an equal opportunity provider and employer.

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Authors and Affiliations

  1. USDA-ARS-WRRC, 800 Buchanan Street, Albany, CA, 94710, USA

    Kurt C. Wagschal & Victor J. Chan

  2. USDA-ARS-NCAUR, 1815 N University Street, Peoria, IL, 61604, USA

    J. Rose Stoller & Douglas B. Jordan

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  1. Kurt C. Wagschal
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Correspondence to Kurt C. Wagschal or Douglas B. Jordan.

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Wagschal, K.C., Rose Stoller, J., Chan, V.J. et al. Expression and Characterization of Hyperthermostable Exopolygalacturonase RmGH28 from Rhodothermus marinus . Appl Biochem Biotechnol 183, 1503–1515 (2017). https://doi.org/10.1007/s12010-017-2518-0

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