cDNA cloning, expression, and enzymatic activity of a cellulase from the mulberry longicorn beetle, Apriona germari.
A novel cellulase [beta-1,4-endoglucanase (EGase), EC 3.2.1.4] cDNA belonging to glycoside hydrolase family (GHF) 45 was cloned from the mulberry longicorn beetle, Apriona germari. The cDNA encoding EGase of A. germari (Ag-EGase) is 711 bp long with an open reading frame of 237 amino acid residues. The Ag-EGase was closely related to another beetle, Phaedon cochleariae, cellulase and one symbiotic protist cellulase in the hindgut of the termite Reticulitermes speratus, those belonging to GHF 45. The catalytic sites of GHF 45 are conserved in Ag-EGase. Southern blot analysis of genomic DNA suggested the presence of Ag-EGase gene as a single copy and Northern blot analysis confirmed midgut-specific expression at transcriptional level. Similarly, the Ag-EGase enzyme assay exhibited high activity only in midgut tissue, suggesting that the midgut is the prime site where large quantities of EGase are synthesized for degrading the absorbed cellulose from the diet. The cDNA encoding Ag-EGase was expressed as a 29-kDa polypeptide in baculovirus-infected insect Sf9 cells and the culture supernatants of the recombinant baculovirus-infected cells showed EGase enzyme activity of 15.25 U/ml of medium containing 0.5 x 10(6) cells at 5 days post-infection (p.i.). The enzyme activity of the purified recombinant Ag-EGase expressed in baculovirus-infected insect cells was approximately 992 U per mg of recombinant Ag-EGase. The purified recombinant Ag-EGase showed the highest enzymatic activity at 50 degrees C and pH 6.0, and was stable at 55 degrees C at least for 10 min.
Lee
Seong Jin
SJ
Division of Biotechnology, College of Natural Resources and Life Science, Dong-A University, Busan 604-714, South Korea.
Kim
Seong Ryul
SR
Yoon
Hyung Joo
HJ
Kim
Iksoo
I
Lee
Kwang Sik
KS
Je
Yeon Ho
YH
Lee
Sang Mong
SM
Seo
Sook Jae
SJ
Dae Sohn
Hung
H
Jin
Byung Rae
BR
D000595:Amino Acid Sequence; D000818:Animals; D002480:Cellulase / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D003001:Cloning, Molecular; D001517:Coleoptera / Q000201:enzymology* / Q000235:genetics*; D018076:DNA, Complementary / Q000235:genetics*; D020869:Gene Expression Profiling; D017344:Genes, Insect / Q000235:genetics; D023281:Genomics; D007814:Larva / Q000201:enzymology / Q000235:genetics; D008969:Molecular Sequence Data; D009928:Organ Specificity; D010802:Phylogeny; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid
D018076:DNA, Complementary; D011994:Recombinant Proteins; D002480:Cellulase