Biochemical analysis of recombinant fungal mutanases: A new family of α1,3-glucanases with novel carbohydrate-binding domains

Abstract

Nucleotide sequence analysis shows that Trichoderma harzianum and Penicillium purpurogenum α1,3-glucanases (mutanases) have homologous primary structures (53% amino acid sequence identity), and are composed of two distinct domains: a NH2-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 α-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 °C at pH 5.5. Also, they exhibit strong binding to insoluble mutan with K(D) around 0.11 and 0.13 μM 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.