title: A combined approach for improving alkaline acetyl xylan esterase production in Pichia pastoris, and effects of glycosylation on enzyme secretion, activity and stability.
abstract: High level expression of axe1, a gene previously cloned from Volvariella volvacea that encodes an acetyl xylan esterase with two potential N-linked glycosylation sites, has been achieved in Pichia pastoris using a codon-optimized axe1 synthesized by the primer extension PCR procedure. The GC content of the codon-optimized axe1 was 48.62% compared with 55.49% in the native gene. Using the codon-optimized construct, AXE1 expression in P. pastoris was increased from an undetectable level to 136.45 U/ml six days after induction of yeast cultures grown in BMMY medium. A further increase (to 463 U/ml) was achieved when conditions for yeast culture were optimized as follows: 2.8% methanol, 0.63% casamino acids, and pH 8.0. This latter value represented a 3.4-fold and 246-fold increase in the enzyme levels recorded in non-optimized P. pastoris cultures and in rice straw-grown cultures of V. volvacea, respectively. N-linked glycosylation played an essential role in AXE1 secretion but had only a slight effect on the catalytic activity and stability of the recombinant enzyme.
authors: State Key Laboratory of Forest Genetics & Biotechnology, Department of Biological Engineering, Nanjing Forestry University, Nanjing, Jiangsu 210037, China.
authors: Bin
authors: B
authors: Tian
authors: Yan
authors: Y
authors: Chen
authors: Shaojun
authors: S
authors: Ding
mesh_terms: D000115:Acetylesterase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular / Q000379:methods*; D003062:Codon; D004795:Enzyme Stability; D006031:Glycosylation; D008969:Molecular Sequence Data; D010843:Pichia / Q000235:genetics* / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D014170:Transformation, Genetic; D014415:Tunicamycin / Q000378:metabolism; D055367:Volvariella / Q000201:enzymology* / Q000235:genetics
chemical_list: D003062:Codon; D011994:Recombinant Proteins; D014415:Tunicamycin; D000115:Acetylesterase; C081851:acetylxylan esterase