A xyloglucan-specific family 12 glycosyl hydrolase from Aspergillus niger: recombinant expression, purification and characterization.
A new GH12 (glycosyl hydrolase 12) family XEG [xyloglucan-specific endo-beta-1,4-glucanase (EC 3.2.1.151)] from Aspergillus niger, AnXEG12A, was overexpressed, purified and characterized. Whereas seven xyloglucanases from GH74 and two xyloglucanases from GH5 have been characterized previously, this is only the third characterized example of a GH12 family xyloglucanase. GH12 enzymes are structurally and mechanistically distinct from GH74 enzymes. Although over 100 GH12 sequences are now available, little is known about the structural and biochemical bases of xyloglucan binding and hydrolysis by GH12 enzymes. Comparison of the AnXEG12A cDNA sequence with the genome sequence of A. niger showed the presence of two introns, one in the coding region and the second one in the 333-nt-long 3'-untranslated region of the transcript. The enzyme was expressed recombinantly in A. niger and was readily purified from the culture supernatant. The isolated enzyme appeared to have been processed by a kexin-type protease, which removed a short prosequence. The substrate specificity was restricted to xyloglucan, with cleavage at unbranched glucose in the backbone. The apparent kinetic parameters were similar to those reported for other xyloglucan-degrading endoglucanases. The pH optimum (5.0) and temperature resulting in highest enzyme activity (50-60 degrees C) were higher than those reported for a GH12 family xyloglucanase from Aspergillus aculeatus, but similar to those of cellulose-specific endoglucanases from the GH12 family. Phylogenetic, sequence and structural comparisons of GH12 family endoglucanases helped to delineate features that appear to be correlated to xyloglucan specificity.
Master
Emma R
ER
Centre for Structural and Functional Genomics, Concordia University, 7141 Sherbrooke Street West, Montreal, QC, Canada H4B 1R6.
Zheng
Yun
Y
Storms
Reginald
R
Tsang
Adrian
A
Powlowski
Justin
J
D001234:Aspergillus niger / Q000201:enzymology*; D005936:Glucans / Q000378:metabolism; D006026:Glycoside Hydrolases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D010802:Phylogeny; D013379:Substrate Specificity; D013696:Temperature; D014990:Xylans / Q000378:metabolism
D005936:Glucans; D014990:Xylans; C029353:xyloglucan; D006026:Glycoside Hydrolases; C072818:xyloglucan endo(1-4)-beta-D-glucanase