Catalase from the silkworm, Bombyx mori: gene sequence, distribution, and overexpression.
Living organisms require mechanisms regulating reactive oxygen species (ROS) such as hydrogen peroxide and superoxide anion. Catalase is one of the regulatory enzymes and facilitates the degradation of hydrogen peroxide to oxygen and water. Biochemical information on an insect catalase is, however, insufficient. Using mRNA from fat body of the silkworm, Bombyx mori, a cDNA encoding a putative catalase was amplified by reverse transcriptase-polymerase chain reaction and sequenced. The deduced amino acid sequence comprised 507 residues with more than seventy residues forming a scaffold for a heme cofactor conserved. The sequence showed 71% and 66% identities to those of the Drosophila melanogaster and Apis mellifera catalases, respectively; the catalase from B. mori was estimated to be phylogenetically close to that from A. mellifera. The transcripts of the gene and the catalase activity were distributed in diverse tissues of B. mori, suggesting its ubiquitous nature. Using the gene, a recombinant catalase (rCAT) was functionally overexpressed in a soluble form using Escherichia coli, purified to homogeneity, and characterized. The pH-optimum of rCAT was broad around pH 8.0. More than 80% of the original rCAT activity was retained after incubation in the following conditions: at pH 8-11 and 4 degrees C for 24 h; at pH 7 and temperatures below 50 degrees C for 30 min. The Michaelis constant for hydrogen peroxide was evaluated to be 28 mM at pH 6.5 and 30 degrees C. rCAT was suggested to be a member of the typical catalase family.
Yamamoto
Kohji
K
Laboratory of Insect Genetic Resources, Faculty of Agriculture, Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka, Fukuoka 812-8581, Japan. yamamok@agr.kyushu-u.ac.jp
Banno
Yutaka
Y
Fujii
Hiroshi
H
Miake
Fumio
F
Kashige
Nobuhiro
N
Aso
Yoichi
Y
D000273:Adipose Tissue / Q000201:enzymology; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D012831:Bombyx / Q000201:enzymology / Q000235:genetics* / Q000254:growth & development; D002374:Catalase / Q000235:genetics*; D003001:Cloning, Molecular; D017931:DNA Primers; D005260:Female; D015971:Gene Expression Regulation, Enzymologic; D006801:Humans; D007814:Larva; D008297:Male; D008969:Molecular Sequence Data; D009928:Organ Specificity; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000235:genetics; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid
D017931:DNA Primers; D012333:RNA, Messenger; D002374:Catalase