title: Molecular characterization and expression of a novel alcohol oxidase from Aspergillus terreus MTCC6324.
abstract: The alcohol oxidase (AOx) cDNA from Aspergillus terreus MTCC6324 with an open reading frame (ORF) of 2001 bp was constructed from n-hexadecane induced cells and expressed in Escherichia coli with a yield of ∼4.2 mg protein g-1 wet cell. The deduced amino acid sequences of recombinant rAOx showed maximum structural homology with the chain B of aryl AOx from Pleurotus eryngii. A functionally active AOx was achieved by incubating the apo-AOx with flavin adenine dinucleotide (FAD) for ∼80 h at 16°C and pH 9.0. The isoelectric point and mass of the apo-AOx were found to be 6.5±0.1 and ∼74 kDa, respectively. Circular dichroism data of the rAOx confirmed its ordered structure. Docking studies with an ab-initio protein model demonstrated the presence of a conserved FAD binding domain with an active substrate binding site. The rAOx was specific for aryl alcohols and the order of its substrate preference was 4-methoxybenzyl alcohol >3-methoxybenzyl alcohol>3, 4-dimethoxybenzyl alcohol > benzyl alcohol. A significantly high aggregation to ∼1000 nm (diameter) and catalytic efficiency (kcat/Km) of 7829.5 min-1 mM-1 for 4-methoxybenzyl alcohol was also demonstrated for rAOx. The results infer the novelty of the AOx and its potential biocatalytic application.
authors.affiliation: Department of Biotechnology, Indian Institute of Technology Guwahati, Assam, India.
authors.forename: Mitun
authors.initials: M
authors.lastname: Chakraborty
authors.affiliation: Department of Biotechnology, Indian Institute of Technology Guwahati, Assam, India.
authors.forename: Manish
authors.initials: M
authors.lastname: Goel
authors.affiliation: Department of Biotechnology, Indian Institute of Technology Guwahati, Assam, India.
authors.forename: Somasekhar R
authors.initials: SR
authors.lastname: Chinnadayyala
authors.affiliation: Department of Biotechnology, Indian Institute of Technology Guwahati, Assam, India.
authors.forename: Ujjwal Ranjan
authors.initials: UR
authors.lastname: Dahiya
authors.affiliation: Department of Biotechnology, Indian Institute of Technology Guwahati, Assam, India.
authors.forename: Siddhartha Sankar
authors.initials: SS
authors.lastname: Ghosh
authors.affiliation: Department of Biotechnology, Indian Institute of Technology Guwahati, Assam, India.
authors.forename: Pranab
authors.initials: P
authors.lastname: Goswami
mesh_terms: D000429:Alcohol Oxidoreductases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D000438:Alcohols / Q000378:metabolism; D000595:Amino Acid Sequence; D001230:Aspergillus / Q000201:enzymology* / Q000235:genetics; D001483:Base Sequence; D055592:Biophysical Phenomena; D002942:Circular Dichroism; D018076:DNA, Complementary / Q000235:genetics; D015180:Electrophoresis, Gel, Two-Dimensional; D004795:Enzyme Stability; D005182:Flavin-Adenine Dinucleotide / Q000378:metabolism; D005784:Gene Amplification; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008027:Light; D008861:Microsomes / Q000378:metabolism; D062105:Molecular Docking Simulation; D008969:Molecular Sequence Data; D010449:Peptide Mapping; D010455:Peptides / Q000737:chemistry; D040901:Proteomics; D011994:Recombinant Proteins / Q000378:metabolism; D012542:Scattering, Radiation; D013050:Spectrometry, Fluorescence; D013379:Substrate Specificity; D013696:Temperature
chemical_list: D000438:Alcohols; D018076:DNA, Complementary; D010455:Peptides; D011994:Recombinant Proteins; D005182:Flavin-Adenine Dinucleotide; D000429:Alcohol Oxidoreductases; C019643:alcohol oxidase
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