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 [Formula: See Text], 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.