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 1) 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 11) from A. germari and also described the gene structure of Ag-EGase I. The AgEGase 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 11 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 11 has 14 conserved cysteine residues and three putative N-glycosylation sites. Northern blot analysis confirmed midgut-specific expression of Ag-EGase 11, suggesting that the midgut is the prime site for cellulase synthesis in A. germari larvae. The cDNA encoding Ag-EGase 11 was expressed as a 36-kDa polypeptide in baculovirus-infected insect Sf9 cells and the enzyme activity of the purified recombinant Ag-EGase 11 was approximately 812 U/mg of recombinant Ag-EGase II. The enzymatic properties of the purified recombinant Ag-EGase 11 showed the highest activity at 50 degrees C and pH 6.0, and were stable at 60 degrees C at least for 10 min. (c) 2004 Elsevier Inc. All rights reserved.