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Int. J. Mol. Sci. 2014, 15(4), 5717-5729; doi:10.3390/ijms15045717
Article

Cloning, Expression and Characterization of a Novel Thermophilic Polygalacturonase from Caldicellulosiruptor bescii DSM 6725

1,2,†
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2,†
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 and
1,*
1 Key Laboratory for Molecular Enzymology & Engineering of the Ministry of Education, School of Life Science, Jilin University, Changchun 130012, China 2 School of Pharmaceutical Sciences, Jilin University, Changchun 130021, China These authors contributed equally to this work.
* Author to whom correspondence should be addressed.
Received: 10 February 2014 / Revised: 25 March 2014 / Accepted: 26 March 2014 / Published: 3 April 2014
(This article belongs to the Section Biochemistry, Molecular Biology and Biophysics)
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Abstract

We cloned the gene ACM61449 from anaerobic, thermophilic Caldicellulosiruptor bescii, and expressed it in Escherichia coli origami (DE3). After purification through thermal treatment and Ni-NTA agarose column extraction, we characterized the properties of the recombinant protein (CbPelA). The optimal temperature and pH of the protein were 72 °C and 5.2, respectively. CbPelA demonstrated high thermal-stability, with a half-life of 14 h at 70 °C. CbPelA also showed very high activity for polygalacturonic acid (PGA), and released monogalacturonic acid as its sole product. The Vmax and Km of CbPelA were 384.6 U·mg−1 and 0.31 mg·mL−1, respectively. CbPelA was also able to hydrolyze methylated pectin (48% and 10% relative activity on 20%–34% and 85% methylated pectin, respectively). The high thermo-activity and methylated pectin hydrolization activity of CbPelA suggest that it has potential applications in the food and textile industry.
Keywords: thermophilic polygalacturonase; CbPelA; exo-PGase; Caldicellulosiruptor bescii thermophilic polygalacturonase; CbPelA; exo-PGase; Caldicellulosiruptor bescii
This is an open access article distributed under the Creative Commons Attribution License (CC BY 3.0).

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Chen, Y.; Sun, D.; Zhou, Y.; Liu, L.; Han, W.; Zheng, B.; Wang, Z.; Zhang, Z. Cloning, Expression and Characterization of a Novel Thermophilic Polygalacturonase from Caldicellulosiruptor bescii DSM 6725. Int. J. Mol. Sci. 2014, 15, 5717-5729.

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