Molecular cloning and expression in Pichia pastoris of a Irpex lacteus exo-beta-(1-->3)-galactanase gene.
A gene encoding exo-beta-(1-->3)-galactanase from Irpex lacteus was cloned by reverse transcriptase-PCR. The deduced amino acid sequence showed high similarity with exo-beta-(1-->3)-galactanases from other sources. The molecular mass of the mature form was calculated to be 45,520 Da. The gene product expressed in Pichia pastoris specifically hydrolyzed beta-(1-->3)-galactooligosaccharides, as did other exo-beta-(1-->3)-galactanases. The recombinant enzyme showed high activity toward arabinogalactan-proteins (AGPs) from radish as well as beta-(1-->3)-galactan. Product analysis revealed that the enzyme released beta-(1-->6)-galactobiose, beta-(1-->6)-galactotriose, and alpha-L-arabinofuranosyl-(1-->3)-beta-galactosyl-(1-->6)-galactose together with Gal from beta-(1-->3)-galactans attached with and without beta-(1-->6)-galactosyl branches prepared from acacia gum. These results indicate that the exo-beta-(1-->3)-galactanase from I. lacteus efficiently hydrolyzes beta-(1-->3)-galactan main chains of AGPs by bypassing beta-(1-->6)-galactosyl side chains.
Kotake
Toshihisa
T
Division of Life Science, Graduate School of Science and Engineering, Saitama University, Japan.
Kitazawa
Kiminari
K
Takata
Ryohei
R
Okabe
Kohei
K
Ichinose
Hitomi
H
Kaneko
Satoshi
S
Tsumuraya
Yoichi
Y
D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D005685:Galactans / Q000378:metabolism; D015870:Gene Expression; D006026:Glycoside Hydrolases / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D008969:Molecular Sequence Data; D009088:Mucoproteins / Q000737:chemistry / Q000378:metabolism; D010843:Pichia / Q000235:genetics*; D010940:Plant Proteins / Q000737:chemistry / Q000378:metabolism; D020072:Polyporales / Q000201:enzymology / Q000235:genetics*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D013696:Temperature
D018076:DNA, Complementary; D005685:Galactans; D009088:Mucoproteins; D010940:Plant Proteins; D011994:Recombinant Proteins; C006619:arabinogalactan proteins; D006026:Glycoside Hydrolases; C063901:exo-beta-(1--3)-D-galactanase