A novel cellulase gene from the mulberry longicorn beetle, Apriona germari: gene structure, expression, and enzymatic activity |
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Authors: | Lee Seong Jin Lee Kwang Sik Kim Seong Ryul Gui Zhong Zheng Kim Yong Soon Yoon Hyung Joo Kim Iksoo Kang Pil Don Sohn Hung Dae Jin Byung Rae |
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Affiliation: | aCollege of Natural Resources and Life Science, Dong-A University, Busan 604-714, Korea;bDepartment of Agricultural Biology, National Institute of Agricultural Science and Technology, Suwon 441-100, Korea |
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Abstract: | We have previously cloned a cellulase [β-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 °C and pH 6.0, and were stable at 60 °C at least for 10 min. |
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Keywords: | Apriona germari Baculovirus cDNA cloning Cellulase Endoglucanase Enzyme Genomic structure Insect cells Mulberry longicorn beetle |
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