Comparison of gene structures and enzymatic properties between two endoglucanases from Humicola grisea |
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| Affiliation: | 1. Bioprocessing Technology Institute, A*STAR (Agency for Science, Technology and Research), 20 Biopolis Way, #06-01 Centros, 138668 Singapore, Singapore;2. Department of Chemical and Biomolecular Engineering, National University of Singapore, 4 Engineering Drive 4, Singapore 117585, Singapore;1. Tobacco Medicine and Tobacco Cessation Center, Center of Respiratory Medicine, China–Japan Friendship Hospital, Beijing, China;2. Department of Pulmonary and Critical Care Medicine, Center of Respiratory Medicine, China–Japan Friendship Hospital, Beijing, China;3. WHO Collaborating Center for Tobacco Cessation and Respiratory Diseases Prevention, Beijing, China;4. National Clinical Research Center for Respiratory Diseases, Beijing, China;5. Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, 100029, China;6. Department of Respiratory Medicine, Capital Medical University, Beijing, China;1. Key Laboratory of Separation Science for Analytical Chemistry, Dalian Institute of Chemical Physics, Chinese Academy of Science, National Chromatographic Research and Analysis Center, 457 Zhongshan Road, Dalian 116023, China;2. Graduate School of Chinese Academy of Sciences, Beijing 100039, China;1. Physique des Polymères, Institut Carnot, CIRIMAT UMR 5085, Université Paul Sabatier, Bat 3R1B2, 118 route de Narbonne, 31062 Toulouse Cedex 04, France;2. Cardiovascular Research Center, CSIC-ICCC, IIB-Sant Pau, Hospital de la Santa Creu i Sant Pau, 08025 Barcelona, Spain;1. Institute of Biomedicine & National Engineering Research Center of Genetic Medicine, Institute of Life and Health Engineering, College of Life Science and Technology, Jinan University, Guangzhou 510632, China;2. Key Laboratory of Functional Protein Research of Guangdong Higher Education Institutes, Institute of Life and Health Engineering, College of Life Science and Technology, Jinan University, Guangzhou 510632, China |
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| Abstract: | We have cloned two endoglucanase genes (egl3 and egl4) from a thermophilic fungus, Humicola grisea. The coding region of the egl3 gene was interrupted by an intron of 56-bp, and the deduced amino acid sequence of the egl3 gene was 305 amino acids in length and showed 98.4% identity with Humicola insolens EGV. The coding region of the egl4 gene was also interrupted by an intron of 173-bp, which contains 34 TTC repeated sequence units, and the deduced amino acid sequence of the egl4 gene was 227 amino acids in length and showed 61.5% identity with H. grisea EGL3. The typical hinge and the cellulose-binding domain were observed in the C-terminal region of EGL3, but they were not observed in EGL4. In the 5′ upstream region of both genes, there were a TATA box or its similar sequence, CAAT motifs, and 6-bp sites which are identical or similar to the consensus sequence for binding a catabolite repressor CREA in Aspergillus nidulans. The egl3 and the egl4 genes were expressed in Aspergillus oryzae, and the translation products were purified. The fusion protein, EGL4CBD, which consists of a catalytic domain of EGL4 and the C-terminal region of EGL3, was also constructed and produced by A. oryzae, and purified. These enzymes showed relatively high activity toward carboxymethyl cellulose (CMC) and could not hydrolyze p-nitrophenyl-β-d-glucoside and p-nitrophenyl-β-d-cellobioside. The positive effect of substituting the C-terminal region of EGL4 with that of EGL3 was observed in the hydrolysis of CMC. |
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