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 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 61200, 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 Ala465Leu and Phe463Trp/Ala465Leu mutants exhibited approximately 10 and 20% increases in azide resistance, respectively.
Takeda
K
K
Research Laboratory, Ichibiki Co., Ltd., Toyohashi, Aichi 441-8019, Japan.
Itoh
H
H
Yoshioka
I
I
Yamamoto
M
M
Misaki
H
H
Kajita
S
S
Shirai
K
K
Kato
M
M
Shin
T
T
Murao
S
S
Tsukagoshi
N
N
D000164:Acremonium / Q000201:enzymology*; D000595:Amino Acid Sequence; D001204:Ascorbate Oxidase / Q000235:genetics*; D001386:Azides / Q000494:pharmacology*; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D003001:Cloning, Molecular; D017124:Conserved Sequence / Q000235:genetics; D003300:Copper / Q000378:metabolism; D004795:Enzyme Stability / Q000235:genetics*; D005656:Fungal Proteins / Q000235:genetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics; D015183:Restriction Mapping; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D013696:Temperature
D001386:Azides; D005656:Fungal Proteins; D011994:Recombinant Proteins; D003300:Copper; D001204:Ascorbate Oxidase