Expression and Characterization of Hyperthermostable Exopolygalacturonase RmGH28 from Rhodothermus marinus

• Published in: Applied biochemistry and biotechnology 2017-12, Vol.183 (4), p.1503-1515
• Main Authors: Wagschal, Kurt C., Rose Stoller, J., Chan, Victor J., Jordan, Douglas B.
• Format: Article
• Language: English
• Subjects: Amino acid sequence; Amino acids; Bacteria; Bacterial Proteins - biosynthesis; Bacterial Proteins - chemistry; Bacterial Proteins - genetics; Biochemistry; Biotechnology; Chemistry; Chemistry and Materials Science; Contamination; E coli; Enzymatic activity; Enzyme activity; Enzyme Stability; Enzymes; Escherichia coli; Esterification; Gene Expression; Genes; Glycoside Hydrolases - biosynthesis; Glycoside Hydrolases - chemistry; Glycoside Hydrolases - genetics; Glycosyl hydrolase; High temperature; Homology; Hot Temperature; Molecular structure; Organisms; Pectin; Polygalacturonase; Polygalacturonic acid; Polymers; Recombinant Proteins - biosynthesis; Recombinant Proteins - chemistry; Recombinant Proteins - genetics; Rhodothermus - enzymology; Rhodothermus - genetics; Rhodothermus marinus; Structure-function relationships; Substrate inhibition
• ISSN: 0273-2289; 1559-0291
• DOI: 10.1007/s12010-017-2518-0

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.