Biochemical analysis of recombinant fungal mutanases. A new family of alpha1,3-glucanases with novel carbohydrate-binding domains

• Published in: The Journal of biological chemistry 2000-01, Vol.275 (3), p.2009-2018
• Main Authors: Fuglsang, C.C, Berka, R.M, Wahleithner, J.A, Kauppinen, S, Shuster, J.R, Rasmussen, G, Halkier, T, Dalboge, H, Henrissat, B
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; amino acid sequences; Aspergillus oryzae; Base Sequence; binding sites; Cloning, Molecular; complementary DNA; enzyme activity; Fungal Proteins - chemistry; Fungal Proteins - genetics; genbank/af214480; genbank/af214481; gene expression; gene transfer; genes; Glycoside Hydrolases - chemistry; Glycoside Hydrolases - genetics; Glycoside Hydrolases - isolation & purification; Hypocrea lixii; Kinetics; Mass Spectrometry; Models, Genetic; Molecular Sequence Data; Multigene Family; mutan; nucleotide sequences; O-glycoside hydrolases; Penicillium - chemistry; Penicillium - genetics; Penicillium purpurogenum; polysaccharides; purification; Recombinant Proteins - chemistry; Recombinant Proteins - genetics; Reverse Transcriptase Polymerase Chain Reaction; Sequence Homology, Amino Acid; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Substrate Specificity; Trichoderma - chemistry; Trichoderma - genetics
• ISSN: 0021-9258; 1083-351X

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.