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Constitutive expression, purification and characterisation of pectin methylesterase from Aspergillus niger in Pichia pastoris for potential application in the fruit juice industry

Authors


Qing Yang, Department of Bioscience and Biotechnology, Dalian University of Technology, No. 2 Linggong Road, Dalian 116024, China. E-mail: qingyang@dlut.edu.cn

Abstract

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.

RESULTS: PME from Aspergillus niger was constitutively expressed to a high level in the yeast Pichia pastoris. 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 Km, Vmax and kcat values of the enzyme with respect to pectin were 8.6 mmol L−1 [equation image], 1.376 mmol min−1 mg−1 and 8.26 × 102 s−1 respectively. Cations such as K+, Mg2+, Ni2+, Mn2+ and Co2+ slightly inhibited its activity, whereas Na+ had no effect.

CONCLUSION: PME from A. niger was constitutively expressed to a high level in P. pastoris 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

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