Characterization of an alpha-L-rhamnosidase from Aspergillus kawachii and its gene.
An alpha-L-rhamnosidase was purified by fractionating a culture filtrate of Aspergillus kawachii grown on L-rhamnose as the sole carbon source. The alpha-L-rhamnosidase had a molecular mass of 90 kDa and a high degree of N-glycosylation of approximately 22%. The enzyme exhibited optimal activity at pH 4.0 and temperature of 50 degrees C. Further, it was observed to be thermostable, and it retained more than 80% of its original activity following incubation at 60 degrees C for 1 h. Its T (50) value was determined to be 72 degrees C. The enzyme was able to hydrolyze alpha-1,2- and alpha-1,6-glycosidic bonds. The specific activity of the enzyme was higher toward naringin than toward hesperidin. The A. kawachii alpha-L-rhamnosidase-encoding gene (Ak-rhaA) codes for a 655-amino-acid protein. Based on the amino acid sequence deduced from the cDNA, the protein possessed 13 potential N-glycosylation recognition sites and exhibited a high degree of sequence identity (up to 75%) with the alpha-L-rhamnosidases belonging to the glycoside hydrolase family 78 from Aspergillus aculeatus and with hypothetical Aspergillus oryzae and Aspergillus fumigatus proteins.
Koseki
Takuya
T
Department of Bioresource Engineering, Faculty of Agriculture, Yamagata University, 1-23 Wakaba-machi, Tsuruoka 997-8555, Japan. tkoseki@tds1.tr.yamagata-u.ac.jp
Mese
Yuichiro
Y
Nishibori
Nahoko
N
Masaki
Kazuo
K
Fujii
Tsutomu
T
Handa
Takashi
T
Yamane
Yuichi
Y
Shiono
Yoshihito
Y
Murayama
Tetsuya
T
Iefuji
Haruyuki
H
D001230:Aspergillus / Q000201:enzymology* / Q000235:genetics; D004271:DNA, Fungal / Q000737:chemistry / Q000235:genetics; D004795:Enzyme Stability; D044950:Flavanones / Q000378:metabolism; D006026:Glycoside Hydrolases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D006031:Glycosylation; D006569:Hesperidin / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D008970:Molecular Weight; D012210:Rhamnose / Q000378:metabolism; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D013696:Temperature; D044366:Transition Temperature
D004271:DNA, Fungal; D044950:Flavanones; D006569:Hesperidin; D006026:Glycoside Hydrolases; C022525:alpha-L-rhamnosidase; C005274:naringin; D012210:Rhamnose