Molecular cloning, expression, and enzymatic activity of a novel endogenous cellulase from the mulberry longicorn beetle, Apriona germari

文献类型： 外文期刊

第一作者： Wei, Ya Dong

作者： Wei, Ya Dong;Lee, Kwang Sik;Gui, Zhong Zheng;Yoon, Hyung Joo;Kim, Iksoo;Zhang, Guo Zheng;Guo, Xijie;Sohn, Hung Dae;Jin, Byung Rae

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关键词： Apriona germari;baculovirus;beetle;cellulase;endoglucanase;enzyme;insect cells;N-glycosylation;N-LINKED GLYCOSYLATION;CDNA CLONING;TERMITE;SYSTEM;SITES;GENE;DEGRADATION;PROTEINS

期刊名称：COMPARATIVE BIOCHEMISTRY AND PHYSIOLOGY B-BIOCHEMISTRY & MOLECULAR BIOLOGY （ 影响因子：2.231； 五年影响因子：2.215 ）

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收录情况： SCI

摘要： 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-EGasc 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 SO cells and with endoglycosidase F of purified recombinant Ag-EGase III, demonstrating that the carbohydrate moieties are not necessary for enzyme activity. (c) 2006 Elsevier Inc. All rights reserved.

分类号： Q5

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