An Aspergillus oryzae acetyl xylan esterase: Molecular cloning and characteristics of recombinant enzyme expressed in Pichia pastoris

• Published in: Journal of biotechnology 2006-02, Vol.121 (3), p.381-389
• Main Authors: Koseki, Takuya, Miwa, Yozo, Akao, Takeshi, Akita, Osamu, Hashizume, Katsumi
• Format: Article
• Language: English
• Subjects: Acetyl xylan esterase; Acetylesterase - analysis; Acetylesterase - chemistry; Acetylesterase - genetics; Acetylesterase - isolation & purification; Acetylesterase - metabolism; Amino Acid Sequence; amino acid sequences; Aoaxe gene; Aspergillus oryzae; Aspergillus oryzae - enzymology; Aspergillus oryzae - genetics; Base Sequence; Biological and medical sciences; Biotechnology; Catalysis; clones; Cloning, Molecular; complementary DNA; Conserved Sequence; Electrophoresis, Polyacrylamide Gel; Enzyme Stability; esterases; Fundamental and applied biological sciences. Psychology; Fungal Proteins - chemistry; Fungal Proteins - genetics; Fungal Proteins - isolation & purification; Fungal Proteins - metabolism; gene expression; gene transfer; genes; Genetic Engineering; Heterologous expression; Hydrogen-Ion Concentration; Immunoblotting; molecular cloning; Molecular Sequence Data; Molecular Weight; nucleotide sequences; Pichia - genetics; Pichia pastoris; Protein Sorting Signals; recombinant proteins; Recombinant Proteins - chemistry; Recombinant Proteins - isolation & purification; Recombinant Proteins - metabolism; Sequence Homology, Amino Acid; Substrate Specificity; Temperature; Time Factors; xylan
• ISSN: 0168-1656; 1873-4863
• DOI: 10.1016/j.jbiotec.2005.07.015

We screened 20,000 clones of an expressed sequence tag (EST) library from Aspergillus oryzae ( http://www.nrib.go.jp/ken/EST/db/index.html) and obtained one cDNA clone encoding a protein with similarity to fungal acetyl xylan esterase. We also cloned the corresponding gene, designated as Aoaxe, from the genomic DNA. The deduced amino acid sequence consisted of a putative signal peptide of 31-amino acids and a mature protein of 276-amino acids. We engineered Aoaxe for heterologous expression in P. pastoris. Recombinant AoAXE (rAoAXE) was secreted by the aid of fused α-factor secretion signal peptide and accumulated as an active enzyme in the culture medium to a final level of 190 mg/l after 5 days. Purified rAoAXEA before and after treatment with endoglycosidase H migrated by SDS-PAGE with a molecular mass of 31 and 30 kDa, respectively. Purified rAoAXE displayed the greatest hydrolytic activity toward α-naphthylacetate (C2), lower activity toward α-naphthylpropionate (C3) and no detectable activity toward acyl-chain substrates containing four or more carbon atoms. The recombinant enzyme catalyzed the release of acetic acid from birchwood xylan. No activity was detectable using methyl esters of ferulic, caffeic or sinapic acids. rAoAXE was thermolabile in comparison to other AXEs from Aspergillus.