title: Engineering the pH-optimum of activity of the GH12 family endoglucanase by site-directed mutagenesis.
abstract: Endo-1,4-β-glucanase from Penicillium verruculosum (PvEGIII) belongs to family 12 of glycoside hydrolases (GH12). Analysis of the enzyme 3D model structure showed that the amino acid residue Asp98 may directly affect the pH-profile of enzyme activity since it is located at the distance of hydrogen bond formation from Glu203 that plays the role of a general acid in catalysis. The gene encoding the PvEGIII was cloned into Escherichia coli. After the deletion of two introns, a plasmid construction was obtained allowing the PvEGIII expression in E. coli. Using site-directed mutagenesis, the Asp98Asn mutant of the PvEGIII was obtained. Both the wild type and mutant PvEGIIIs were expressed in E. coli with a yield of up to 1 g/L and then isolated in a highly purified form. The enzyme specific activity against soluble carboxymethylcellulose was not changed after a single amino acid substitution. However, the pH-optimum of activity of the mutant PvEGIII was shifted from pH 4.0 to 5.1, compared to the wild type enzyme. The shift in the enzyme pH-optimum to more neutral pH was also observed on insoluble cellulose, in the process of enzymatic depigmentation of denim fabric. Similar situation featuring the effect of the Asp/Asn residue, located near the Glu catalytic residue, on the enzyme activity pH-profile has previously been described for xylanases of the GH11 family. Thus, the glycoside hydrolases belonging to the GH11 and GH12 families function by a rather similar mechanism of catalysis.
authors.affiliation: Department of Chemistry, M. V. Lomonosov Moscow State University, Leninskie Gory 1/11, Moscow 119991, Russia.
authors.forename: Vladimir I
authors.initials: VI
authors.lastname: Tishkov
authors.forename: Alexander V
authors.initials: AV
authors.lastname: Gusakov
authors.forename: Anna S
authors.initials: AS
authors.lastname: Cherkashina
authors.forename: Arkady P
authors.initials: AP
authors.lastname: Sinitsyn
mesh_terms: D000595:Amino Acid Sequence; D001224:Aspartic Acid; D020134:Catalytic Domain; D002480:Cellulase / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed / Q000379:methods*; D009154:Mutation; D010407:Penicillium / Q000201:enzymology / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
chemical_list: D011994:Recombinant Proteins; D001224:Aspartic Acid; D002480:Cellulase
keywords: CMC; Cloning; EG; Endoglucanase; PCR; Penicillium verruculosum; Pv; SDS-PAGE; Site-directed mutagenesis; Tr; Trichoderma reesei; carboxymethylcellulose; endo-1,4-β-glucanase; pH-optimum; polymerase chain reaction; sodium dodecylsulfate polyacrylamide gel electrophoresis; wild type; wt