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

A gene encoding a thermostable ascorbate oxidase (ASOM) was cloned fro m Acremonium sp. HI-25 and sequenced. The gene comprised 1709 bp and w as interrupted by a single intron of 57 bp. ASOM consisted of 551 amin o acids including a signal peptide with a molecular mass of 61 200, an d contained four histidine-rich regions with high sequence homology to the corresponding regions of other multicopper oxidases. The ASOM gen e was expressed in Aspergillus nidulans under the Aspergillus oryzae T aka-amylase A gene promoter. The recombinant enzyme (An-ASOM) exhibite d almost the same enzymatic properties as ASOM. The ASOM gene was muta ted by site-directed mutagenesis with reference to the amino acid sequ ences of plant enzymes to generate enzymes with altered azide sensitiv ity. Site-directed mutagenesis at the trinuclear active copper site re sulted in an increase in azide resistance; the Ala(465)Leu and Phe(463 )Trp/Ala(465)Leu mutants exhibited approximately 10 and 20% increases in azide resistance, respectively. (C) 1998 Elsevier Science B.V. All rights reserved.