Functional characterization and oligomerization of a recombinant xyloglucan-specific endo-β-1,4-glucanase (GH12) from Aspergillus niveus.
Xyloglucan is a major structural polysaccharide of the primary (growing) cell wall of higher plants. It consists of a cellulosic backbone (beta-1,4-linked glucosyl residues) that is frequently substituted with side chains. This report describes Aspergillus nidulans strain A773 recombinant secretion of a dimeric xyloglucan-specific endo-β-1,4-glucanohydrolase (XegA) cloned from Aspergillus niveus. The ORF of the A. niveus xegA gene is comprised of 714 nucleotides, and encodes a 238 amino acid protein with a calculated molecular weight of 23.5kDa and isoelectric point of 4.38. The optimal pH and temperature were 6.0 and 60°C, respectively. XegA generated a xyloglucan-oligosaccharides (XGOs) pattern similar to that observed for cellulases from family GH12, i.e., demonstrating that its mode of action includes hydrolysis of the glycosidic linkages between glucosyl residues that are not branched with xylose. In contrast to commercial lichenase, mixed linkage beta-glucan (lichenan) was not digested by XegA, indicating that the enzyme did not cleave glucan β-1,3 or β-1,6 bonds. The far-UV CD spectrum of the purified enzyme indicated a protein rich in β-sheet structures as expected for GH12 xyloglucanases. Thermal unfolding studies displayed two transitions with mid-point temperatures of 51.3°C and 81.3°C respectively, and dynamic light scattering studies indicated that the first transition involves a change in oligomeric state from a dimeric to a monomeric form. Since the enzyme is a predominantly a monomer at 60°C, the enzymatic assays demonstrated that XegA is more active in its monomeric state.
Departamento de Bioquímica e Imunologia, Universidade de São Paulo, Ribeirão Preto, Brazil.
André R L
AR
Damásio
Liliane F C
LF
Ribeiro
Lucas F
LF
Ribeiro
Gilvan P
GP
Furtado
Fernando
F
Segato
Fausto B R
FB
Almeida
Augusto C
AC
Crivellari
Marcos S
MS
Buckeridge
Tatiana A C B
TA
Souza
Mário T
MT
Murakami
Richard J
RJ
Ward
Rolf A
RA
Prade
Maria L T M
ML
Polizeli
D000595:Amino Acid Sequence; D001230:Aspergillus / Q000737:chemistry* / Q000201:enzymology; D001233:Aspergillus nidulans / Q000235:genetics; D002473:Cell Wall / Q000737:chemistry* / Q000201:enzymology; D002480:Cellulase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D002942:Circular Dichroism; D005656:Fungal Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D005936:Glucans / Q000737:chemistry* / Q000378:metabolism; D006026:Glycoside Hydrolases / Q000737:chemistry / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007526:Isoelectric Point; D007700:Kinetics; D008027:Light; D008969:Molecular Sequence Data; D008970:Molecular Weight; D055503:Protein Multimerization; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012542:Scattering, Radiation; D013379:Substrate Specificity; D013696:Temperature; D014990:Xylans / Q000737:chemistry* / Q000378:metabolism
D005656:Fungal Proteins; D005936:Glucans; D011994:Recombinant Proteins; D014990:Xylans; C029353:xyloglucan; D006026:Glycoside Hydrolases; D002480:Cellulase; C090774:endoglucanase EGL1; C028020:licheninase