Biochemical Analysis of Recombinant Fungal Mutanases

Abstract

Nucleotide sequence analysis shows that<i>Trichoderma harzianum</i> and <i>Penicillium purpurogenum</i> 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<i>-</i>terminal polysaccharide-binding domain separated by a <i>O-</i>glycosylated Pro-Ser-Thr-rich linker peptide. Each mutanase was expressed in <i>Aspergillus oryzae</i> 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 <i>K</i> _D around 0.11 and 0.13 μm at pH 7 for the <i>P. purpurogenum</i> and<i>T. harzianum</i> mutanases, respectively. Partial hydrolysis showed that the COOH<i>-</i>terminal domain of the <i>T. harzianum</i> 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.