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
K. Takeda et al., 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, Biochimica et biophysica acta. Protein structure and molecular enzymology, 1388(2), 1998, pp. 444-456
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
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