Characterization of a new oxygen-insensitive azoreductase from Brevibacillus laterosporus TISTR1911: Toward dye decolorization using a packed-bed metal affinity reactor

• Published in: Bioresource technology 2013-12, Vol.150, p.298-306
• Main Authors: Lang, Weeranuch, Sirisansaneeyakul, Sarote, Ngiwsara, Lukana, Mendes, Sónia, Martins, Lígia O., Okuyama, Masayuki, Kimura, Atsuo
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Azo Compounds - isolation & purification; Azoreductase; Bacteria; Biodegradation, Environmental - drug effects; Biological and medical sciences; Bioreactors - microbiology; Brevibacillus - drug effects; Brevibacillus - enzymology; Brevibacillus - genetics; Brevibacillus laterosporus; Color; Coloring Agents - isolation & purification; Degradation; Dyes; Enzymes; Fundamental and applied biological sciences. Psychology; Genes, Bacterial - genetics; Hydrogen-Ion Concentration - drug effects; Immobilization; Mathematical models; Metals - chemistry; Methyl orange; Molecular Sequence Data; NADH; NADH, NADPH Oxidoreductases - chemistry; NADH, NADPH Oxidoreductases - isolation & purification; NADH, NADPH Oxidoreductases - metabolism; Oxygen - pharmacology; Phylogeny; Reactors; Recombinant; Recombinant Proteins - isolation & purification; Recycle packed-bed reactor; RNA, Ribosomal, 16S - genetics; Sequence Alignment; Substrate Specificity; Temperature
• ISSN: 0960-8524; 1873-2976
• DOI: 10.1016/j.biortech.2013.09.124

[Display omitted] •A recombinant oxygen-insensitive azoreductase BrAzo was characterized.•His6-tagged BrAzo was successfully immobilized onto an affinity packed-bed reactor.•The set-up of a recycling process with continuous flow PBR is promising.•Coupling NADH recycling produced a shorter degradation period and increased cycles. This study reports the identification of a new bacterial azoreductase from Brevibacillus laterosporus TISTR1911, its heterologous production in Escherichia coli, the biochemical characterization and immobilization for use in dye biodegradation processes. The recombinant azoreductase (BrAzo) is a monomeric FMN oxygen-insensitive enzyme with a molecular mass of 23kDa showing a broad specificity for the reduction of synthetic azo dyes. Double hexahistidine-tagged BrAzo was immobilized onto a nickel chelating column and methyl orange was used to assess its degradation potential using a packed-bed reactor. The dye degradation is described by an exponential model in a downstream batchwise continuous flow mode operated with recycling. The complete degradation of methyl orange (170μM at 600mL/h) was achieved in 3h and continued over 9 cycles. Coupling the immobilized BrAzo with glucose dehydrogenase for NADH regeneration yielded a shorter 1.5h-degradation period that was maintained throughout 16 cycles.