Characterization of recombinant rhamnogalacturonan alpha-L-rhamnopyranosyl-(1,4)-alpha-D-galactopyranosyluronide lyase from Aspergillus aculeatus. An enzyme that fragments rhamnogalacturonan I regions of pectin.
The four major oligomeric reaction products from saponified modified hairy regions (MHR-S) from apple, produced by recombinant rhamnogalacturonan (RG) alpha-L-rhamnopyranosyl-(1, 4)-alpha-D-galactopyranosyluronide lyase (rRG-lyase) from Aspergillus aculeatus, were isolated and characterized by 1H-nuclear magnetic resonance spectroscopy. They contain an alternating RG backbone with a degree of polymerization of 4, 6, 8, and 10 and with an alpha-Delta-(4,5)-unsaturated D-galactopyranosyluronic acid at the nonreducing end and an L-rhamnopyranose at the reducing end. L-Rhamnopyranose units are substituted at C-4 with beta-galactose. The maximum reaction rate of rRG-lyase toward MHR-S at pH 6.0 and 31 degreesC was 28 units mg-1. rRG-lyase and RG-hydrolase cleave the same alternating RG I subunit in MHR. Both of these enzymes fragment MHR by a multiple attack mechanism. The catalytic efficiency of rRG-lyase for MHR increases with decreasing degree of acetylation. Removal of arabinose side chains improves the action of rRG-lyase toward MHR-S. In contrast, removal of galactose side chains decreased the catalytic efficiency of rRG-lyase. Native RG-lyase was purified from A. aculeatus, characterized, and found to be similar to the rRG-lyase expressed in Aspergillus oryzae.
Mutter
M
M
Wageningen Agricultural University, Department of Food Chemistry, Bomenweg 2, 6703 HD Wageningen, The Netherlands.
Colquhoun
I J
IJ
Beldman
G
G
Schols
H A
HA
Bakx
E J
EJ
Voragen
A G
AG
D000107:Acetylation; D001230:Aspergillus / Q000737:chemistry / Q000201:enzymology*; D002240:Carbohydrate Sequence; D004789:Enzyme Activation; D009682:Magnetic Resonance Spectroscopy; D008968:Molecular Conformation; D008969:Molecular Sequence Data; D010368:Pectins / Q000378:metabolism*; D011133:Polysaccharide-Lyases / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry* / Q000378:metabolism
D011994:Recombinant Proteins; C042491:rhamnogalacturonan I; D010368:Pectins; D011133:Polysaccharide-Lyases; C098240:rhamnogalacturonase B