Biochemical characterization of a glycoside hydrolase family 61 endoglucanase from Aspergillus kawachii.
The glycoside hydrolase family 61 endoglucanase from Aspergillus kawachii (AkCel61) is a modular enzyme that consists of a catalytic domain and a carbohydrate-binding module belonging to family 1 (CBM1) that are connected by a Ser-Thr linker region longer than 100 amino acids. We expressed the recombinant AkCel61, wild-type enzyme (rAkCel61), and a truncated enzyme consisting of the catalytic domain (rAkCel61DeltaCBM) in Pichia pastoris and analyzed their biochemical properties. Purified rAkCel61 and rAkCel61DeltaCBM migrated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and were demonstrated to have apparent molecular masses of 81,000 and 34,000 Da, respectively. After treatment with endoglycosidase H, both proteins showed an increase in mobility, thus, demonstrating estimated molecular masses of 78,000 and 28,000 Da, respectively. Mass spectrometry analysis revealed that rAkCel61 and rAkCel61DeltaCBM expressed in P. pastoris are heterogeneous due to protein glycosylation. The rAkCel61 protein bound to crystalline cellulose but not to arabinoxylan. The rAkCel61 and rAkCel61DeltaCBM proteins produced small amounts of oligosaccharides from soluble carboxymethylcellulose. They also exhibited a slight hydrolytic activity toward laminarin. However, they showed no detectable activity toward microcrystalline cellulose, arabinoxylan, and pectin. Both recombinant enzymes also showed no detectable activity toward p-nitrophenyl beta-D: -glucoside, p-nitrophenyl beta-D: -cellobioside, and p-nitrophenyl beta-D -cellotrioside.
Koseki
Takuya
T
Department of Bioresource Engineering, Yamagata University, Tsuruoka, Japan. tkoseki@tds1.tr.yamagata-u.ac.jp
Mese
Yuichiro
Y
Fushinobu
Shinya
S
Masaki
Kazuo
K
Fujii
Tsutomu
T
Ito
Kiyoshi
K
Shiono
Yoshihito
Y
Murayama
Tetsuya
T
Iefuji
Haruyuki
H
D000595:Amino Acid Sequence; D001230:Aspergillus / Q000201:enzymology*; D050260:Carbohydrate Metabolism; D002480:Cellulase / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D003001:Cloning, Molecular; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D006868:Hydrolysis; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D005810:Multigene Family; D011108:Polymers / Q000378:metabolism; D011485:Protein Binding; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D016415:Sequence Alignment; D017384:Sequence Deletion; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
D005656:Fungal Proteins; D011108:Polymers; D011994:Recombinant Proteins; D002480:Cellulase