Molecular cloning, expression, and enzymatic activity of a novel endogenous cellulase from the mulberry longicorn beetle, Apriona germari.
A novel endogenous beta-1,4-endoglucanase (Ag-EGase III) gene belonging to the glycoside hydrolase family (GHF) 5 was cloned from the mulberry longicorn beetle, Apriona germari. The Ag-EGase III gene spans 1061 bp and consists of a single exon coding for 325 amino acid residues. The Ag-EGase III showed 89% protein sequence identity to another beetle, Psacothea hilaris, cellulase belonging to GHF 5. The Ag-EGase III has the potential proton donor and nucleophile amino acids conserved in GHF 5 and two putative N-glycosylation sites. Northern blot and Western blot analyses showed that Ag-EGases were expressed in the gut; Ag-EGase III and Ag-EGase I were expressed in three gut regions, and no Ag-EGase II was found in hindgut, indicating that the foregut and midgut are the prime sites for cellulase synthesis in A. germari larvae. The cDNA encoding Ag-EGase III was expressed as a 47-kDa polypeptide in baculovirus-infected insect Sf9 cells and the enzyme activity of the purified recombinant Ag-EGase III was approximately 1037 U per mg of recombinant Ag-EGase III. The enzymatic property of the purified recombinant Ag-EGase III showed the highest activity at 55 degrees C and pH 6.0, and was stable at 60 degrees C at least for 10 min. In addition, the N-glycosylation of Ag-EGase III was revealed by treatment with tunicamycin of recombinant virus-infected insect Sf9 cells and with endoglycosidase F of purified recombinant Ag-EGase III, demonstrating that the carbohydrate moieties are not necessary for enzyme activity.
Wei
Ya Dong
YD
Department of Applied Biotechnology, College of Natural Resources and Life Science, Dong-A University, Busan 604-714, Korea.
Lee
Kwang Sik
KS
Gui
Zhong Zheng
ZZ
Yoon
Hyung Joo
HJ
Kim
Iksoo
I
Zhang
Guo Zheng
GZ
Guo
Xijie
X
Sohn
Hung Dae
HD
Jin
Byung Rae
BR
D000818:Animals; D002480:Cellulase / Q000235:genetics* / Q000378:metabolism; D003001:Cloning, Molecular; D001517:Coleoptera / Q000201:enzymology* / Q000235:genetics; D015971:Gene Expression Regulation, Enzymologic; D006031:Glycosylation / Q000187:drug effects; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase / Q000494:pharmacology; D031625:Morus; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D016415:Sequence Alignment; D013045:Species Specificity; D014415:Tunicamycin / Q000494:pharmacology
D011994:Recombinant Proteins; D014415:Tunicamycin; D002480:Cellulase; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase