Endo-xylogalacturonan hydrolase, a novel pectinolytic enzyme.
We screened an Aspergillus tubingensis expression library constructed in the yeast Kluyveromyces lactis for xylogalacturonan-hydrolyzing activity in microwell plates by using a bicinchoninic acid assay. This assay detects reducing carbohydrate groups when they are released from a carbohydrate by enzymatic activity. Two K. lactis recombinants exhibiting xylogalacturonan-hydrolyzing activity were found among the 3,400 colonies tested. The cDNA insert of these recombinants encoded a 406-amino-acid protein, designated XghA, which was encoded by a single-copy gene, xghA. A multiple-sequence alignment revealed that XghA was similar to both polygalacturonases (PGs) and rhamnogalacturonases. A detailed examination of conserved regions in the sequences of these enzymes revealed that XghA resembled PGs more. High-performance liquid chromatography and matrix-assisted laser desorption ionization-time of flight mass spectrometry of the products of degradation of xylogalacturonan and saponified modified hairy regions of apple pectin by XghA demonstrated that this enzyme uses an endo type of mechanism. XghA activity appeared to be specific for a xylose-substituted galacturonic acid backbone.
van der Vlugt-Bergmans
C J
CJ
Industrial Microbiology Group, Department of Food Technology and Nutritional Sciences, Wageningen Agricultural University, NL-6700 EV Wageningen, The Netherlands. cecile.vandervlugt@imb.ftns.wau.nl
Meeuwsen
P J
PJ
Voragen
A G
AG
van Ooyen
A J
AJ
D000595:Amino Acid Sequence; D001230:Aspergillus / Q000201:enzymology* / Q000235:genetics; D001483:Base Sequence; D015139:Blotting, Southern; D002851:Chromatography, High Pressure Liquid; D018076:DNA, Complementary / Q000235:genetics; D005656:Fungal Proteins; D015870:Gene Expression; D015723:Gene Library; D006026:Glycoside Hydrolases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D007716:Kluyveromyces / Q000201:enzymology / Q000235:genetics*; D008969:Molecular Sequence Data; D010368:Pectins / Q000378:metabolism*; D011096:Polygalacturonase / Q000235:genetics / Q000378:metabolism; D017422:Sequence Analysis, DNA; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D018076:DNA, Complementary; D005656:Fungal Proteins; D010368:Pectins; D006026:Glycoside Hydrolases; C403662:XghA protein, Aspergillus tubingensis; D011096:Polygalacturonase