Meg. Suykerbuyk et al., CLONING AND CHARACTERIZATION OF 2 RHAMNOGALACTURONAN HYDROLASE GENES FROM ASPERGILLUS-NIGER, Applied and environmental microbiology, 63(7), 1997, pp. 2507-2515
A rhamnogalacturonan hydrolase gene of Aspergillus aculeatus was used
as a probe for the cloning of two rhamnogalacturonan hydrolase genes o
f Aspergillus niger. The corresponding proteins, rhamnogalacturonan hy
drolases A and B, are 78 and 72% identical, respectively, with the A.
aculeatus enzyme, In A. niger cultures which were shifted from growth
on sucrose to growth on apple pectin as a carbon source, the expressio
n of the rhamnogalacturonan hydrolase A gene (rhgA) was transiently in
duced after 3 h of growth on apple pectin, The rhamnogalacturonan hydr
olase B gene was not induced by apple pectin, but the rhgB gene was de
repressed after 18 h of growth on either apple pectin or sucrose, Gene
fusions of the A. niger rhgA and rhgB coding regions with the strong
and inducible Aspergillus awamori exlA promoter were used to obtain hi
gh-producing A. awamori transformants which were then used for the pur
ification of the two A. niger rhamnogalacturonan hydrolases. High-perf
ormance anion-exchange chromatography of oligomeric degradation produc
ts showed that optimal degradation of an isolated highly branched pect
in fraction by A. niger rhamnogalacturonan hydrolases A and B occurred
at pH 3.6 and 4.1, respectively. The specific activities of rhamnogal
acturonan hydrolases A and B were then 0.9 and 0.4 U/mg, respectively,
which is significantly lower than the specific activity of A. aculeat
us rhamnogalacturonan hydrolase (2.5 U/mg at an optimal pH of 4.5), Co
mpared to the A enzymes, the A. niger B enzyme appears to have a diffe
rent substrate specificity, since additional oligomers are formed.