title: High-level expression of a specific beta-1,3-1,4-glucanase from the thermophilic fungus Paecilomyces thermophila in Pichia pastoris.
abstract: In this study, a novel beta-1,3-1,4-glucanase gene (designated as PtLic16A) from Paecilomyces thermophila was cloned and sequenced. PtLic16A has an open reading frame of 945 bp, encoding 314 amino acids. The deduced amino acid sequence shares the highest identity (61%) with the putative endo-1,3(4)-beta-glucanase from Neosartorya fischeri NRRL 181. PtLic16A was cloned into a vector pPIC9K and was expressed successfully in Pichia pastoris as active extracellular beta-1,3-1,4-glucanase. The recombinant beta-1,3-1,4-glucanase (PtLic16A) was secreted predominantly into the medium which comprised up to 85% of the total extracellular proteins and reached a protein concentration of 9.1 g l(-1) with an activity of 55,300 U ml(-1) in 5-l fermentor culture. The enzyme was then purified using two steps, ion exchange chromatography, and gel filtration chromatography. The purified enzyme had a molecular mass of 38.5 kDa on SDS-PAGE. It was optimally active at pH 7.0 and a temperature of 70 degrees C. Furthermore, the enzyme exhibited strict specificity for beta-1,3-1,4-D: -glucans. This is the first report on the cloning and expression of a beta-1,3-1,4-glucanase gene from Paecilomyces sp.
authors.affiliation: Department of Biotechnology, China Agricultural University, Beijing, China.
authors.forename: Chengwei
authors.initials: C
authors.lastname: Hua
authors.forename: Qiaojuan
authors.initials: Q
authors.lastname: Yan
authors.forename: Zhengqiang
authors.initials: Z
authors.lastname: Jiang
authors.forename: Yinan
authors.initials: Y
authors.lastname: Li
authors.forename: Priti
authors.initials: P
authors.lastname: Katrolia
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D044644:Endo-1,3(4)-beta-Glucanase / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D005800:Genes, Fungal / Q000235:genetics; D008969:Molecular Sequence Data; D010143:Paecilomyces / Q000201:enzymology*; D010843:Pichia / Q000378:metabolism*; D015202:Protein Engineering; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D016415:Sequence Alignment; D013379:Substrate Specificity
chemical_list: D011994:Recombinant Proteins; D044644:Endo-1,3(4)-beta-Glucanase