Endo-xylogalacturonan hydrolase, a novel pectinolytic enzyme

We screened an Aspergillus tubingensis expression library constructed in th e yeast Kluyveromyces lactis for xylogalacturonan-hydrolyzing activity in m icron ell plates by using a bicinchoninic acid assay. This assay detects re ducing carbohydrate groups when they are released from a carbohydrate by en zymatic activity. Two K. lactis recombinants exhibiting xylogalacturonan-hy drolyzing activity were found among the 3,400 colonies tested. The cDNA ins ert of these recombinants encoded a 406-amino-acid protein, designated XghA which was encoded by a single-copy gene, xghA. A multiple-sequence alignme nt revealed that XghA was similar to both polygalacturonases (PGs) and rham nogalacturonases. A detailed examination of conserved regions in the sequen ces of these enzymes revealed that XghA resembled PGs more. High-performanc e liquid chromatography and matrix-assisted laser desorption ionization-tim e of flight mass spectrometry of the products of degradation of xylogalactu ronan and saponified modified hairy regions of apple pectin by XghA demonst rated that this enzyme uses an endo type of mechanism. XghA activity appear ed to be specific for a xylose-substituted galacturonic acid backbone.