title: Characterization of Aspergillus niger endo-1,4-β-glucanase ENG1 secreted from Saccharomyces cerevisiae using different expression vectors.
abstract: Heterologous expression of Aspergillus niger endo-1,4-β-glucanase (ENG1) in Saccharomyces cerevisiae was tested both with an episomal plasmid vector (YEGAp/eng1) and a yeast vector capable of integration into the HO locus of the S. cerevisiae chromosome (pHO-GAPDH-eng1-KanMX4-HO). In both cases, eng1 gene expression in yeast, with its native signal sequence for secretion, was under the control of the strong glyceraldehyde 3-phosphate dehydrogenase (GAPDH) promoter. We aimed to verify how each expression system affects protein expression, posttranslational modification, and biochemical properties. Expression of eng1 from the episomal plasmid vector YEGAp/eng1 significantly slowed the growth of a yeast cell culture. However, expression of eng1 from the vector integrated into the HO locus of the chromosome did not cause growth suppression, and the enzyme activity in a culture supernatant was maintained throughout the incubation time. ENG1 has optimum catalytic activity at pH 6.0, and is stable in the pH range 5.0-9.0. The enzyme's optimum temperature for catalytic activity at pH 6.0 is 70°C; importantly, more than 95% of the enzyme's initial activity remained after a 2-h incubation at 60°C. The biochemical characterization of ENG1 confirmed the correct expression of the protein and showed that ENG1 expressed by the pHO-GAPDH-eng1-KanMX4-HO vector, in addition to its N-linked sites, is overglycosylated at its O-glycosylation sites compared with ENG1 expressed by the YEGAp/eng1 vector. It is likely that the O-glycosylated form of the A. niger ENG1 retains more stable activity during continuous cultivation of recombinant yeasts than the form that is only N-glycosylated.
authors.affiliation: Institute of Biology and Biotechnology Problems, al-Farabi Kazakh National University, Almaty, Kazakhstan.
authors.forename: S M
authors.initials: SM
authors.lastname: Taipakova
authors.affiliation: Institute of Biology and Biotechnology Problems, al-Farabi Kazakh National University, Almaty, Kazakhstan.
authors.forename: I T
authors.initials: IT
authors.lastname: Smekenov
authors.affiliation: DNA Repair Group, Institut Gustave Roussy, Villejuif, France.
authors.forename: M K
authors.initials: MK
authors.lastname: Saparbaev
authors.affiliation: Institute of Biology and Biotechnology Problems, al-Farabi Kazakh National University, Almaty, Kazakhstan Amangeldy.Bisenbaev@kaznu.kz.
authors.forename: A K
authors.initials: AK
authors.lastname: Bissenbaev
mesh_terms: D001234:Aspergillus niger / Q000201:enzymology / Q000235:genetics*; D002480:Cellulase / Q000096:biosynthesis* / Q000235:genetics; D003001:Cloning, Molecular; D015252:Deoxyribonucleases, Type II Site-Specific / Q000235:genetics; D015966:Gene Expression Regulation, Fungal; D005822:Genetic Vectors; D006031:Glycosylation; D010957:Plasmids / Q000235:genetics; D011401:Promoter Regions, Genetic; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics
chemical_list: D029701:Saccharomyces cerevisiae Proteins; C530845:HO protein, S cerevisiae; D015252:Deoxyribonucleases, Type II Site-Specific; D002480:Cellulase