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.


CONCLUSIONS
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.
Department of Bioscience and Biotechnology, Dalian University of Technology, No. 2 Linggong Road, Dalian 116024, China.
Xiuping
X
Jiang
Peng
P
Chen
Maolu
M
Yin
Qing
Q
Yang
D001234:Aspergillus niger / Q000201:enzymology*; D001628:Beverages / Q000032:analysis; D002265:Carboxylic Ester Hydrolases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D002412:Cations / Q000494:pharmacology; D005591:Chemical Fractionation; D002852:Chromatography, Ion Exchange; D004790:Enzyme Induction; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005511:Food Handling; D005638:Fruit / Q000737:chemistry; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis / Q000187:drug effects; D007700:Kinetics; D008745:Methylation / Q000187:drug effects; D008970:Molecular Weight; D010368:Pectins / Q000378:metabolism; D010843:Pichia / Q000201:enzymology / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
D002412:Cations; D004791:Enzyme Inhibitors; D005656:Fungal Proteins; D011994:Recombinant Proteins; D010368:Pectins; D002265:Carboxylic Ester Hydrolases; C022323:pectinesterase