Biochemical analysis of recombinant fungal mutanases. A new family of alpha1,3-glucanases with novel carbohydrate-binding domains.
Nucleotide sequence analysis shows that Trichoderma harzianum and Penicillium purpurogenum alpha1,3-glucanases (mutanases) have homologous primary structures (53% amino acid sequence identity), and are composed of two distinct domains: a NH(2)-terminal catalytic domain and a putative COOH-terminal polysaccharide-binding domain separated by a O-glycosylated Pro-Ser-Thr-rich linker peptide. Each mutanase was expressed in Aspergillus oryzae host under the transcriptional control of a strong alpha-amylase gene promoter. The purified recombinant mutanases show a pH optimum in the range from pH 3.5 to 4.5 and a temperature optimum around 50-55 degrees C at pH 5.5. Also, they exhibit strong binding to insoluble mutan with K(D) around 0.11 and 0.13 microM at pH 7 for the P. purpurogenum and T. harzianum mutanases, respectively. Partial hydrolysis showed that the COOH-terminal domain of the T. harzianum mutanase binds to mutan. The catalytic domains and the binding domains were assigned to a new family of glycoside hydrolases and to a new family of carbohydrate-binding domains, respectively.
Fuglsang
C C
CC
Novo Nordisk A/S, Bagsvaerd DK-2880, Denmark. ccf@novo.dk
Berka
R M
RM
Wahleithner
J A
JA
Kauppinen
S
S
Shuster
J R
JR
Rasmussen
G
G
Halkier
T
T
Dalboge
H
H
Henrissat
B
B
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D005656:Fungal Proteins / Q000737:chemistry* / Q000235:genetics; D006026:Glycoside Hydrolases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D007700:Kinetics; D013058:Mass Spectrometry; D008957:Models, Genetic; D008969:Molecular Sequence Data; D005810:Multigene Family; D010407:Penicillium / Q000737:chemistry / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017386:Sequence Homology, Amino Acid; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013379:Substrate Specificity; D014242:Trichoderma / Q000737:chemistry / Q000235:genetics
D005656:Fungal Proteins; D011994:Recombinant Proteins; D006026:Glycoside Hydrolases; C021780:exo-1,3-alpha-glucanase