Cloning of a thermostable ascorbate oxidase gene from Acremonium sp. HI-25 and modification of the azide sensitivity of the enzyme by site-directed mutagenesis

Abstract

A gene encoding a thermostable ascorbate oxidase (ASOM) was cloned from Acremonium sp. HI-25 and sequenced. The gene comprised 1709 bp and was interrupted by a single intron of 57 bp. ASOM consisted of 551 amino acids including a signal peptide with a molecular mass of 61 200, and contained four histidine-rich regions with high sequence homology to the corresponding regions of other multicopper oxidases. The ASOM gene was expressed in Aspergillus nidulans under the Aspergillus oryzae Taka-amylase A gene promoter. The recombinant enzyme (An-ASOM) exhibited almost the same enzymatic properties as ASOM. The ASOM gene was mutated by site-directed mutagenesis with reference to the amino acid sequences of plant enzymes to generate enzymes with altered azide sensitivity. Site-directed mutagenesis at the trinuclear active copper site resulted in an increase in azide resistance; the Ala 465Leu and Phe 463Trp/Ala 465Leu mutants exhibited approximately 10 and 20% increases in azide resistance, respectively.