title: A kinetic study of Trichoderma reesei Cel7B catalyzed cellulose hydrolysis.
abstract: One prominent feature of Trichoderma reesei (Tr) endoglucanases catalyzed cellulose hydrolysis is that the reaction slows down quickly after it starts (within minutes). But the mechanism of the slowdown is not well understood. A structural model of Tr- Cel7B catalytic domain bound to cellulose was built computationally and the potentially important binding residues were identified and tested experimentally. The 13 tested mutants show different binding properties in the adsorption to phosphoric acid swollen cellulose and filter paper. Though the partitioning parameter to filter paper is about 10 times smaller than that to phosphoric acid swollen cellulose, a positive correlation is shown for two substrates. The kinetic studies show that the reactions slow down quickly for both substrates. This slowdown is not correlated to the binding constant but anticorrelated to the enzyme initial activity. The amount of reducing sugars released after 24h by Cel7B in phosphoric acid swollen cellulose, Avicel and filter paper cellulose hydrolysis is correlated with the enzyme activity against a soluble substrate p-nitrophenyl lactoside. Six of the 13 tested mutants, including N47A, N52D, S99A, N323D, S324A, and S346A, yield ∼15-35% more reducing sugars than the wild type (WT) Cel7B in phosphoric acid swollen cellulose and filter paper hydrolysis. This study reveals that the slowdown of the reaction is not due to the binding of the enzyme to cellulose. The activity of Tr- Cel7B against the insoluble substrate cellulose is determined by the enzyme's capability in hydrolyzing the soluble substrate.
authors.affiliation: Shandong Provincial Key Laboratory of Synthetic Biology, Laboratory of Biofuels, Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences, Qingdao 266061, China.
authors.forename: Xiangfei
authors.initials: X
authors.lastname: Song
authors.affiliation: Shandong Provincial Key Laboratory of Synthetic Biology, Laboratory of Biofuels, Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences, Qingdao 266061, China.
authors.forename: Shujun
authors.initials: S
authors.lastname: Zhang
authors.affiliation: Shandong Provincial Key Laboratory of Synthetic Biology, Laboratory of Biofuels, Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences, Qingdao 266061, China.
authors.forename: Yefei
authors.initials: Y
authors.lastname: Wang
authors.affiliation: Shandong Provincial Key Laboratory of Synthetic Biology, Laboratory of Biofuels, Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences, Qingdao 266061, China.
authors.forename: Jingwen
authors.initials: J
authors.lastname: Li
authors.affiliation: Shandong Provincial Key Laboratory of Synthetic Biology, Laboratory of Biofuels, Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences, Qingdao 266061, China.
authors.forename: Chunyan
authors.initials: C
authors.lastname: He
authors.affiliation: Shandong Provincial Key Laboratory of Synthetic Biology, Laboratory of Biofuels, Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences, Qingdao 266061, China. Electronic address: yaols@qibebt.ac.cn.
authors.forename: Lishan
authors.initials: L
authors.lastname: Yao
mesh_terms: D020134:Catalytic Domain / Q000235:genetics; D002480:Cellulase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002482:Cellulose / Q000378:metabolism*; D003001:Cloning, Molecular; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006868:Hydrolysis; D007700:Kinetics; D056004:Molecular Dynamics Simulation; D016297:Mutagenesis, Site-Directed; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity; D014242:Trichoderma / Q000201:enzymology* / Q000235:genetics
chemical_list: D005656:Fungal Proteins; D011994:Recombinant Proteins; D002482:Cellulose; D002480:Cellulase
keywords: Adsorption; Cel7B; Cellulose; Kinetics; Trichoderma reesei