Kinetic properties and substrate inhibition of α-galactosidase from Aspergillus niger

Abbreviations: BSA, bovine serum albumin; GH, glycoside hydrolase family; GM2, 6^I-α-d-galactosylmannobiose; GM3, 6^I-α-d-galactosylmannotriose; G2M5, 6^III,6^IV-α-d-galactosylmannopentaose; HPAEC-PAD, high-performance anion-exchange chromatography-pulsed amperometric detection; PNPG, p-nitrophenyl α-galactoside; rAglB, recombinant AglB.

Abstract

The recombinant AglB produced by Pichia pastoris exhibited substrate inhibition behavior for the hydrolysis of p-nitrophenyl α-galactoside, whereas it hydrolyzed the natural substrates, including galactomanno-oligosaccharides and raffinose family oligosaccharides, according to the Michaelian kinetics. These contrasting kinetic behaviors can be attributed to the difference in the dissociation constant of second substrate from the enzyme and/or to the ability of the leaving group of the substrates. The enzyme displays the grater k_cat/K_m values for hydrolysis of the branched α-galactoside in galactomanno-oligosaccharides than that of raffinose and stachyose. A sequence comparison suggested that AglB had a shallow active-site pocket, and it can allow to hydrolyze the branched α-galactosides, but not linear raffinose family oligosaccharides.

Graphical abstract

AglB exhibits substrate inhibition for the hydrolysis of PNPG (left). Substrate specificity of AglB (right).

Key words:

• glycoside hydrolase family 27
• α-galactosidase
• substrate inhibition
• substrate specificity

Acknowledgments

We thank the staff of the Instrumental Analysis Division of the Creative Research Institution at Hokkaido University for the amino acid analysis. The authors would like to thank Enago (www.enago.jp) for the English language review.

Notes

Abbreviations: BSA, bovine serum albumin; GH, glycoside hydrolase family; GM2, 6^I-α-d-galactosylmannobiose; GM3, 6^I-α-d-galactosylmannotriose; G2M5, 6^III,6^IV-α-d-galactosylmannopentaose; HPAEC-PAD, high-performance anion-exchange chromatography-pulsed amperometric detection; PNPG, p-nitrophenyl α-galactoside; rAglB, recombinant AglB.