Constitutive expression, purification and characterisation of pectin methylesterase from Aspergillus niger in Pichia pastoris for potential application in the fruit juice industry. (17th July 2012)

• Record Type: Journal Article
• Authors: Jiang, Xiuping; Chen, Peng; Yin, Maolu; Yang, Qing
• Is Part Of: Journal of the science of food and agriculture. Volume 93:Number 2(2013:Jan. 30)
• Page Start: 375
• Page End: 381
• Publication Date: 2012-07-17
• Subjects: Food -- Periodicals; Agriculture -- Periodicals; 664
• DOI: 10.1002/jsfa.5771
• Languages: English
• ISSNs: 0022-5142

Abstract

<abstract abstract-type="main" xml:lang="en"> <title>Abstract</title> <p> <bold>BACKGROUND: Pectin methylesterase (PME) catalyses the hydrolysis of the methyl ester of pectin, yielding free carboxyl groups and methanol. PME is widely used in the food, cosmetic and pharmaceutical industries.</bold> </p> <p> <bold>RESULTS: PME from <italic>Aspergillus niger</italic> was constitutively expressed to a high level in the yeast <italic>Pichia pastoris</italic>. The recombinant PME was purified by a combination of ammonium sulfate fractionation and ion exchange chromatography, giving an overall yield of 28.0%. It appeared as a single band in sodium dodecyl sulfate polyacrylamide gel electrophoresis, with a molecular mass of about 45 kDa. Optimal activity of the enzyme occurred at a temperature of 50 °C and a pH of 4.7. The <italic>K</italic><sub>m</sub>, <italic>V</italic><sub>max</sub> and <italic>k</italic><sub>cat</sub> values of the enzyme with respect to pectin were 8.6 mmol L<sup>−1</sup> [</bold> <inline-graphic xlink:href="ark:/27927/pgg1n93b6vb" mimetype="image" xlink:type="simple" xmlns:xlink="http://www.w3.org/1999/xlink" /> <bold>], 1.376 mmol min<sup>−1</sup> mg<sup>−1</sup> and 8.26 × 10<sup>2</sup> s<sup>−1</sup> respectively. Cations such as K<sup>+</sup>, Mg<sup>2+</sup>, Ni<sup>2+</sup>, Mn<sup>2+</sup> and Co<sup>2+</sup> slightly inhibited its activity, whereas Na<sup>+</sup> had no effect.</bold> </p> <p> <bold>CONCLUSION: PME from <italic>A. niger</italic> was constitutively expressed to a high level in <italic>P. pastoris</italic> without methanol induction. The recombinant PME was purified and characterised and shown to be a good candidate for potential application in the fruit juice industry. Copyright © 2012 Society of Chemical Industry</bold> </p> </abstract>