title: Expression and characterization of two secreted His6-tagged endo-beta-1,4-glucanases from the mollusc Ampullaria crossean in Pichia pastoris.
abstract: Two endo-beta-1,4-glucanase cDNAs, eg27I and eg27II, from the mollusc Ampullaria crossean were expressed in Pichia pastoris cells. The secreted His6-tagged proteins were purified in a single chromatography step. The purified recombinant EG27I and EG27II showed enzymatic activity on carboxylmethyl cellulose sodium salt at 15.31 U/mg and 12.40 U/mg, respectively. The optimum pH levels of the recombinant EG27I and EG27II were 5.5 and 5.5-6.0, respectively, and the optimum temperatures were 50 degrees C and 50 degrees C-55 degrees C, respectively. The pH stability study revealed that both EG27I and EG27II showed their highest stability at pH 8.0. Analysis of their thermostability indicated that both EG27I and EG27II were relatively stable up to 40 degrees C. Site-directed mutagenesis of Asp43 and Asp153 of both EG27I and EG27II showed that the two Asp residues are critical for the enzymatic activity.
authors.affiliation: Key Laboratory of Proteomics, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 20031, China.
authors.forename: Rui
authors.initials: R
authors.lastname: Guo
authors.forename: Ming
authors.initials: M
authors.lastname: Ding
authors.forename: Siliang
authors.initials: S
authors.lastname: Zhang
authors.forename: Genjun
authors.initials: G
authors.lastname: Xu
authors.forename: Fukun
authors.initials: F
authors.lastname: Zhao
mesh_terms: D000818:Animals; D044644:Endo-1,3(4)-beta-Glucanase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004789:Enzyme Activation; D004795:Enzyme Stability; D008974:Mollusca / Q000201:enzymology* / Q000235:genetics; D010843:Pichia / Q000201:enzymology* / Q000235:genetics; D015202:Protein Engineering / Q000379:methods*
chemical_list: D044644:Endo-1,3(4)-beta-Glucanase