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

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 K m , V max and k cat values of the enzyme with respect to pectin were 8.6 mmol L −1 [ ], 1.376 mmol min −1 mg −1 and 8.26 × 10 2 s −1 respectively. Cations such as K + , Mg 2+ , Ni 2+ , Mn 2+ and Co 2+ 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