cDNA cloning,expression, and enzymatic activity of a novel endogenous cellulase from the beetle Batocera horsfieldi |
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| Authors: | Dingguo Xia Yadong Wei Guozheng Zhang Qiaoling Zhao Yeshun Zhang Zhonghuai Xiang Cheng Lu |
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| Institution: | 1. Institute of Sericulture and System Biology, Southwest University, Chongqing 400716, China;2. Sericultural Research Institute, Chinese Academy of Agricultural Sciences, Zhenjiang, Jiangsu 212018, China |
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| Abstract: | In this study, we report a novel cellulase β-1,4-endoglucanase (EGase), EC 3.2.1.4] cDNA (Bh-EGase II) belonging to the glycoside hydrolase family (GHF) 45 from the beetle Batocera horsfieldi. The Bh-EGase II gene spans 720 bp and consists of a single exon coding for 239 amino acid residues. Bh-EGase II showed 93.72% protein sequence identity to Ag-EGase II from the beetle Apriona germari. The GHF 45 catalytic site is conserved in Bh-EGase II. Bh-EGase II has three putative N-glycosylation sites at 56–58 (N–K–S), 99–101 (N–S–T), and 237–239 (N–Y–S), respectively. The cDNA encoding Bh-EGase II was expressed in baculovirus-infected insect BmN cells and Bombyx mori larvae. Recombinant Bh-EGase II from BmN cells and larval hemolymph had an enzymatic activity of approximately 928 U/mg. The enzymatic catalysis of recombinant Bh-EGase II showed the highest activity at 50 °C and pH 6.0. |
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| Keywords: | Ag-Egase I β-1 4-endoglucanase I from the beetle Apriona germari Ag-Egase II β-1 4-endoglucanase II from the beetle Apriona germari Bh Batocera horsfieldi Bh-EGase II β-1 4-endoglucanase II from the beetle Batocera horsfieldi Bm Bombyx mori BmN cell Bombyx mori N cell BmNPV Bombyx mori nucleopolyhedrovirus BSA bovine serum albumin CMC carboxymethyl cellulose C tremulae Chrysomela tremula DNS dinitrosalicylic D ponderosae Dendroctonus ponderosae EGase endoglucanase ELISA enzyme-linked immunosorbent assay FBS fetal bovine serum GHF glycoside hydrolase family G zeae Gibberella zeae LB Luria Bertani L decemlineata Leptinotarsa decemlineata M circinelloides Mucor circinelloides O albomarginata chamela Oncideres albomarginata chamela ORF open reading frame PBS phosphate buffer solution PFU plaque forming units RACE rapid amplification of cDNA ends R oryzae Rhizopus oryzae R speratus Reticulitermes speratus SDS-PAGE SDS-polyacrylamide gel electrophoresis SOC super optimal broth with catabolite repression S oryzae Sitophilus oryzae TMB tetramethylbenzidine |
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