Constitutive expression,purification and characterisation of pectin methylesterase from Aspergillus niger in Pichia pastoris for potential application in the fruit juice industry |
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Authors: | Xiuping Jiang Peng Chen Maolu Yin Qing Yang |
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Affiliation: | Department of Bioscience and Biotechnology, Dalian University of Technology, No. 2 Linggong Road, Dalian 116024, China |
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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 [ ], 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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Keywords: | pectin methylesterase constitutive expression purification Pichia pastoris fruit juice |
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