A novel cellulase gene from the mulberry longicorn beetle, Apriona germari: gene structure, expression, and enzymatic activity.
We have previously cloned a cellulase [beta-1,4-endoglucanase (EGase), EC 3.2.1.4] cDNA (Ag-EGase I) belonging to glycoside hydrolase family (GHF) 45 from the mulberry longicorn beetle, Apriona germari. We report here the gene structure, expression and enzyme activity of an additional celluase (Ag-EGase II) from A. germari and also described the gene structure of Ag-EGase I. The Ag-EGase II gene spans 1033 bp and consisted of two introns and three exons coding for 239 amino acid residues. The 2713-bp-long genomic DNA of Ag-EGase I also consisted of two introns and three exons. The Ag-EGase II showed 61% protein sequence identity to Ag-EGase I and 51% to another beetle, Phaedon cochleariae, cellulase, belonging to GHF 45. The catalytic sites of GHF 45 are conserved in Ag-EGase II. The Ag-EGase II has 14 conserved cysteine residues and three putative N-glycosylation sites. Northern blot analysis confirmed midgut-specific expression of Ag-EGase II, suggesting that the midgut is the prime site for cellulase synthesis in A. germari larvae. The cDNA encoding Ag-EGase II was expressed as a 36-kDa polypeptide in baculovirus-infected insect Sf9 cells and the enzyme activity of the purified recombinant Ag-EGase II was approximately 812 U/mg of recombinant Ag-EGase II. The enzymatic properties of the purified recombinant Ag-EGase II showed the highest activity at 50 degrees C and pH 6.0, and were stable at 60 degrees C at least for 10 min.
Lee
Seong Jin
SJ
College of Natural Resources and Life Science, Dong-A University, Busan 604-714, Korea.
Lee
Kwang Sik
KS
Kim
Seong Ryul
SR
Gui
Zhong Zheng
ZZ
Kim
Yong Soon
YS
Yoon
Hyung Joo
HJ
Kim
Iksoo
I
Kang
Pil Don
PD
Sohn
Hung Dae
HD
Jin
Byung Rae
BR
D000595:Amino Acid Sequence; D000818:Animals; D002480:Cellulase / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D003001:Cloning, Molecular; D001517:Coleoptera / Q000201:enzymology* / Q000235:genetics*; D018076:DNA, Complementary / Q000235:genetics; D015971:Gene Expression Regulation, Enzymologic; D017344:Genes, Insect / Q000235:genetics; D023281:Genomics; D006863:Hydrogen-Ion Concentration; D019476:Insect Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D008969:Molecular Sequence Data; D010802:Phylogeny; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013045:Species Specificity; D013696:Temperature
D018076:DNA, Complementary; D019476:Insect Proteins; D011994:Recombinant Proteins; D002480:Cellulase